WO2026013165A1 - Combination of ship2 inhibitor and plk1 inhibitor for use in the treatment of cancer - Google Patents

Abstract

This application discloses a SH2 domain-containing inositol 5'-phosphatase 2 (SHIP2) inhibitor and a Polo-like kinase I (PLK1) inhibitor for use in medicine, in particular for use in the treatment of cancer, more in particular of oesophageal cancer and colorectal cancer. Also disclosed is a kit of parts comprising a SHIP2 inhibitor and a PLK1 inhibitor for use in the treatment of cancer. Further disclosed are a kit of parts comprising a dosage form of a SHIP2 inhibitor and a dosage form of a PLK1 inhibitor and a pharmaceutical composition comprising a SHIP2 inhibitor and a PLK1 inhibitor.

Description

COMBINATION OF SHIP2 INHIBITOR AND PLK1 INHIBITOR FOR USE IN THE TREATMENT OF CANCER

FIELD OF THE INVENTION

The invention is broadly in the medical field, particularly in the field of treatment or prevention of cancer.

BACKGROUND OF THE INVENTION

The phosphoinositide 3-kinase (PI3K)/mTOR/AKT pathway ranks as the most frequently activated pathway in human cancer. Operating downstream of several common receptor families, the PI3K/AKT signalling pathway assumes a central role in fostering cancer cell growth, survival, and metabolic processes. Frequent cancer-associated alterations in this pathway encompass (i) activating mutations in PIK3C, the gene encoding the p110ct catalytic subunit of Class 1 A PI3K, and (ii) loss-of-function mutations and deletions in PTEN. Both mutations stand among the most prevalent drivers of human cancer, leading to constitutive PI3K/mTOR/AKT pathway activation. Given the critical role of the PI3K/AKT pathway in cancer progression, multiple inhibitors have been designed to selectively dampen its activation in cancer cells. However, due to the pathway’s ubiquity and its influence on vital physiological functions, these inhibitors elicit side effects that can compromise treatment efficacy and patients’ quality of life.

In the PI3K pathway, PI 5-phosphatases, and particularly SH2-containing 5’ inositol phosphates 1 and 2 (SHIP1/2) emerge as pivotal enzymes regulating the PI3K pathway, and thereby having crucial roles in cancer. P5 5-phosphates in general are capable of catalysing phosphatidylinositol 3,4 biphosphate by acting on phosphatidylinositol 3,4,5-triphosphate thus affecting and controlling the pool of phosphatidylinositol 3,4,5-triphosphate, which serve as crucial signalling molecules, notably influencing (cancer) cell adhesion, migration and invasion. Notably, SHIP2 engages in scaffolding complexes with multiple proteins, thereby governing critical cellular mechanisms such as receptordockingatthe plasma membrane and Erk pathway activation. Interestingly, the role of SHIP2 in cancer cells exhibits context dependent characteristics. While SHIP2’s role appears pro-tumorigenic in some cancer types, it serves an opposing role in other cancer types. SUMMARY

The present invention is at least in part based on the inventor’s discovery that oesophageal cancer, and in particular oesophageal squamous cell carcinoma, represents the cancer type with the highest frequency of INPPL1 amplification, encoding SHIP2. Moreover, a positive correlation between INPPL1 amplification and upregulated SHIP2 transcript levels was found. Leveraging SHIP2 knockdown in vitro further showed the regulatory influence of SHIP2 expression on oesophageal squamous cell carcinoma cell survival and adhesion. The inventors further demonstrate that pharmacological inhibition of SHIP2 phosphatase activity effectively suppressed oesophageal squamous cell carcinoma cell survival both in vitro and in vivo. Finally, it is shown that inhibition of both SHIP2 and PLK1 results in a synergistic effect on not only oesophageal squamous cell carcinoma cells, but also in various colorectal cancer (CRC) cells. The teaching of these illustrative embodiments is thus generally applicable to cancers, and in particularto cancers overexpressing SHIP2, such as due to INPPL1 amplification (i.e., cancers in which at least some cancerous cells contain more than two alleles of INPPL1 ).

Accordingly, an aspect of the invention provides a SH2 domain-containing inositol 5’- phosphatase 2 (SHIP2) inhibitor and a Polo-like kinase I (PLK1 ) inhibitor for use in the treatment of cancer in a subject.

Another aspect provides a SHIP2 inhibitorfor use in the treatment of cancer in a subject, wherein the SHIP2 inhibitor is ad ministered to the subject simultaneously or sequentially in any order with a PLK1 inhibitor.

Still another aspect provides a PLK1 inhibitor for use in the treatment of cancer in a subject, wherein the PLK1 inhibitor is administered to the subject simultaneously or sequentially in any orderwith a SHIP2 inhibitor.

Further provided is a kit of parts comprising a SHIP2 inhibitor and a PLK1 inhibitor for use in the treatment of cancer in a subject.

Another aspect of the invention is a kit of parts comprising a dosage form of a SHI P2 inhibitor and a dosage form of a PLK1 inhibitor.

Also provided is a pharmaceutical composition comprisinga SHIP2 inhibitor a nd a PLK1 inhibitor.

The present application further provides a method of treating cancer in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a SHIP2 inhibitor and a PLK1 inhibitor, or a pharmaceutical composition comprising a SHIP2 inhibitor and a PLK1 inhibitor. In some embodiments, the cancer is a cancer that overexpresses SHIP2.

In some embodiments, the cancer is a solid tumour; preferably selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/CNS cancer, cervical cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, endometrial cancer, pancreatic cancer, sarcoma, and melanoma; more preferably the cancer is selected from oesophageal cancer, head and neck cancer, and colorectal cancer; even more preferably the cancer is oesophageal cancer or colorectal cancer. In some embodiments, the cancer is oesophageal cancer. In some embodiments, the cancer is colorectal cancer.

In some embodiments, the subject is a human subject.

These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of the appended claims is hereby specifically incorporated in this specification.

BRIEF DESCRIPTION OF DRAWINGS

These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of the appended claims is hereby specifically incorporated in this specification.

Figure 1. INPPL1 Encoding SHIP2 Is Frequently Amplified in Oesophageal Squamous Cell Carcinoma eSCC. A. Heatmap showing the frequent amplification of the 11 q13 locus (n=96 eSCC). B. Histogram summarizing the copy number alteration frequency of INPPL1 (encoding SHIP2) in the 15 cancer types in which the INPPL1 CAN frequency is the highest among 32 studies from the cancer genome atlas (TCGA). C. Box plot showing the INPPL1 mRNA expression compared to INPPL1 copy number alteration. D. Scheme summarizing the role of SHIP2 in the PI3K/AKT signalling pathway. E. Bar plot summarizing the copy number alteration frequency of 11 genes related to the PI3K/AKT signalling pathway, as well as CCND1 in eSCC (n=96). F. Immunostaining of SHIP2 in human eSCC sample. These data highlight SHIP2 expression in eSCC human tumour epithelial cells. G. SHIP2 staining intensity in tissue microarray. Samples were divided into non-differentiated tumours, moderately differentiated tumours and well- differentiated tumours (n=60). H. SHIP2 staining intensity depending on tumour stage (n=60). I. Positive correlation between INPPL1 absolute copy number and INPPL1 mRNA expression. All omics data from human eSCC samples were obtained from The Cancer Genome Atlas. All the omics data from the human eSCC cell lines were obtained from the Cancer Cell Line Encyclopaedia. J. Violin plot depicting the expression of the different PI 5-phosphatases in human eSCC cells. These data show that SHIP2 is the most abundant PI 5-phophatase in eSCC cells. On the opposite, INPP5D mRNA encoding SHIP1 is virtually absent from these cells.

Figure 2. INPPL1 Knockdown Decreases Cell Survival and AKT Phosphorylation in eSCC. A. Experimental design to measure the impact of SHIP2 KD on eSCC cells survival. B. Knockdown (KD) of SHIP2 using two different siRNAs targeting SHIP2 (siSHIP2 and siSHIP2#2) resulted in a reduced survival of KYSE-410 cells compared to a control siRNA (siCT). C. Western blot illustrating SHIP2 KD following transfection with a control siRNA (siCT) and a siRNA specifically targeting SHIP2 (siSHIP2). D. Western blot analysis of SHIP2 expression on the selected human eSCC cell lines. Two different SHIP2 antibodies have been used to validate the data. E. Cell survival measured by MTS assay in 6 different cell lines transfected with siCT or siSHIP2. Data obtained in at least 3 independent assays. Data are represented as Box plot and whiskers (Min to Max). F. SHIP2, total AKT, pAKT S473 and GAPDH protein expression measured by Western blot in siCT and siSHIP2 condition, with or without 50ng/ml EGF stimulation (5 min for KYSE-30 cells or 10min for COLO-680 cells) after cell starvation. G. Venn diagram depicting the overlap between the transcripts measured by RNA-seq that are significantly (FDR < 5%) upregulated or downregulated following SHIP2 KD compared to control condition in 3 different eSCC cell lines (KYSE-520, KYSE-30, and KYSE-410). H. Volcano plot showing the transcripts that are significantly modified (FDR < 5%) in KYSE-30 and KYSE-410 lines. Most downregulated genes are highlighted in blue. I. Top 5 most significantly enriched gene sets in KYSE-410 RNA-seq data. These data highlight the down-expression of transcripts related to cell division and to Myc targets following SHIP2 KD. Altogether, these data show that SHIP2 KD decreases eSCC cell proliferation, whether they have a high or low SHIP2 expression level.

Figure 3. SHIP2 Pharmacological Inhibition Represses eSCC Survival In Vitro In Cells and In Vivo In Mice

A. Experimental design to measure the impact of SHIP2 inhibition on eSCC cells survival. B. Histogram summarizing the impact of SHIP2 inhibition by K149 compared to vehicle alone (DMSO) on eSCC cells survival measured by MTS assay in multiple eSCC cells. C. Histogram summarizing the impact of SHIP2 inhibition by K149 compared to DMSO on eSCC cells proliferation measured by EdU incorporation in multiple eSCC cells. D. Total AKT, pAKT S473 and GAPDH protein expression measured by Western blot in DMSO and K149 conditions, with or without 50 ng/ml EGF stimulation (5 or 10 min) after starvation. E. Experimental design to measure the impact of K149-mediated SHIP2 inhibition in xenotransplants in vivo. F. Immunostaining for E-cadherin and the proliferation marker Ki67 or apoptosis marker active- Casp3 (Caspase3) in KYSE-410 derived eSCC xenotranslants treated with K149 or DMSO. G. Quantification of the proportion of E-cadherin+/Ki67+ or E-cadherin+/active-Caspase3+ in KYSE- 410 derived eSCC xenotransplants treated with K149 or DMSO for a week. H. Experimental design to measure by RNA-seq the impact of K149-mediated SHIP2 inhibition on KYSE-410 eSCC cells’ transcriptome in vitro. I. Volcano plot showing the transcripts that are significantly modified (FDR < 5%) in KYSE-410 cells following SH I P2 inhibition. Most downregulated genes are highlighted in blue. J. Top 4 most significantly enriched gene sets in KYSE-410 RNA-seq data. These data highlight the down-expression of transcripts related to cell division following SHIP2 pharmacological inhibition. K. Heatmap summarizing the top 30 most downregulated genes upon SHIP2 inhibition in KYSE-410 cells. 16 out 30 transcripts are related to cell division. In (B), (C) and (G), P-values were measured using Mann-Whitney non-parametric test.

Figure 4. SHIP2 pharmacological inhibition decreases eSCC cell proliferation in vitro.

A. Experimental design to measure the impact of K149- orAS1949490-mediated SHIP2 inhibition in the eSCC cell line KYSE-410. Vehicle (DMSO) treatment is used as control treatment.

B. SHIP2 inhibition byAS1949490 significantly downregulated 15 out of 16 transcripts related to cell division that are downregulated following K149 mediated SHIP2 inhibition (see Fig.3K).

C and D. Heatmap analysis (C) and Venn diagram (D) representing gene expression analysis by RNA-seq in KYSE-410 cells and showing the overlap between the transcripts that are significantly (FDR < 1%) upregulated or downregulated following treatment of cells with AS1949490 or K149.

Figure 5. SHIP2 Knockout Induces Compensatory Mechanisms To Maintain AKT Signalling pathway

A. Experimental design to knockout SHIP2 using CRISPR/Cas9 in KYSE-410 cells. 4 distinct KO lines were generated and compared to a control line (same procedure but without gRNA). B. SHIP2, total AKT, phosphorylated AKT (Ser473) and GAPDH protein expression measured by Western blot in a wild-type and 4 different SHIP2 KO eSCC cells. C. Brightfield pictures of KYSE- 410 cells under control conditions or following SHIP2 knockout (clones G4 and G10) reveal no morphological differences between these conditions (left). Additionally, cell survival measured usingthe MTS assay also showed no difference between these conditions, suggestingthat SHIP2 knockout has been compensated. D. Principal component analysis on RNA-seq data from wildtype and 4 SHIP2 KO eSCC cells. E. Venn diagram depicting the overlap between the transcripts measured by RNA-seq that are significantly (FDR < 5%) upregulated or downregulated following SHIP2 KO compared to control condition i.e. native/original cells in 4 different KO clones (G4, H3, D4 and G10). F. KMean analysis of RNA-seq from wild-type and 4 different SHIP2 KO eSCC lines and table summarizing the gene sets enriched in clusters associated to H3, D4 and G10 SHIP2 KO eSCC clones compared to G4 and control clones. G. Volcano plot showing the transcripts that are significantly modified (FDR < 5%) in SHIP2 KO clone G4 cells compared to control KYSE- 410 eSCC cells. Most downregulated genes are highlighted in blue and upregulated genes in red. H. Top 5 most significantly enriched gene sets in G4 SHIP2 KO clone RNA-seq data. These data highlight the upregulation of transcripts related to cell division ('E2F_targets' and 'G2/M_checkpoint') and the Myc pathway.

Figure 6. SHIP2 Inhibition In eSCC Cells Enhances Sensitivity To PLK1 Inhibitor. A. Top 10 most significantly downregulated transcripts in KYSE-410 eSCC cells upon SHIP2 pharmacological inhibition with K149. These data highlight the strong downregulation of the cell cycle regulator Polo-like kinase-1 (PLK1) mRNA. B. PLK1 mRNA expression measured by RNA sequencing in 3 different samples in control condition (siCT) or 3 days following SHIP2 KD (siSHIP2). C. Experimental design to test K149 and Volasertib, alone or in combination on eSCC cell survival in vitro. D. MTS assay showing Volasertib dose response in KYSE-410 cells. E. Box plot showing the percentage of eSCC cell survival measured after a 72-hour treatment with DMSO, K149, Volasertib or the combination of K149 and Volasertib. 5 different cell lines were tested (KYSE-520 ; COLO-680; KYSE-410 ; TE-11 and KYSE-30). F. Synergy maps calculated based on the combination of 8 concentrations of K149 and Volasertib. Mean ZIP synergy scores are indicated above the maps. Gray squares delineate the most synergistic areas. ZIP scores in these areas are indicated on the right of the maps. 3 different eSCC cell lines were analyzed : KYSE-30 ; KYSE-410 and COLO-680N. G. Box plot showing the percentage of KYSE-410 eSCC cell survival measured after a 72-hour treatment with DMSO, K149, Volasertib or the combination of K149 and Volasertib (limiting concentrations identified in the synergy map shown in (F)). H. SHIP2, total AKT, pAKT S473 and GAPDH protein expression measured by Western blot in eSCC cells treated (1) DMSO, (2) BYL719, (3) Volasertib, (4) K149, (5) BYL719 and K149, and (6) K149 and Volasertib. These data show that Volasertib on its own decreases S473 pAKT expression level and that the combination of K149 and Volasertib virtually abolishes S473 pAKT. These data suggest that synergy between SHIP2 inhibitor and PLK1 inhibitor results, at least partially, in the regulation of AKT phosphorylation and PLK1 expression by SHIP2 and in the regulation of AKT phosphorylation by the PLK1 inhibitor. Figure 7. Synergistic effect between K149 and the PLK1 inhibitor GSK461364. Synergy maps calculated based on the combination of 8 concentrations of K149 and GSK461364 in KYSE-410 eSCC cells. ZIP energy scores are indicated above the map. Gray squares delineate the most synergistic areas. ZIP scores in this area are indicated on the right of the map.

Figure 8. A and B Synergy maps calculated based on the combination of 8 concentrations of the SHIP2 inhibitor AS1949490 and 7 concentrations of the PLK1 inhibitor Volasertib in KYSE-410 eSCC cells. Synergy scores ZIP (A) or Bliss (B) in this area are indicated on the right of the map. Scores >10 suggest synergy between the two drugs tested.

Figure 9. SHIP2 inhibitor K149 synergizes with PLK1 inhibitor Volasertib to inhibit human colorectal cancer cell growth in vitro. A.. Experimental design for the screening of synergy in 4 different colorectal cancer cell lines (HCT116, DLD1 , LOVO, and HT29). Cells were treated with increasing concentrations of the SHIP2 inhibitor K149 and the PLK1 inhibitor Volasertib, either alone or in combination, for 72h. Cell viability was assessed using the MTS assay. B. Synergy maps calculated based on the combination of 8 concentrations of K149 and Volasertib in 4 different colorectal cancer cell types: HCT116, DLD1 , LOVO, and HT29. Synergy scores ZIP are indicated for each cell type. Synergy scores (ZIP - Zero Interaction Potency) were calculated using SynergyFinder+. ZIP scores >10 suggest synergy between the two drugs tested.

DESCRIPTION OF EMBODIMENTS

As used herein, the singularforms “a”, “an”, and “the” include both singularand plural referents unless the context clearly dictates otherwise.

The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of” and “consisting essentially of”, which enjoy well-established meanings in patent terminology.

The recitation of numerical ranges by endpoints includes all intervening values between the lower and upper endpoints, as well as the recited endpoints. Intervening values may be integers or, where applicable, fractions, i.e., more broadly any real numbers such as any rational numbers. This applies to numerical ranges irrespective of whether they are introduced by the expression “from... to...” or the expression “between... and...” or another expression. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, each sub-range between any stated value in a stated range and any other stated value in that stated range is also specifically disclosed. Each sub-range between any stated value in a stated range and either the lower endpoint or the upper endpoint of the stated range is also specifically disclosed. The stated value may be an isolated value or an endpoint of a range subsumed by or overlapping with the stated range. For example, for a stated range with lower endpoint L1 and upper endpoint U1 (i.e., stated range L1 -U1 ) and a stated sub-range nested within the stated range with lower endpoint L2 and upper endpoint U2 (i.e., stated sub-range L2- U2), also specifically disclosed are the subranges L1 -L2, L1 -U2, L2-U1 , and U2-U1 .

The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1 % or less, and still more preferably +/-0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed.

Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1 , 2, 3, 4, 5, 6, 7 or more.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention.

The present inventors experimentally demonstrated inter alia that oesophageal cancer, and in particular oesophageal squamous cell carcinoma, shows the highest frequency of INPPL1 amplification, encoding SHIP2. Moreover, a positive correlation between INPPL1 amplification and upregulated SHIP2 transcript levels was found. Leveraging SHIP2 knockdown in vitro further showed the regulatory influence of SHIP2 expression on oesophageal squamous cell carcinoma cell survival and adhesion. The inventors further demonstrate that pharmacological inhibitor of SHIP2 phosphatase activity effectively suppressed oesophageal squamous cell carcinoma cell survival both in vitro and in vivo. The inventors also found that inhibition of both SHIP2 and PLK1 results in a synergistic effect on both oesophageal squamous cell carcinoma cells and colorectal cancer cells. The inventors further realised the relevance of these experimental findings to cancers in general, and to cancers overexpressing SHIP2 in particular, such cancers overexpressing SHIP2 due to INPPL1 amplification (i.e., cancers in which at least some cancerous cells contain more than two alleles of INPPL1 ).

Accordingly, an aspect of the invention relates to a SHIP2 inhibitor and a PLK1 inhibitor for use in the treatment of cancer in a subject. In some embodiments, a SHIP2 inhibitor and a PLK1 inhibitor are provided for use of the treatment of a solid tumour, preferably selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/CNS cancer, cervical cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, endometrial cancer, pancreatic cancer, sarcoma, and melanoma. In some embodiments, a SHIP2 inhibitor and a PLK1 inhibitor are provided for use of the treatment of cancer selected from the group consisting of oesophageal cancer, colorectal cancer and head and neck cancer. In some embodiments, a SHIP2 inhibitor and a PLK1 inhibitor are provided for use of the treatment of cancer selected from the group consisting of oesophageal cancer, and colorectal cancer. In some embodiments, a SHIP2 inhibitor and a PLK1 inhibitor are provided for use of the treatment of oesophageal cancer, preferably oesophageal squamous cell carcinoma (eSCC). In some embodiments, a SHIP2 inhibitor and a PLK1 inhibitor are provided for use of the treatment of colorectal cancer.

Also provided is a SHIP2 inhibitor for use in the treatment of cancer in a subject wherein the SHIP2 inhibitor is administered to the subject simultaneously or sequentially in any order with a PLK1 inhibitor. In some embodiments, the SHIP2 inhibitor is for use in the treatment of a solid tumour, preferably selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/CNS cancer, cervical cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, endometrial cancer, pancreatic cancer, sarcoma, and melanoma; more preferably for use in the treatment of cancer selected from the group consisting of oesophageal cancer, colorectal cancer and head and neck cancer; even more preferably for use in the treatment of oesophageal cancer, such as oesophageal squamous cell carcinoma (eSCC), or colorectal cancer, wherein the SHIP2 inhibitor is ad ministered to the subject simultaneously or sequentially in any order with a PLK1 inhibitor. In some embodiments, a SHIP2 inhibitor for use in the treatment of oesophageal cancer, such as oesophageal squamous cell carcinoma (eSCC), in a subject is provided, wherein the SHIP2 inhibitor is ad ministered to the subject simultaneously or sequentially in any order with a PLK1 inhibitor. In some embodiments, a SHIP2 inhibitor for use in the treatment of colorectal cancer, in a subject is provided, wherein the SHIP2 inhibitor is administered to the subject simultaneously or sequentially in any order with a PLK1 inhibitor.

Further provided is a PLK1 inhibitor for use in the treatment of cancer in a subject wherein the PLK1 inhibitor is administered to the subject simultaneously or sequentially in any order with a SHIP2 inhibitor. In some embodiments, the PLK1 inhibitor is for use in the treatment of a solid tumour, preferably selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/CNS cancer, cervical cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, endometrial cancer, pancreatic cancer, sarcoma, and melanoma; more preferably for use in the treatment of cancer selected from the group consisting of oesophageal cancer, colorectal cancer and head and neck cancer; even more preferably for use in the treatment of oesophageal cancer, such as oesophageal squamous cell carcinoma (eSCC), or colorectal cancer, wherein the PLK1 inhibitor is administered to the subject simultaneously or sequentially in any order with a SHIP2 inhibitor. In some embodiments, a PLK1 inhibitorfor use in the treatment of oesophageal cancer, such as oesophageal squamous cell carcinoma (eSCC), in a subject is provided, wherein the PLK1 inhibitor is administered to the subject simultaneously or sequentially in any order with a SHIP2 inhibitor. In some embodiments, a PLK1 inhibitor for use in the treatment of colorectal cancer, in a subject is provided, wherein the PLK1 inhibitor is administered to the subject simultaneously or sequentially in any order with a SHIP2 inhibitor.

Another aspect of the invention provides a kit of parts comprising a SHIP2 inhibitor and a PLK1 inhibitor for use in the treatment of cancer in a subject. In some embodiments, said kit of parts comprises a dosage form of the SHIP2 inhibitor and a dosage form of the PLK1 inhibitorfor use in the treatment of cancer in a subject. In some embodiments, the dosage form of the SHIP2 inhibitor and the dosage form of the PLK1 inhibitor are separate and allow either simultaneous or sequential in any order administration of both dosage forms. In some embodiments, the kit of parts comprises a pharmaceutical composition comprising a SHIP2 inhibitor and a pharmaceutical composition comprising a PLK1 inhibitor. In some further embodiments, the pharmaceutical composition comprising the SHIP2 inhibitor and the pharmaceutical composition comprising the PLK1 inhibitor are separate and allow either simultaneous or sequential in any order administration of both pharmaceutical compositions. In some embodiments, the kit of parts comprises a pharmaceutical composition comprising a SHIP2 inhibitor and a PLK1 inhibitor.

The term “SHIP2” or “SH2 domain-containing inositol 5’-posphatase 2” is also known as “Skicontaining inositol 5’-posphatase 2”, “phosphatidylinositol 3.4.5-triphosphate 5-phosphatase 2”, “Inositol polyphosphate phosphatase-like protein 1 ” or “SHIP-2”. SHIP2 is one of several human enzymes that can dephosphorylate at the 5-position of phosphoinositides or inositol phosphates. It specifically catalyses the dephosphorylation at the 5-position of mainly phosphatidylinositol 3,4,5-triphosphate to generate phosphatidylinositol 3.4-bisphosphate. The SHIP2 protein is encoded by the INPPL1 gene, also known as the “inositol polyphosphate phosphatase like 1 ” gene.

By means of further guidance, the human INPPL1 gene, that encodes the SHIP2 protein, is annotated and available under NCBI Gene ID No. 3636. The messenger RNA (mRNA) reference sequence of Homo sapiens INPPL1 is annotated in NCBI Genbank Accession number NM_001567.4, with transcript variants XM_047426888.1 (isoform XI ), XM_047426890.1 (isoform X2), XM_047426891 .1 (isoform X3), XM_047426892.1 (isoform X4), XM_047426893.1 (isoform X5).

The protein encoded by the INPPL1 gene is the SHIP2 protein. The reference human SHI P2 protein sequence is annotated under NCBI Genbank accession number NP_001558.3 with transcript variants XP_047282844.1 (isoform X1 ), XP_047282846.1 (isoform X2), XP_047282847.1 (isoform X3), XP_047282848.1 (isoform X4), and XP_047282849.1 (isoform X5). The canonical human SHIP2 protein sequence annotated in Uniprot (www.uniprot.org) is accession number 015357, and is by means of example reproduced below (SEQ ID NO: 1 ).

MASACGAPGPGGALGSQAPSWYHRDLSRAAAEELLARAGRDGSFLVRDSESVAGAFALCVLYQKHVH TYRILPDGEDFLAVQTSQGVPVRRFQTLGELIGLYAQPNQGLVCALLLPVEGEREPDPPDDRDASDGED EKPPLPPRSGSTSISAPTGPSSPLPAPETPTAPAAESAPNGLSTVSHDYLKGSYGLDLEAVRGGASHLPH LTRTLATSCRRLHSEVDKVLSGLEILSKVFDQQSSPMVTRLLQQQNLPQTGEQELESLVLKLSVLKDFLS GIQKKALKALQDMSSTAPPAPQPSTRKAKTIPVQAFEVKLDVTLGDLTKIGKSQKFTLSVDVEGGRLVLLR RQRDSQEDWTTFTHDRIRQLIKSQRVQNKLGWFEKEKDRTQRKDFIFVSARKREAFCQLLQLMKNKHS KQDEPDMISVFIGTWNMGSVPPPKNVTSWFTSKGLGKTLDEVTVTIPHDIYVFGTQENSVGDREWLDLL RGGLKELTDLDYRPIAMQSLWNIKVAVLVKPEHENRISHVSTSSVKTGIANTLGNKGAVGVSFMFNGTSF GFVNCHLTSGNEKTARRNQNYLDILRLLSLGDRQLNAFDISLRFTHLFWFGDLNYRLDMDIQEILNYISR KEFEPLLRVDQLNLEREKHKVFLRFSEEEISFPPTYRYERGSRDTYAWHKQKPTGVRTNVPSWCDRILW KSYPETHIICNSYGCTDDIVTSDHSPVFGTFEVGVTSQFISKKGLSKTSDQAYIEFESIEAIVKTASRTKFFIE FYSTCLEEYKKSFENDAQSSDNINFLKVQWSSRQLPTLKPILADIEYLQDQHLLLTVKSMDGYESYGECV VALKSMIGSTAQQFLTFLSHRGEETGNIRGSMKVRVPTERLGTRERLYEWISIDKDEAGAKSKAPSVSRG SQEPRSGSRKPAFTEASCPLSRLFEEPEKPPPTGRPPAPPRAAPREEPLTPRLKPEGAPEPEGVAAPPPK NSFNNPAYYVLEGVPHQLLPPEPPSPARAPVPSATKNKVAITVPAPQLGHHRHPRVGEGSSSDEESGGT LPPPDFPPPPLPDSAIFLPPSLDPLPGPWRGRGGAEARGPPPPKAHPRPPLPPGPSPASTFLGEVASGD DRSCSVLQMAKTLSEVDYAPAGPARSALLPGPLELQPPRGLPSDYGRPLSFPPPRIRESIQEDLAEEAPC LQGGRASGLGEAGMSAWLRAIGLERYEEGLVHNGWDDLEFLSDITEEDLEEAGVQDPAHKRLLLDTLQ LSK (SEQ ID NO: 1 )

The term “PLK1 ” or “Polo-like kinase 1 ” refers to a Ser/Thr protein kinase that belongs to the CDC5/Polo subfamily. It is highly expressed during mitosis and elevated levels are found in many different types of cancer.

By means of further guidance, the human PLK1 gene is annotated and available under NCBI Gene ID No. 5347. The messenger RNA (mRNA) reference sequence of Homo sapiens PLK1 is annotated in NCBI Genbank Accession number NM_005030.6.

The reference human PLK1 protein sequence is annotated under NCBI Genbank accession number NP_005021.2. The canonical human PLK1 protein sequence annotated in Uniprot (www.uniprot.org) is accession number P53350, and is by means of example reproduced below (SEQ ID NO: 2).

MSAAVTAGKLARAPADPGKAGVPGVAAPGAPAAAPPAKEIPEVLVDPRSRRRYVRGRFLGKGGFAKCFE ISDADTKEVFAGKIVPKSLLLKPHQREKMSMEISIHRSLAHQHWGFHGFFEDNDFVFWLELCRRRSLL ELHKRRKALTEPEARYYLRQIVLGCQYLHRNRVIHRDLKLGNLFLNEDLEVKIGDFGLATKVEYDGERKKT LCGTPNYIAPEVLSKKGHSFEVDVWSIGCIMYTLLVGKPPFETSCLKETYLRIKKNEYSIPKHINPVAASLIQ I<MLQTDPTARPTINELLNDEFFTSGYIPARLPITCLTIPPRFSIAPSSLDPSNRI<PLTVLNI<GLENPLPERPR EKEEPWRETGEWDCHLSDMLQQLHSVNASKPSERGLVRQEEAEDPACIPIFWVSKWVDYSDKYGLG YQLCDNSVGVLFNDSTRLILYNDGDSLQYIERDGTESYLTVSSHPNSLMKKITLLKYFRNYMSEHLLKAG ANITPREGDELARLPYLRTWFRTRSAIILHLSNGSVQINFFQDHTKLILCPLMAAVTYIDEKRDFRTYRLSLL EEYGCCKELASRLRYARTMVDKLLSSRSASNRLKAS (SEQ ID NO: 2) A skilled person can appreciate that any sequences represented in sequence databases or in the present specification may be of precursors of peptides, polypeptides, proteins, or nucleic acids ad may include parts which are processed away from mature molecules.

The term “protein” as used throughout this specification generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. As used herein, the term may encompass proteins that carry one or more co- or post-expression-type modifications of the polypeptide chains(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes protein variants or mutants which carry amino acid sequence variations vis-a-vis corresponding native proteins, such as, e.g. amino acid deletions, additions and/or substitutions. The term contemplates both full-length proteins and protein parts or fragments, e.g., naturally-occurring protein parts that ensue form processing of such full-length proteins.

The term “polypeptide” as used throughout this specification generailyencompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. Without limitation, protein, polypeptides or peptides can be produced recombinantly by a suitable host or host cell expression system and isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free transcription and/or translation, or non-biological protein, polypeptide or peptide synthesis.

The term “nucleic acid” as used throughout this specification typically refers to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units. A nucleoside unit commonly includes a heterocyclic base and a sugar group. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases.

The term “nucleic acid” further preferably encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA/RNA hybrids. RNA is inclusive of RNAi (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), tRNA (transfer RNA, whether charged or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA). A nucleic acid can be naturally occurring, e.g., present in or isolated from nature, can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesized. A naturally occu rring variant of a given sequence refers to all variants of the sequence which encode the same functional protein and that are present in or can be isolated from nature. Typically, this includes all variants of the sequence encountered in mammals, more particularly humans. It will be understood that variants from closely related species will have a higher sequence identity than variants from evolutionary more distant species. In particular embodiments, the natural variant of a given sequence has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%; 90% or 95% sequence identity with the given sequence.

Without limitation, nucleic acids can be produced recombinantly by a suitable host or host cell expression system and isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free transcription, or non-biological nucleic acid synthesis. A nucleic acid can be double-stranded, partly double-stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, the nucleic acid can be circular or linear.

The reference to any peptide, polypeptide, or nucleic acid, corresponds to the peptide, polypeptide, protein, or nucleic acid, commonly known under the respective designations in the art. The terms encompass such peptides, polypeptides, proteins, or nucleic acids, of any organism where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and non-human mammals, still more preferably of humans.

Depending on the nature of the subject under consideration, the SHIP2 inhibitor targets a SHIP2 peptide, polypeptide, protein, or nucleic acid which is of animal origin, preferably warm-blooded animal origin, more preferablyvertebrate origin, yet more preferably mammalian origin, including human origin and non-human mammalian origin, still more preferably human origin.

Depending on the nature of the subject under consideration, the PLK1 inhibitor targets a PLK1 peptide, polypeptide, protein, or nucleic acid which is of animal origin, preferably warm-blooded animal origin, more preferablyvertebrate origin, yet more preferably mammalian origin, including human origin and non-human mammalian origin, still more preferably human origin. Unless otherwise apparent from the context, reference herein to any peptide, polypeptide, protein, or nucleic acid, or fragment thereof may generally also encompass modified forms of said marker, peptide, polypeptide, protein, or nucleic acid, or fragment thereof, such as bearing post-expression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, and the like.

The terms “SHIP2 inhibitor” or “inhibitor of SHI P2” may be used interchangeably herein and refer to any agent that can be regarded to have an inhibitory effect on SHIP2. It is known to a skilled person that an inhibitor can act in different ways and methods to determine whether an agent has an inhibitory effect on SHIP2 are within the skill set of a person skilled in the art. Hence, a SHIP2 inhibitor according to the invention may be effective in any possible manner, i.e. said inhibitor can either act on DNA level, RNA level, or protein level. In preferred embodiments, the SHIP2 inhibitor acts on protein level. By means of guidance and not limitation, the inhibitor may inhibit, reduce, or decrease the activity of the SHIP2 inhibitor.

In some embodiments, the SHIP2 inhibitor as disclosed herein can also have an inhibitory effect on another target, for example on SHIP1 . Such inhibitor may be denoted as “non-specific” or “non-selective” in that it inhibits at least SHIP2 but not necessarily substantially only SHIP2, i.e., it may inhibit one or more other targets, such as one or more SHIP proteins other than SHIP2, such as both SHIP1 and SHIP2. In some embodiments, the SHIP2 inhibitor can thus be an inhibitor that has an inhibitory effect on both SHIP1 and SHIP2. SHIP1 as used herein refers to “SH2 domain-containing inositol 5’-phosphatase 1 ” or “SH2-containing inositol 5’-phosphatase 1 ”. The human SHIP1 protein is encoded by the human “Inositol Polyphosphate-5-phosphatase D” gene, also known as the “Inpp5d” gene.

By means of further guidance, the human INPP5D gene, that encodes the SHIP1 protein, is annotated and available under NCBI Gene ID No. 3635. The messenger RNA (mRNA) reference sequence of Homo sapiens INNP5D is annotated in NCBI Genbank Accession number NM_00107915.3, with transcript variants NM_005541 .5 (isoform b)

The protein encoded by the INPP5D gene is the SHIP1 protein. The reference human SHIP1 protein sequence is annotated under NCBI Genbank accession number NP_001017915.1 with transcript variant NP_005532.2 (isoform b). The canonical human SHIP1 protein sequence annotated in Uniprot (www.uniprot.org) is accession number Q92835, and is by means of example reproduced below (SEQ ID NO: 3). MVPCWNHGNITRSKAEELLSRTGKDGSFLVRASESISRAYALCVLYRNCVYTYRILPNEDDKFTVQASEG VSMRFFTKLDQLIEFYKKENMGLVTHLQYPVPLEEEDTGDDPEEDTVESWSPPELPPRNIPLTASSCEAK EVPFSNENPRATETSRPSLSETLFQRLQSMDTSGLPEEHLKAIQDYLSTQLAQDSEFVKTGSSSLPHLKKL TTLLCKELYGEVIRTLPSLESLQRLFDQQLSPGLRPRPQVPGEANPINMVSKLSQLTSLLSSIEDKVKALLH EGPESPHRPSLIPPVTFEVKAESLGIPQKMQLKVDVESGKLIIKKSKDGSEDKFYSHKKILQLIKSQKFLNK LVILVETEKEKILRKEYVFADSKKREGFCQLLQQMKNKHSEQPEPDMITIFIGTWNMGNAPPPKKITSWFL SKGQGKTRDDSADYIPHDIYVIGTQEDPLSEKEWLEILKHSLQEITSVTFKTVAIHTLWNIRIWLAKPEHE NRISHICTDNVKTGIANTLGNKGAVGVSFMFNGTSLGFVNSHLTSGSEKKLRRNQNYMNILRFLALGDK KLSPFNITHRFTHLFWFGDLNYRVDLPTWEAETIIQKIKQQQYADLLSHDQLLTERREQKVFLHFEEEEIT FAPTYRFERLTRDKYAYTKQKATGMKYNLPSWCDRVLWKSYPLVHWCQSYGSTSDIMTSDHSPVFATF EAGVTSQFVSKNGPGTVDSQGQIEFLRCYATLKTKSQTKFYLEFHSSCLESFVKSQEGENEEGSEGELV VKFGETLPKLKPIISDPEYLLDQHILISIKSSDSDESYGEGCIALRLEATETQLPIYTPLTHHGELTGHFQGEI KLQTSQGKTREKLYDFVKTERDESSGPKTLKSLTSHDPMKQWEVTSRAPPCSGSSITEIINPNYMGVGPF GPPMPLHVKQTLSPDQQPTAWSYDQPPKDSPLGPCRGESPPTPPGQPPISPKKFLPSTANRGLPPRTQ ESRPSDLGKNAGDTLPQEDLPLTKPEMFENPLYGSLSSFPKPAPRKDQESPKMPRKEPPPCPEPGILSP SIVLTKAQEADRGEGPGKQVPAPRLRSFTCSSSAEGRAAGGDKSQGKPKTPVSSQAPVPAKRPIKPSRS EINQQTPPTPTPRPPLPVKSPAVLHLQHSKGRDYRDNTELPHHGKHRPEEGPPGPLGRTAMQ (SEQ ID NO: 3)

In some embodiments, the SHIP2 inhibitor as disclosed herein is a specific SHIP2 inhibitor, also referred to as a selective SHIP2 inhibitor. A “selective SHIP2 inhibitor” or a “specific SHIP2 inhibitor” is an inhibitor that selectively inhibits SHIP2, while it does not inhibit any other target, in particular any other SHIP, or inhibits one or more other targets, in particular one or more other SHIPs, to a much lower extent. For example, a specific SHIP2 inhibitor will inhibit SHIP2 but not SHIP1 , or it will be more active towards SHIP2. Particularly, a selective or specific SHIP2 inhibitor will be, for example in an in vitro experiment with all conditions other than the SHIP target being the same , at least 2 times more active towards the targeted SHIP2 than to other SHIP molecules, such as for example SHIP1 ; at least 5 times more active towards the targeted SHIP2 than to other SHIP molecules, such as for example SHIP1 ; or at least 10 times more active, or at least 50 times more active, or at least 100 times more active, or at least 1000 times more active, or even 1 x10⁴ or more times more active towards the targeted SHIP2 than to other SHIP molecules, such as for example SHIP1 . The SHIP2 inhibitor of the present invention preferably specifically acts on SHIP2 and/or on the INPPL1 gene, in particular to inhibit SHIP2. In preferred embodiments, the SHIP2 inhibitor of the invention acts on the SHIP2 protein level. In some preferred embodiments, the SHIP2 inhibitor of the invention inhibits, reduces, or decreases the activity of the SHIP2 protein. In some further embodiments, the SHIP2 inhibitor of the invention is a small molecule inhibitor of SHIP2.

Any meaningful extent of inhibition of the expression and/or activity of SHIP2 is envisaged. Hence, the terms “inhibit” or “inhibited”, or “downregulate” or “downregulated”, or “reduce” or “reduced”, or “decrease” or “decreased” may in appropriate contexts, such as in experimental or therapeutic contexts, denote a statistically significant decrease relative to a reference. The skilled person is able to select such a reference. An example of a suitable reference may be the SHIP2 or INPPL1 expression and/or activity when exposed to a ‘negative control’ molecule, such as a molecule of similar composition but known to have no effects on SHIP2. For example, such decrease may fall outside of error margins for the reference (as expressed, for example, by standard deviation or standard error, or by a predetermined multiple thereof, e.g., ±1xSD, ±2xSD, or ±1xSE, ±2xSE). By means of an illustration, the expression and/or activity of SHIP2 may be considered reduced when it is decreased by at least 10%, such as by at least 20% or by at least 30%, preferably by at least 40%, such as by at least 50% or by at least 60%, more preferably by at least 70%, such as by at least 80% or by at least 90% or more, as compared to the reference, up to and including a 100% decrease (i.e., absent activity as compared to the reference).

SHIP2 is a phosphatase with catalytic activity. Accordingly, the present SHIP2 inhibitor may in certain embodiments inhibit one or more of the various molecular functions and activities of SHIP2. By means of example and not limitation, one or more of the following can be inhibited: translocation capacity of SHIP2 to the plasma membrane, or the catalytic activity of SHIP2. A skilled person can design in vitro or cell assays to measure such one or more activity of SHIP2.

The terms “PLK1 inhibitor” or “inhibitor of PLK1 ” may be used interchangeably herein and refer to any agent that can be regarded to have an inhibitory effect on PLK1. It is known to a skilled person that an inhibitor can act in different ways and methods to determine whether an agent has an inhibitory effect on PLK1 are within the skill set of a person skilled in the art. Hence, a PLK1 inhibitor according to the invention may be effective in any possible manner, i.e. said inhibitor can either act on DNA level, RNA level, or protein level. In preferred embodiments, the PLK1 inhibitor acts on protein level. By means of guidance and not limitation, the inhibitor may inhibit, reduce, or decrease the activity of the PLK1 inhibitor. In some embodiments, the PLK1 inhibitor as disclosed herein can also have an inhibitory effect on another target, for example on PLK2. Such inhibitor may be denoted as “non-specific” or“non- selective” in that it inhibits at least PLK1 but not necessarily substantially only PLK1 , i.e. , it may inhibit one or more other targets, such as one or more PLK proteins other than PLK1 , such as both PLK1 and PLK2. In some embodiments, the PLK1 inhibitor can thus be an inhibitor that has an inhibitory effect on both PLK1 and PLK2. PLK2 as used herein refers to “plo-like kinase 2”. The human PLK2 protein is encoded by the human “polo-like kinase 2” gene, also known as the “PLK2” gene.

By means of further guidance, the human PLK2 gene is annotated and available under NCBI Gene ID No. 10769. The messenger RNA (mRNA) reference sequence of Homo sapiens PLK2 is annotated in NCBI Genbank Accession number NM_006622.4 for isoform 1 and NM_001252226.2 for isoform 2.

The reference human PLK2 protein sequence is annotated under NCBI Genbank accession number NP_006613.2 for isoform 1 and NP_001239155.1 for isoform 2. The canonical human PLK2 protein sequence annotated in Uniprot (www.uniprot.org) is accession number Q9NYY3, and is by means of example reproduced below (SEQ ID NO: 4).

MELLRTITYQPAASTKMCEQALGKGCGADSKKKRPPQPPEESQPPQSQAQVPPAAPHHHHHHSHSG PEISRIIVDPTTGKRYCRGKVLGKGGFAKCYEMTDLTNNKVYAAKIIPHSRVAKPHQREKIDKEIELHRILH HKHWQFYHYFEDKENIYILLEYCSRRSMAHILKARKVLTEPEVRYYLRQIVSGLKYLHEQEILHRDLKLG NFFINEAMELKVGDFGLAARLEPLEHRRRTICGTPNYLSPEVLNKQGHGCESDIWALGCVMYTMLLGR PPFETTNLKETYRCIREARYTMPSSLLAPAKHLIASMLSKNPEDRPSLDDIIRHDFFLQGFTPDRLSSSCC HTVPDFHLSSPAKNFFKKAAAALFGGKKDKARYIDTHNRVSKEDEDIYKLRHDLKKTSITQQPSKHRTDE ELQPPTTTVARSGTPAVENKQQIGDAIRMIVRGTLGSCSSSSECLEDSTMGSVADTVARVLRGCLENMP EADCIPKEQLSTSFQWVTKWVDYSNKYGFGYQLSDHTVGVLFNNGAHMSLLPDKKTVHYYAELGQCS VFPATDAPEQFISQVTVLKYFSHYMEENLMDGGDLPSVTDIRRPRLYLLQWLKSDKALMMLFNDGTFQV NFYHDHTKIIICSQNEEYLLTYINEDRISTTFRLTTLLMSGCSSELKNRMEYALNMLLQRCN (SEQ ID NO: 4)

In some embodiments, the PLK1 inhibitor as disclosed herein is a specific PLK1 inhibitor, also referred to as a selective PLK1 inhibitor. A “selective PLK1 inhibitor” or a “specific PLK1 inhibitor” is an inhibitor that selectively inhibits PLK1 , while it does not inhibit any other target, in particular any other PLK, or inhibits one or more other targets, in particular one or more other PLKs, to a much lower extent. For example, a specific PLK1 inhibitor will inhibit PLK1 but not PLK2, or it will be more active towards PLK1. Particularly, a selective or specific PLK1 inhibitor will be, for example in an in vitro experimentwith all conditions otherthan the PLK target being the same, at least 2 time more active towards the targeted PLK1 than to other PLK molecules, such as for example PLK2; at least 5 times more active towards the targeted PLK1 than to other PLK molecules, such as for example PLK2, or at least 10 times more active, or at least 50 times more active, or at least 100 times more active, or at least 1000 times more active, or even 1x 10⁴ times more active towards the targeted PLK1 than to other PLK molecules, such as for example PLK2.

The PLK1 inhibitor of the present invention preferably specifically acts on PLK1 and/or on the PLK1 gene, in particular to inhibit PLK1. In preferred embodiments, the PLK1 inhibitor of the invention acts on the PLK1 protein level. In some preferred embodiments, the PLK1 inhibitor of the invention inhibits, reduces, or decreases the activity of the PLK1 protein. In some further embodiments, the PLK1 inhibitor of the invention is a small molecule inhibitor of PLK1 .

Any meaningful extent of inhibition of the expression and/or activity of PLK1 is envisaged. Hence, the terms “inhibit” or “inhibited”, or “downregulate” or “downregulated”, or “reduce” or “reduced”, or “decrease” or “decreased” may in appropriate contexts, such as in experimental or therapeutic contexts, denote a statistically significant decrease relative to a reference. The skilled person is able to select such a reference. An example of a suitable reference may be the PLK1 expression and/or activity when exposed to a ‘negative control’ molecule, such as a molecule of similar composition but known to have no effects on PLK1. For example, such decrease may fall outside of error margins for the reference (as expressed, for example, by standard deviation or standard error, or by a predetermined multiple thereof, e.g., ±1xSD, ±2xSD, or ±1xSE, ±2xSE). By means of an illustration, the expression and/or activity of SHIP2 may be considered reduced when it is decreased by at least 10%, such as by at least 20% or by at least 30%, preferably by at least 40%, such as by at least 50% or by at least 60%, more preferably by at least 70%, such as by at least 80% or by at least 90% or more, as compared to the reference, up to and including a 100% decrease (i.e., absent activity as compared to the reference).

PLK1 is a microtubule-associated protein essential for correct behaviour of spindle poles and M- phase microtubules. Accordingly, the present PLK1 inhibitor may in certain embodiments inhibit one or more of the various molecular functions and activities of PLK1 . By means of example and not limitation, one or more of the following can be inhibited: regulation of cell division or phosphorylation of downstream targets that regulate mitosis. A skilled person can design in vitro or cell assays to measure such one or more activity of PLK1 . As used herein, the term “small molecule inhibitor”, such as a small molecular inhibitor of SHIP2 or a small molecular inhibitor of PLK1 , refers to a small molecule drug inhibitor typically having a molecular weight of less than about 1000. The small molecule inhibitors as used herein are thus small molecule drugs having a molecular weight of less than about 1000 Daltons. Exemplary molecular weights of small molecular inhibitors include molecular weights of: less than about 950 Daltons; less than about 900 Daltons; less than about 950 Daltons; less than about 900 Daltons; less than about 950 Daltons; less than about 900 Daltons; less than about 850 Daltons; less than about 800 Daltons; less than about 750 Daltons; less than about 700 Daltons; less than about 650 Daltons; less than about 600 Daltons; less than about 550 Daltons; less than about 500 Daltons; and less than about 450 Daltons.

In some embodiments, the SHIP2 inhibitor as disclosed herein is a compound of Formula I or Formula II, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof: wherein X" represents a pharmaceutically acceptable anion, such as for example Cl’, F’, Br or I’.

In preferred embodiments, X’ in Formula I is Cl’, and the compound is represented by Formula IV:

Formula IV

In some embodiments, the SHIP2 inhibitor is a compound of Formula I, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prod rugthereof. In some embodiments, the SHIP2 inhibitor is a compound of Formula I.

In some embodiments, the SHIP2 inhibitor is a compound of Formula IV, and said compound is also referred to herein as compound K149. Compound K149 is known as a pan-SHIP1/2 inhibitor.

In some embodiments, the SHIP2 inhibitor is a compound of Formula II, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prod ru thereof. In some embodiments, the SHIP2 inhibitor is a compound of Formula II. The compound of Formula II as used herein, is also referred to herein as compound AS1949490. Compound AS1949490 is known as a selective SHIP2 inhibitor.

In some embodiments, the PLK1 inhibitor as disclosed herein is a compound of Formula III, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof:

Formula III In some embodiments, the PLK1 inhibitor as disclosed herein is a compound of Formula III. The compound of Formula III is also referred to herein as volasertib. Volasertib is a highly potent PLK1 inhibitor with IC50 of 0.87 nM in a cell-free assay.

In some embodiments, the PLK1 inhibitor as disclosed herein is a compound of Formula V. The compound of Formula V is also referred to herein as GSK461364. GSK461364 is a selective, reversible and ATP-competitive PLK1 inhibitorwith a K, value of 2.2 nM.

Formula V

As used herein and unless otherwise stated, the term "stereoisomer" refers to all possible different isomeric as well as conformational forms which the compounds of structural formula herein may possess, in particular all possible stereochemically and conformationally isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Some compounds of the present invention may exist in different tautomeric forms, all of the latter being included within the scope of the present invention.

The present invention includes all possible stereoisomers of compounds of formula I, II, III or V and any subgroup thereof. When a compound is desired as a single enantiomer, such may be obtained by stereospecific synthesis, by resolution of the final product or any convenient intermediate, or by chiral chromatographic methods as each are known in the art. Resolution of the final product, an intermediate, or a starting material may be effected by any suitable method known in the art. See, for example, Stereochemistry of Organic Compounds by E. L. Eliel, S. H. Wilen, and L. N. Mander (Wiley- Interscience, 1994), incorporated by reference with regard to stereochemistry. A structural isomer is a type of isomer in which molecules with the same molecular formula have different bonding patterns and atomic organization. Where structural isomers are interconvertible via a low energy barrier, tautomeric isomerism ('tautomerism') can occur. This can take the form of proton tautomerism in compounds of the invention containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety.

The term “prodrug” as used herein means the pharmacologically acceptable derivatives such as esters, amides and phosphates, such that the resulting in vivo biotransformation product of the derivative is the active drug. The reference by Goodman and Gilman (The Pharmacological Basis of Therapeutics, 8th Ed, McGraw-Hill, Int. Ed. 1992, “Biotransformation of Drugs”, p 13-15) describing pro-drugs generally is hereby incorporated. Prodrugs of the compounds of the invention can be prepared by modifying functional groups present in said component in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent component. Typical examples of prodrugs are described for instance in WO 99/33795, WO 99/33815, WO 99/33793 and WO 99/33792 all incorporated herein by reference. Prodrugs are characterized by increased bio-availability and are readily metabolized into the active inhibitors in vivo. The term “prodrug”, as used herein, means any compound that will be modified to form a drug species, wherein the modification may take place either inside or outside of the body, and either before or after the pre-drug reaches the area of the body where administration of the drug is indicated.

The compounds of the invention may be in the form of salts, preferably pharmaceutically acceptable salts, as generally described below. Some preferred, but non-limiting examples of suitable pharmaceutically acceptable organic and/or inorganic acids are as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citric acid, as well as other pharmaceutically acceptable acids known per se (for which reference is made to the prior art referred to below).

When the compounds of the invention contain an acidic group as well as a basic group the compounds of the invention may also form internal salts, and such compounds are within the scope of the invention. When the compounds of the invention contain a hydrogen-donating heteroatom (e.g. NH), the invention also covers salts and/or isomers formed by transfer of said hydrogen atom to a basic group or atom within the molecule.

Pharmaceutically acceptable salts of the compounds of Formula I, II, III or V and any subgroup thereof include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. Fora reviewon suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporated herein by reference.

The compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. The term 'amorphous' refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid properties occurs which is characterized by a change of state, typically second order ('glass transition'). The term 'crystalline' refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterized by a phase change, typically first order ('melting point').

Pharmaceutically acceptable salts of compounds of Formula I, II, III, or V may be prepared by one or more of these methods:

(i) by reactingthe compound of Formula I, II, III, or Vwith the desired acid;

(ii) by reactingthe compound of Formula I, II, III, or Vwith the desired base;

(iii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound of formula I, II, III, or V, or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, usingthe desired acid; or

(iv) by converting one salt of the compound of Formula I, II, III, or Vto another by reaction with an appropriate acid or by means of a suitable ion exchange column. All these reactions are typically carried out in solution. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the salt may vary from completely ionized to almost non-ionized.

The compounds of the invention may also exist in unsolvated and solvated forms. The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water.

A currently accepted classification system for organic hydrates is one that defines isolated site, channel, or metal-ion coordinated hydrates - see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Britain, Marcel Dekker, 1995), incorporated herein by reference. Isolated site hydrates are ones in which the water molecules are isolated from direct contact with each other by intervening organic molecules. In channel hydrates, the water molecules lie in lattice channels where they are next to other water molecules. In metal-ion coordinated hydrates, the water molecules are bonded to the metal ion.

When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water/solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.

Also included within the scope of the invention are multi-component complexes (other than salts and solvates) wherein the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. Complexes of this type include clathrates (drug-host inclusion complexes) and co-crystals. The latter are typically defined as crystalline complexes of neutral molecular constituents which are bound together through non-covalent interactions, but could also be a complex of a neutral molecule with a salt. Co-crystals may be prepared by melt crystallization, by recrystallization from solvents, or by physically grinding the components together - see Chem Commun, 17, 1889-1896, by O. Almarsson and M. J. Zaworotko (2004), incorporated herein by reference. For a general review of multi-component complexes, see J Pharm Sci, 64 (8), 1269-1288, by Haleblian (August 1975), incorporated herein by reference.

The compounds of the invention may also exist in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (either melt or solution). Mesomorphism arising as the result of a change in temperature is described as 'thermotropic' and that resulting from the addition of a second component, such as water or another solvent, is described as 'lyotropic'. Compounds that have the potential to form lyotropic mesophases are described as 'amphiphilic' and consist of molecules which possess an ionic (such as -COO-Na+, -COO-K+, or -SO3-Na+) or non-ionic (such as -N-N+(CH3)3) polar head group. For more information, see Crystals and the Polarizing Microscope by N. H. Hartshorne and A. Stuart, 4th Edition (Edward Arnold, 1970), incorporated herein by reference.

All references to compounds of Formula I, II, III, IV, V or any subgroups thereof include references to salts, solvates, multi-component complexes and liquid crystals thereof and to solvates, multicomponent complexes and liquid crystals of salts thereof.

The compounds of the invention include compounds of Formula I, II, III, IV orV or any subgroups thereof as hereinbefore defined, including all polymorphs and crystal habits thereof, prodrugs and isomers thereof (including optical, geometric and tautomeric isomers) as hereinafter defined and isotopically-labelled compounds of Formula I, II, III, IV or V.

In addition, although generally, with respect to the salts of the compounds of the invention, pharmaceutically acceptable salts are preferred, it should be noted that the invention in its broadest sense also included non-pharmaceutically acceptable salts, which may for example be used in the isolation and/or purification of the compounds of the invention.

In some embodiments, the SHIP2 inhibitor can be a SHIP2-binding protein, such as an antibody, an antibody fragment, an antibody-like protein scaffold, or a INPPL1 gene targeting nucleic acid.

In certain embodiments, the SHIP2 inhibitor is a INPPL1 gene targeting nucleic acid.

In certain embodiments, the SHIP2 inhibitor is a SHIP2 binding antibody.

In certain embodiments, the SHIP2 inhibitor is a INPPL1 gene targeting nucleic acid.

In some embodiments, the PLK1 inhibitor can be a PLK1 -binding protein, such as an antibody, an antibody fragment, an antibody-like protein scaffold, or a PLK1 gene targeting nucleic acid.

In certain embodiments, the PLK1 inhibitor is a PLK1 binding antibody.

In certain embodiments, the PLK1 inhibitor is a PLK1 gene targeting nucleic acid.

The term “antibody” as used herein is to be interpreted its broadest sense and generally refers to any immunologic binding agent. The term specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent) and/or multi-specific antibodies (e.g., bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest, i.e., antigen-binding fragments), as well as multivalent and/or multi-specific composites of such fragments. The term “antibody” is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Furthermore, the term “antibody” is indicative for antibodies described herein, regardless of whether they are produced in vitro or in vivo. In certain embodiments, the SHIP2 inhibitor is a SHIP2 binding antibody that directly binds at least one functional domain, or an epitope comprised in the SHIP2 protein. In certain embodiments wherein the inhibitor of SHIP2 is an antibody, binding to the SHIP2 protein by the inhibitor induces SHIP2 protein sequestering. In alternative embodiments wherein the inhibitor of SHIP2 is an antibody, binding to the SHIP2 protein by the inhibitor induces SHIP2 protein precipitation. In particular embodiments, the antibody binds to a protein having SEQ ID NO:1 . In certain embodiments, the PLK1 inhibitor is a PLK1 binding antibody that directly binds at least one functional domain, or an epitope comprised in the PLK1 protein. In certain embodiments wherein the inhibitor of PLK1 is an antibody, binding to the PLK1 protein by the inhibitor induces PLK1 protein sequestering. In alternative embodiments wherein the inhibitor of PLK1 is an antibody, binding to the PLK1 protein by the inhibitor induces PLK1 protein precipitation. In particular embodiments, the antibody binds to a protein having SEQ ID NO: 2.

An antibody may be any of IgA, IgD, IgE, IgG and IgM classes, and preferably IgG class antibody. An antibody may be a polyclonal antibody, e.g., an antiserum or immunoglobulins purified there from (e.g., affinity-purified). An antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies can target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility. By means of example and not limitation, monoclonal antibodies may be made by the hybridoma method described in the art and known to a skilled person (Kohler et al., Continuous cultures of fused cells secreting antibody of predefined specificity., Nature, 1975). Alternatively, a skilled person is aware that antibodies can be made by recombinant DNA methods (Boss et al., Assembly of functional antibodies from immunoglobulin heavy and light chains synthesised in E. coli, Nucleic Acids Research, 1984). As a further non-limiting example, monoclonal antibodies can also be generated by relying on the use of phage display libraries (Clarckson et al., Making antibody fragments using phage display libraries, Nature 1991 ). In certain embodiments, the SHIP2 inhibitor or PLK1 inhibitor is an antibody fragment. The term “antibody fragments” comprises a portion of an intact antibody, comprising the antigen-binding or variable region thereof. Methods to produce and purify antibody fragments are well established in the art (Bates and Power, David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments, Antibodies (Basel), 2019). By means of guidance and not limitation, examples of antibody fragments include Fab, Fab’, F(ab’)2, Fv and scFv fragments, single domain (sd) Fv, such as VH domains, VL domains and VHH domains; diabodies; linear antibodies; single-chain antibody molecules, in particular heavy-chain antibodies; and multivalent and/or multispecific antibodies formed from antibody fragment(s), e.g., dibodies, tribodies, and multibodies. The above designations Fab, Fab’, F(ab’)2, Fv, scFv etc. are intended to have their art-established meaning. In certain embodiments, the SHIP2 inhibitor or PLK1 inhibitor is an antibody fragment that directly binds at least one functional domain, or an epitope comprised in the SHIP2 protein or the PLK1 protein respectively. In particular embodiments, the SHIP2 protein is a protein having SEQ ID NO:1 . In particular embodiments, the PLK1 protein is a protein having SEQ ID NO:2.

The term antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g., birds and mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, the antibodies may be human, murine (e.g., mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel (e.g., Camelus bactrianus and Camelus dromaderius), llama (e.g., Lama paccos, Lama glama or Lama vicugna), horse, or shark.

A skilled person will understand that an antibody can include one or more amino acid deletions, additions and/or substitutions (e.g., conservative substitutions), insofar such alterations preserve its binding of the respective antigen. An antibody may also include one or more native or artificial modifications of its constituent amino acid residues (e.g., glycosylation, etc.).

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art, as are methods to produce recombinant antibodies or fragments thereof (see for example, Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory, New York, 1988; Harlow and Lane, “Using Antibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory, New York, 1999, ISBN 0879695447; “Monoclonal Antibodies: A Manual of Techniques”, by Zola, ed., CRC Press 1987, ISBN 0849364760; “Monoclonal Antibodies: A Practical Approach”, by Dean & Shepherd, eds., Oxford University Press 2000, ISBN 0199637229; Methods in Molecular Biology, vol. 248: “Antibody Engineering: Methods and Protocols”, Lo, ed., Humana Press 2004, ISBN 1588290921 ).

In certain embodiments, the agent may be a Nanobody. The terms “Nanobody” and “Nanobodies” are trademarks of Ablynx NV (Belgium). The term “Nanobody” is well-known in the art and as used herein in its broadest sense encompasses an immunological binding agent obtained (1) by isolating the VHH domain of a heavy-chain antibody, preferably a heavy-chain antibody derived from camelids; (2) by expression of a nucleotide sequence encoding a VHH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by “camelization” of a VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelization” of a “domain antibody” or “dAb” as described in the art, or by expression of a nucleic acid encoding such a camelized dAb; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing. “Camelids” as used herein comprise old world camelids (Camelus bactrianus and Camelus dromaderius) and new world camelids (for example Lama paccos, Lama glama and Lama vicugna). It is known to a person skilled in the art that, depending on the specific situation, nanobodies may display favourable characteristics compared to “traditional” antibodies, including but not limited to a high production yield in a broad variety of expression systems, minimal size, great stability, reversible refolding, and solubility in aqueous solutions.

In further embodiments the inhibitor of SHIP2 or PLK1 is a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a primatized antibody, a human antibody, a Nanobody, an intrabody, or any combination thereof. In further embodiments, the inhibitor of SHIP2 or PLK1 is a concatenation of antibodies.

In certain embodiments, the SHIP2 or PLK1 inhibitor is an antibody-like scaffold or antibody mimetic. The term “antibody-like protein scaffolds” or “engineered protein scaffolds” broadly encompasses proteinaceous non-immunoglobulin specific-binding agents, typically obtained by combinatorial engineering (such as site-directed random mutagenesis in combination with phage display or other molecular selection techniques). Usually, such scaffolds are derived from robust and small soluble monomeric proteins (such as Kunitz inhibitors or lipocalins) or from a stably folded extra-membrane domain of a cell surface receptor (such as protein A, fibronectin or the ankyrin repeat). Methods and protocols to generate antibody-like protein scaffolds have been extensively reported in the art and are therefore known to a skilled person (inter alia in Skerra, Alternative non-antibody scaffolds for molecular recognition, Current opinion in biotechnology, 2007). Non-limiting examples of antibody-like protein scaffolds include affibodies, based on the Z-domain of staphylococcal protein A (Nygren, Alternative binding proteins: affibody binding proteins developed from a small three-helix bundle scaffold, Federation of European Biochemical Societies (FEBS) journal, 2008); engineered Kunitz domains based on a small (ca. 58 residues) and robust, disulphide-crosslinked serine protease inhibitor (Nixon and Wood, Engineered protein inhibitors of proteases, Current opinion in drug discovery & development, 2006); monobodies or adnectins based on the 10th extracellular domain of human fibronectin III (10Fn3) that adopt an Ig-like beta-sandwich fold with 2 to 3 exposed loops, but lack the central disulphide bridge (Koide and Koide, Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain, Methods in molecular biology, 2007); anticalins derived from the lipocalins, a diverse family of eight-stranded beta-barrel proteins that naturally form binding sites for small ligands by means of four structurally variable loops at the open end (Skerra, Alternative binding proteins: anticalins - harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities, FEBS journal, 2008); DARPins, which are designed ankyrin repeat domains (Stumpp et al., DARPins: a new generation of protein therapeutics, Drug DiscoveryToday, 2008); avimers (Silverman etal., Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains, Nature Biotechnology, 2005); and cysteine-rich knottin peptides (Kolmar, Alternative binding proteins: Biological activity and therapeutic potential of cystine-knot miniproteins, FEBS journal, 2008).

In certain embodiments wherein the SHIP2 or PLK1 inhibitor is a protein such as a SHIP2 or PLK1 binding antibody, antibody fragment, or antibody-like scaffold, the inhibitor may further comprise additional amino acid sequences corresponding to a peptide or protein tag sequence, which optionally is a regulatory sequence, or a localization signal, preferably a nuclear localization signal. Tag sequences are routinely used in molecular biology and therefore their merits are known to a skilled person. Non-limiting examples of commonly used peptide tag sequences are the AviTag, C-tag, calmodulin-tag, polyglutamate tag, E-tag, Flag-tag, HA-tag, His-tag, Myc-tag, NE-tag, Rho1 D4-tag, S-tag, SBP-tag, Softag 1 , Softag3, Spot-tag, Strep-tag, TC tag, Ty tag, V5 tag, VSV-tag, Xpress tag, isopeptag, SpyTag, SnoopTag, DogTag, and the SdyTag. In certain embodiments, the inhibitor comprises multiple tag sequences. In further embodiments, the inhibitor comprises at least two distinct peptide or protein tag sequences. Likewise (nuclear) localization signals and methods to identify them have been reported in the art (Cokol et al., Finding nuclear localization signals, EMBO reports, 2000). In certain embodiments, the inhibitor of SHIP2 or PLK1 inhibits SHIP2 or PLK1 respectively by direct binding to the SHIP2 protein or PLK1 protein respectively and inducing precipitation of the protein. In certain embodiments, the inhibitor of SHIP2 or PLK1 inhibits SHIP2 or PLK1 respectively by direct binding to the SHIP2 protein or PLK1 protein respectively and inducing oligomerization of the protein.

In certain embodiments, the inhibitor as disclosed herein can be a gene targeting nucleic acid, such as a INPPL1 targeting nucleic acid. As used herein, introduction of such a gene targeting nucleic acid into the cell can result in downregulation of one or more RNA products or one or more proteins that are endogenously produced in the cell. By means of example, gene targeting nucleic acids include antisense oligonucleotides, RNA interference agents such as siRNA or shRNA, or polynucleotides comprising or encoding components of a gene editing system (such as CRISPR/Cas). For example, one or more antisense or RNA interference polynucleotides may be introduced into a cell, in particular a cancer cell, to reduce the amount or translation of an RNA molecule, such as an mRNA molecule, produced by the cell. In another example, one or more polynucleotides comprising or encoding components of a gene editing system, such as CRISPR/Cas, may be introduced into a cell, in particular a cancer cell, in order to effect a gene editing event, such as SHIP2 ablation or PLK1 ablation, in the cell’s genomic material.

In some embodiments, SHIP2 inhibitors may include, but are not limited to, K149, AS1949490, or AS1938909. In one embodiment, the SHIP2 inhibitor is the compound K149. In another embodiment, the SHIP2 inhibitor is the compound AS1949490.

In some embodiments, PLK1 inhibitors may include, but are not limited to, volasertib (also known as B6727), BI2536, onvansertib (also known as NMS-1286937), GSK461364, SBE 13 HCl, MLN905. In one embodiment, the PLK1 inhibitor is the volasertib. In one embodiment, the PLK1 inhibitor is GSK461364.

In an aspect of the invention, the SHIP2 inhibitor or combinations of SHIP2 inhibitors, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof, as described herein may be formulated in a pharmaceutical composition. Such composition may contain, in addition to one or more active pharmaceutical ingredients, at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant. In an aspect of the invention, the PLK1 inhibitor or combinations of PLK1 inhibitors, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof, as described herein may be formulated in a pharmaceutical composition. Such composition may contain, in addition to one or more active pharmaceutical ingredients, at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant.

In an aspect of the invention, the SHIP2 inhibitor or combinations of SHIP2 inhibitors, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof, and the PLK1 inhibitor or combinations of PLK1 inhibitors, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof, as described herein may be formulated in a pharmaceutical composition. Such composition may contain, in addition to one or more active pharmaceutical ingredients, at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant.

The present invention provides a SHIP2 inhibitor and a PLK1 inhibitor for use in the treatment of cancer in a subject. In some embodiments, the SHIP2 inhibitor and the PLK1 inhibitor as disclosed herein are administered to the subject simultaneously or sequentially in any order. In some embodiments, the SHIP2 inhibitor and the PLK1 inhibitor as disclosed herein are administered to the subject simultaneously. In some embodiments, the SHIP2 inhibitor and the PLK1 inhibitor as disclosed herein are administered to the subject sequentially in any order, such as for example administration of the SHIP2 inhibitor followed by administration of the PLK1 inhibitor, or administration of the PLK1 inhibitor followed by administration of the SHI P2 inhibitor. When the SHIP2 inhibitor and the PLK1 inhibitor are administered sequentially, the time interval in between administration of the first inhibitor and administration of the second inhibitor can be anytime, for example anytime above 5 minutes.

Also provided is a SHIP2 inhibitor for use in the treatment of cancer in a subject wherein the SHIP2 inhibitor is administered to the subject simultaneously or sequentially in any order with a PLK1 inhibitor. Further provided is a PLK1 inhibitor for use in the treatment of cancer in a subject wherein the PLK1 inhibitor is administered to the subject simultaneously or sequentially in any order with a SHIP2 inhibitor. In some embodiments, the SHIP2 inhibitor and the PLK1 inhibitor are administered simultaneously to the subject. In some other embodiments, the SHIP2 inhibitor and the PLK1 inhibitor are administered sequentially in any order, such as for example administration of the SHIP2 inhibitor followed by administration of the PLK1 inhibitor, or administration of the PLK1 inhibitor followed by administration of the SHIP2 inhibitor. When the SHIP2 inhibitor and the PLK1 inhibitor are administered sequentially, the time interval in between administration of the first inhibitor and administration of the second inhibitor can be any time, for example anytime above 5 minutes.

In an aspect of the invention, a kit of parts comprising a SHIP2 inhibitor and a PLK1 inhibitor is provided. In another aspect, a kit of parts comprising a SHIP2 inhibitor and a PLK1 inhibitor for use in the treatment of cancer in a subject is provided. In some embodiments, the SHI P2 inhibitor and the PLK1 inhibitor in said kit of parts are configured for simultaneous or sequential administration in any order. In some embodiments, the kits of parts as disclosed herein comprises a dosage form of the SHIP2 inhibitor as disclosed herein and a dosage form of the PLK1 inhibitor as disclosed herein. In some embodiments, the dosage form of the SHIP2 inhibitor and the dosage form of the PLK1 inhibitor are separate and allow simultaneous or sequential in any order administration of both dosage forms. In some embodiments, the dosage form of the SHIP2 inhibitor and the dosage form of the PLK1 inhibitor are separate and allow simultaneous administration of both dosage forms to the subject. In some embodiments, the dosage form of the SHIP2 inhibitor and the dosage form of the PLK1 inhibitor are separate and allow sequential in any order administration of both dosage forms. In some embodiments, the kit of parts as disclosed herein comprises pharmaceutical composition comprising a SHIP2 inhibitor and a PLK1 inhibitor as disclosed herein, that allows simultaneous administration of both the SHIP2 inhibitor and the PLK1 inhibitor. In some embodiments, the kit of parts as disclosed herein comprises a pharmaceutical composition comprising a SHIP2 inhibitor as disclosed herein and a pharmaceutical composition comprising a PLK1 inhibitor as disclosed herein such that the pharmaceutical composition comprising the SHIP2 inhibitor and the pharmaceutical composition comprising the PLK1 inhibitor can be administered sequential in any order.

The term “dosage form” as used herein refers to a composition that contains predetermined dose of a compound.

The term “simultaneous administration” as used herein, means that a first inhibitor, composition or dosage form and a second inhibitor, composition or dosage form are administered to the subject at the same time or with a short time separation such as no more than about 5 minutes. When the inhibitors are administered simultaneously, the first and second inhibitors may be contained in the same composition (e.g., a composition comprising both a SHIP2 inhibitor and a PLK1 inhibitor), or in separate compositions (e.g., a SHIP2 inhibitorcontained in one composition and a PLK1 inhibitor contained in another composition). In general, agents beingadministered in combination do not necessarily have to be administered in the sample pharmaceutical composition, and may, e.g., because of different physical and chemical characteristics, be administered by different routes.

The term “sequential administration” as used herein means that the first inhibitor, composition or dosage form and the second inhibitor, composition, or dosage form are not administered to the subject at the same time but rather one of the inhibitors, compositions or dosage forms is administered first, and the other inhibitor, composition or dosage form is administered after administration of the first inhibitor, composition or dosage form with a time separation of, for example, more than 5 minutes. Either the SHIP2 inhibitor or composition or dosage form comprising the SHIP2 inhibitor or the PLK1 inhibitor or composition or dosage form comprising the PLK1 inhibitor may be administered first. The first inhibitor, composition or dosage form and the second inhibitor, composition or dosage form are contained in separate compositions, which may be contained in the same or different packages or kits.

As shown in the examples, a synergistic effect was found for the combination of a SHI P2 inhibitor and a PLK1 inhibitor on the survival of oesophageal cancer cells. In some embodiments, combination of the SHIP2 inhibitor and the PLK1 inhibitor for use according to any of the embodiments of the invention is synergistic. In certain embodiments, the combination has a synergistic effect. In certain embodiments, the combination has a synergistic anti-cancer effect. In certain embodiments, the combination has a synergistic therapeutic effect.

As used herein, the term “synergistic” or “synergy” means that the effect achieved with the combinations of compounds, in particular with the combination of SHIP2 inhibitor and PLK1 inhibitor encompassed in this invention, is greater than the sum of the effects that result from using the compounds separately as a monotherapy. Advantageously, such synergy provides greater efficacy at the same doses than administration of the compounds as monotherapy.

As used herein, “cancer” refers to a describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumours or micrometastases. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. The term “tumour” as used herein, refers to abnormal growth of tissues that may or may not be cancerous. In some embodiments, the cancer is a cancer that overexpresses SHIP2. As used herein, the term “overexpression” of a gene or protein is to be understood as an increased expression level of a gene or protein in a cancer sample as compared to the expression level of the same gene or protein in a control sample, such as a tissue sample that is obtained from a healthy subject. The increased expression level can be an increase in the expression the gene or protein of at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or even at least 100% or more, as compared to the control sample.

In some embodiments, the cancer is a solid tumour. In some further embodiments, the solid tumour is selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/central nervous system (CNS) cancer, cervical cancer, ovarian cancer, endometrial cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, pancreatic cancer, stomach cancer, and melanoma. In some embodiments, the cancer is selected from the group consisting of oesophageal cancer, colorectal cancer and head and neck cancer, such as head and neck squamous cell carcinoma. In some embodiments, the cancer is oesophageal cancer or colorectal cancer. In preferred embodiments, the cancer is oesophageal cancer, preferably oesophageal squamous cell carcinoma (eSCC). In other preferred embodiments, the cancer is colorectal cancer.

Examples of cancer may include primary tumours of any of the above types of cancer or metastatic tumours at alternative (non-original) sites derived from any of the above types of cancer.

Except when noted, the terms “subject”, “patient” or “individual” are used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates. Preferred subjects or patients are human subjects including both genders and all age categories thereof. In some embodiments, the subjects are adult subjects. The term “adult subject” as used herein refers to an individual 18 years of age or older. In certain embodiments, the subject is a senior adult, e.g., 65 years or older.

The subject or patients as envisaged herein may in particular require a treatment as taught herein. Particularly intended are subjects that are diagnosed with or are suspected of having cancer, in particular a solid tumour, more in particular wherein the solid tumour is selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/central nervous system (CNS) cancer, cervical cancer, ovarian cancer, endometrial cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, pancreatic cancer, stomach cancer, and melanoma. In some embodiments, the subjects are diagnosed with or are suspected of having oesophageal cancer, colorectal cancer or head and neck cancer. In some embodiments, the subjects are diagnosed with or are suspected of having oesophageal cancer or colorectal cancer. In certain embodiments, the subjects are diagnosed with or are suspected of having oesophageal cancer; preferably oesophageal squamous cell carcinoma (eSCC). In certain embodiments, the subjects are diagnosed with or are suspected of having colorectal cancer.

As used herein, a phrase such as “a subject in need of treatment” includes subjects that would benefit from treatment of a given condition, in particular cancer. Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to contract or develop said condition and/or those in whom said condition is to be prevented.

The terms “treat” or “treatment” encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed disease condition, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent the chances of incidence of an undesired affliction, such as to prevent the chances of progression of cancer. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In a preferred embodiment, “treatment” in the light of the present invention implies reduction of cancer biomarkers or cancer progression. Phrased differently; “treatment” covers any treatment of a disease in a mammal, particular a human and includes: (a) preventing the disease or symptoms but has not yet been diagnosed as having it; (b) inhibiting the disease symptoms, i.e. arresting its development; or (c) relieving the disease symptoms, i.e. causing regressing of the disease or symptom. Non-limiting examples of already available therapeutic treatment options for the treatment of cancer are radiotherapy, chemotherapy, targeted drug therapy, immunotherapy and surgery.

In some embodiments, the SHIP2 inhibitor and/or PLK1 inhibitor for use according to any of the embodiments, or the kits of parts or the pharmaceutical composition according to any of the embodiments can be combined with any other available therapeutic treatment option for the treatment of cancer, such as for example radiotherapy, chemotherapy, targeted drug therapy, immunotherapy, surgery, or a combination thereof. In some embodiments, the SHIP2 inhibitor and/or the PLK1 inhibitor for use in the treatment of cancer according to any of the embodiments of the invention are administered to the subject in a therapeutically effective amount. In some embodiments, the SHIP2 inhibitor and/or the PLK1 inhibitor for use in the treatment of cancer according to any of the embodiments of the invention are administered to the subject in a prophylactically effective amount.

The term “therapeutically effective amount” as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. Methods are known in the art for determining therapeutically or prophylactically effective doses for the present compounds.

The term “prophylactically effective amount” refers to an amount of an active compound or pharmaceutical agent that inhibit or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician.

In some embodiments, the subject has been selected by assaying a sample from the subject for a mutation or amplification in one or more of the following genes: CCND1 , PIK3CA, INPPL1 . In some embodiments, the subject has been selected by assaying a tumour sample from the subject for a mutation or amplification in one or more of the following genes: CCND1 , PIK3CA, INPPL1.

In some embodiments, the SHIP2 inhibitor, the PLK1 inhibitor, the kits of parts, or any of the pharmaceutical compositions as disclosed herein are for use in the treatment of cancer in a subject, wherein the cancer, preferably oesophageal cancer, is characterized by the presence of a mutation, amplification, or both in one or more of the following genes: CCND1 , PIK3CA, INPPL1 . In some embodiments, the cancer is characterized by a mutation in one or more of the genes CCND1 , PI3KCA and INPPL1 . In some embodiments, the cancer is characterized by an amplification in one or more of the genes CCND1 , PI3KCA and INPPL1 . In some embodiments, the cancer is characterized by a mutation and an amplification in one or more of the genes CCND1 , PI3KCA and INPPL1 . In some embodiments, the cancer is characterized by a mutation in the gene CCND1 . In some embodiments, the cancer is characterized by an amplification in the gene CCND1. In some embodiments, the cancer is characterized by a mutation in the gene PIK3CA. In some embodiments, the cancer is characterized by an amplification in the gene PIK3CA. In some embodiments, the cancer is characterized by a mutation in the gene INPPL1 . In some embodiments, the cancer is characterized by an amplification in the gene INPPL1 . In some embodiments, the presence or absence of a mutation or amplification in any one of the genes CCND1 , PIK3CA, INPPL1 can be assessed by evaluating the presence of such mutation or amplification in a sample obtained from the subject. In some embodiments, the sample obtained from the subject is a sample that contains or is suspected to contain tumour cells. In some embodiments, the sample contains tumour cells. In some embodiments, the sample is a tumour tissue sample or a fluid sample comprising tumour cells. In some embodiments, the sample is a tumour tissue sample derived from subjects diagnosed with cancer. In some embodiments, the sample is a fluid sample, such as a blood sample, comprising tumour cells, in particular circulating tumour cells. In some embodiments, the sample is a sample comprising circulating tumour cells. The tissue sample may be, for example, a tissue resection, a tissue biopsy, or metastatic lesion obtained from a patient suffering from, suspected to suffer from, or diagnosed with cancer. Preferably, the sample is a sample of tissue, a resection or biopsy of a tumour, known or suspected metastatic cancer lesion or section.

Methods of obtaining biological samples including tissue resections, biopsies, and body fluids, e.g., blood samples comprising cancer/tumour cells, are well known in the art.

In some embodiments, the sample is a tumour tissue sample, such as a fresh-frozen tumour tissue sample, a fresh tumour tissue sample, or a formalin-fixed paraffin-embedded (FFPE) tumour tissue sample. Isolation of nucleic acids, such as RNA or DNA, from tumour tissue samples can be performed using any technique known in the art. For example, RNA isolation from a tumour tissue sample, such as an FFPE tumour tissue sample, can be performed using the QIAGEN RNeasy FFPE kit (standard kit) following Manufacturer’s instructions (Qiagen).

In some embodiments, the sample is a fluid sample comprising tumour cells, in particular circulating tumour cells. In some embodiments, the fluid sample is a blood sample, e.g. a peripheral blood sample, known or suspected to comprise circulating cancer cells. The sample may comprise both cancer cells, i.e. tumour cells, and non-cancerous cells, and, in certain embodiments, comprises both cancerous and non-cancerous cells. In some embodiments, the sample is a sample of isolated circulating tumour cells. Isolation of circulating tumour cells from liquid samples, such as a blood sample, can be performed using any technique known in the art. For example, isolation of circulating tumour can be performed using the EasySepTM Direct Human CTC enrichment kit (Stemcell Technologies), or the CTC ready-to-use CTC AdnaTest of Qiagen (Qiagen). RNA and/or DNA isolation from the isolated CTCs can for example be performed using the AUPrep RNA/mRNA Nano kit (Qiagan), or the RNAeasy Tissue/Cells Advances mini kit (Qiagen). In an aspect of the invention, the SHIP2 inhibitor, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof, as described herein may be formulated in a pharmaceutical composition. Such composition may contain, in addition to one or more pharmaceutical ingredients, at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant.

In an aspect of the invention, the PLK1 inhibitor, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof, as described herein may be formulated in a pharmaceutical composition. Such composition may contain, in addition to one or more pharmaceutical ingredients, at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant.

In an aspect of the invention, the SHIP2 inhibitor and the PLK1 inhibitor, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof, as described herein may be formulated in a pharmaceutical composition. Such composition may contain, in addition to one or more pharmaceutical ingredients, at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant.

A further aspect of the present invention provides a pharmaceutical composition comprising a SHIP2 inhibitor and a PLK1 inhibitor as disclosed herein.

Further provided is a pharmaceutical composition comprising a compound of Formula I or Formula II or a combination thereof, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt or prod rug thereof, and a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising a compound of Formula III, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising a compound of Formula IV, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising a compound of Formula V, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier.

In an aspect, a combination of a SHI P2 inhibitor, forexample a SHIP2 inhibitor that is a compound of Formula I, II or IV, or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof and a PLK1 inhibitor, for example a PLK1 inhibitor that is a compound of Formula III or V, or a or a stereoisomer, enantiomer, tautomer, solvate, hydrate, pharmaceutically acceptable salt, or prodrug thereof, is provided. In some embodiments, said combination is comprised in a pharmaceutical composition. In some embodiments, the combination can be configured to allow for administration of the two compounds simultaneously, or to allow for administration of the two compounds sequentially in any order.

The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

As used herein, “carrier” or “excipient” includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active substance, its use in the therapeutic compositions may be contemplated.

Illustrative, non-limiting carriers for use in formulating the pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents suitable for intravenous (IV) use, liposomes or surfactant-containing vesicles, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to one of ordinary skill in the art.

Pharmaceutical compositions as intended herein may be formulated for essentially any route of administration, such as without limitation, oral administration (such as, e.g., oral ingestion or inhalation), intranasal administration (such as, e.g., intranasal inhalation or intranasal mucosal application), parenteral administration (such as, e.g., subcutaneous, intravenous (I.V.), intramuscular, intraperitoneal or intrasternal injection or infusion), transdermal or transmucosal (such as, e.g., oral, sublingual, intranasal) administration, topical administration, rectal, vaginal or intra-tracheal instillation, and the like. In this way, the therapeutic effects attainable by the methods and compositions can be, for example, systemic, local, tissue-specific, etc., depending of the specific needs of a given application.

In some embodiments, the compound or the pharmaceutical composition as taught herein is administered parenterally. Preferably, the compound or the pharmaceutical composition as taught herein is administered intravenously, for example by infusion.

In some embodiments, the compound or the pharmaceutical composition as taught herein is administered orally.

Suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is made to for instance USA-6,372, 778, US-A-6,369,086, US-A-6,369,087 and US-A6,372,733, as well as the standard handbooks, such as the latest edition of Remington’s Pharmaceutical Sciences.

Some preferred, but non-limiting examples of such preparations include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, cremes, lotions, soft and hard gelatin capsules, suppositories, drops, sterile injectable solutions and sterile packaged powders (which are usually reconstituted prior to use) for administration as a bolus and/or for continuous administration, which may be formulated with carriers, excipients, and diluents that are suitable per se for such formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, edible oils, vegetable oils and mineral oils or suitable mixtures thereof. The formulations can optionally contain other pharmaceutically active substances (which may or may not lead to a synergistic effect with the compounds of the invention) and other substances that are commonly used in pharmaceutical formulations, such as lubricating agents, wetting agents, emulsifying and suspending agents, dispersing agents, disintegrants, bulkingagents, fillers, preserving agents, sweetening agents, flavoringagents, flow regulators, release agents, etc.. The compositions may also be formulated so as to provide rapid, sustained or delayed release of the active compound(s) contained therein, for example using liposomes or hydrophilic polymeric matrices based on natural gels or synthetic polymers. In order to enhance the solubility and/or the stability of the compounds of a pharmaceutical composition according to the invention, it can be advantageous to employ a-, 0- or y- cyclodextrins or their derivatives. In addition, co-solvents such as alcohols may improve the solubility and/or the stability of the compounds. The preparations may be prepared in a manner known per se, which usually involves mixing the at least one compound according to the invention with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is again made to US-A- 6,372,778, US-A-6,369,086, US-A-6,369,087 and US-A-6,372,733 and the further prior art mentioned above, as well as to the standard handbooks, such as the latest edition of Remington’s Pharmaceutical Sciences.

The pharmaceutical preparations of the invention are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Generally, such unit dosages will contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the invention, e.g. about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

The compounds, dosage forms or pharmaceutical compositions as disclosed herein can be administered bya variety of routes including the oral, ocular, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes, depending mainly on the specific preparation used and the condition to be treated or prevented, and with oral and intravenous administration usually being preferred. The at least one compound of the invention will generally be administered in an “effective amount”, by which is meant any amount of a compound of the formula as taught herein that, upon suitable administration, is sufficient to achieve the desired therapeutic or prophylactic effect in the subject to which it is administered. The dosage or amount of the agent as taught herein, optionally in combination with one or more other active compounds to be administered, and therapeutic efficacy of the agent as described herein or pharmaceutical compositions comprising the same can be determined by known pharmaceutical procedures in, for example, cell cultures or experimental animals. These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Pharmaceutical compositions that exhibit high therapeutic indices are preferred. While pharmaceutical compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to normal cells (e.g., non-target cells) and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in appropriate subjects. The dosage of such pharmaceutical compositions lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilised. For a pharmaceutical composition used as described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the pharmaceutical composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

The unit dose and regimen depend on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, the unit dose and regimen depend on the nature and the severity of the disorder to be treated, and also on factors such as the species of the subject, the sex, age, body weight, general health, diet, mode and time of administration, immune status, and individual responsiveness of the human or animal to be treated, efficacy, metabolic stability and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the agent of the invention. In order to optimize therapeutic efficacy, the compound or the pharmaceutical composition as taught herein can be first administered at different dosing regimens. Typically, levels of the agent in a tissue can be monitored using appropriate screening assays as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. The frequency of dosing is within the skills and clinical judgement of medical practitioners (e.g., doctors, veterinarians or nurses). Typically, the administration regime is established by clinical trials which may establish optimal administration parameters. However, the practitioner may vary such administration regimes according to the one or more of the aforementioned factors, e.g., subject’s age, health, weight, sex and medical status. The frequency of dosing can be varied depending on whether the treatment is prophylactic or therapeutic.

Usually, depending on the condition to be prevented or treated and the route of administration, such an effective amount will usually be between 0.01 to 1000 mg per kilogram, more often between 0.1 and 500 mg, such as between 1 and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg, per kilogram body weight day of the patient per day, which may be administered as a single daily dose, divided over one or more daily doses, or essentially continuously, e.g. using a drip infusion. The amount(s) to be administered, the route of administration and the further treatment regimen may be determined by the treating clinician, depending on factors such as the age, gender and general condition of the patient and the nature and severity of the disease/symptoms to be treated. Reference is again made to US-A- 6,372,778,US-A-6,369,086, US-A-6,369,087 and US-A-6,372,733 and the further prior art mentioned above, as well as to the standard handbooks, such as the latest edition of Remington’s Pharmaceutical Sciences.

In accordance with the present invention, said pharmaceutical composition can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The present invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly.

For an oral administration form, the compositions of the present invention can be mixed with suitable additives, such as excipients, stabilizers or inert diluents, and brought by means of the customary methods into the suitable administration forms, such as tablets, coated tablets, hard capsules, aqueous, alcoholic, or oily solutions. Examples of suitable inert carriers are gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose, or starch, in particular, corn starch. In this case, the preparation can be carried out both as dry and as moist granules. Suitable oily excipients or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil. Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions, or mixtures thereof. Polyethylene glycols and polypropylene glycols are also useful as further auxiliaries for other administration forms. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, these compositions may be prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compounds of the invention in a pharmaceutically acceptable solvent, such as ethanol orwater, or a mixture of such solvents. If required, the formulation can also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant.

For subcutaneous or intravenous administration, the compound according to the invention, if desired with the substances customary therefore such as solubilizers, emulsifiers or further auxiliaries are brought into solution, suspension, or emulsion. The compounds of the invention can also be lyophilized and the lyophilizates obtained used, for example, for the production of injection or infusion preparations. Suitable solvents are, for example, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose or mannitol solutions, or alternatively mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1 ,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, these formulations may be prepared by mixing the compounds according to the invention with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.

In some embodiments the composition is administered once a day, twice a day, three times a day or four times a day. In one embodiment, the administration of a dosage of 50 mg to 2 g/day/patient can be envisioned. More particularly, a dosage of about 250 mg to 750 mg/day, for example 500 mg/day, can be envisioned. It is particularly advantageous to formulate the pharmaceutical compositions envisioned in unit dosage regime form in order to facilitate administration and uniformity of the dosage regime. The unit dosage regime form in the present document refers to physically distinct units that can serve as unit doses, each unit containing a predetermined amount of active ingredient.

Afurtheraspectof the invention provides a method of treating cancer in a subject in need thereof, wherein the method comprises administering to said subject a therapeutically effective amount of a SHIP2 inhibitor and a PLK1 inhibitor as disclosed herein.

Further provided is a method of treating cancer in a subject in need thereof, wherein the method comprises administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising a SHIP2 inhibitor and a therapeutically effective amount of a pharmaceutical composition comprising a PLK1 inhibitor as disclosed herein.

Also provided is a method of treating cancer in a subject in need thereof, wherein the method comprises administering to said subject a dosage form comprising a SHIP2 inhibitor and a dosage form comprising a pharmaceutical composition comprising a PLK1 inhibitor as disclosed herein.

In some embodiments, the methods of treating cancer as disclosed herein are for treating a cancer that overexpresses SHIP2. In some embodiments, the methods of treating cancer as disclosed herein are for treating a solid tumour in a subject. In some further embodiments, the solid tumour is selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/central nervous system (CNS) cancer, cervical cancer, ovarian cancer, endometrial cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, pancreatic cancer, stomach cancer, and melanoma. In some further embodiments, the solid tumour is selected from the group consisting of oesophageal cancer, colorectal cancer and head and neck cancer. In some embodiments, the solid tumour is oesophageal cancer or colorectal cancer. In preferred embodiments, the cancer is oesophageal cancer, preferably oesophageal squamous cell carcinoma (eSCC). In some embodiments, the cancer is colorectal cancer. Examples of cancer may include primary tumours of any of the above types of cancer or metastatic tumours at alternative (non-original) sites derived from any of the above types of cancer.

In some embodiments, the methods of treating cancer as disclosed herein a re for treating cancer in a human subject, in particular a human subject that is diagnosed with or is suspected of having cancer, in particular a solid tumour, more in particular wherein the solid tumour is selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/central nervous system (CNS) cancer, cervical cancer, ovarian cancer, endometrial cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, pancreatic cancer, stomach cancer, and melanoma. In some further embodiments, the solid tumour is selected from the group consisting of oesophageal cancer, colorectal cancer and head and neck cancer. In some embodiments, the solid tumour is oesophageal cancer or colorectal cancer. In certain embodiments, the subjects is diagnosed with or is suspected of having oesophageal cancer; preferably oesophageal squamous cell carcinoma (eSCC). In some embodiments, the cancer is colorectal cancer. In some embodiments, the methods of treating cancer according to the invention further comprise the administration of any other available therapeutic treatment option, such as for example radiotherapy, chemotherapy, targeted drug therapy, immunotherapy, surgery, or a combination thereof.

In some embodiments, the methods of treating cancer as disclosed herein are for treating cancer in a subject in need thereof, wherein the subject has been selected by assaying a sample from the subject for a mutation or amplification in one or more of the following genes: CCND1 , PIK3CA, INPPL1 . In some embodiments, the subject has been selected by assaying a tumour sample from the subject for a mutation or amplification in one or more of the following genes: CCND1 , PIK3CA, INPPL1.

Further provided is the use of a SHIP2 inhibitor and a PLK1 inhibitor as disclosed herein for the manufacture of a medicamentforthe treatment of cancer. In some embodiments, said use is for the manufacture of a medicamentforthe treatment of cancer wherein the cancer overexpresses SHIP2. In some embodiments, the cancer is a solid tumour. In some further embodiments, the solid tumour is selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/central nervous system (CNS) cancer, cervical cancer, ovarian cancer, endometrial cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, pancreatic cancer, stomach cancer, and melanoma. In some further embodiments, the solid tumour is selected from the group consisting of oesophageal cancer, colorectal cancer and head and neck cancer. In some embodiments, the solid tumour is oesophageal cancer or colorectal cancer. In preferred embodiments, the cancer is oesophageal cancer, preferably oesophageal squamous cell carcinoma (eSCC). In some embodiments, the cancer is colorectal cancer. Examples of cancer may include primary tumours of any of the above types of cancer or metastatic tumours at alternative (non-original) sites derived from any of the above types of cancer.

Also provided is the use of a SHIP2 inhibitor and a PLK1 inhibitor as disclosed herein, or the use of a pharmaceutical composition or dosage form as disclosed herein, for the treatment of cancer. In some embodiments, said use is for the treatment of cancer wherein the cancer overexpresses SHIP2. In some embodiments, the cancer is a solid tumour. In some further embodiments, the solid tumour is selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/central nervous system (CNS) cancer, cervical cancer, ovarian cancer, endometrial cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, pancreatic cancer, stomach cancer, and melanoma. In some further embodiments, the solid tumour is selected from the group consisting of oesophageal cancer, colorectal cancer and head and neck cancer. In some embodiments, the solid tumour is oesophageal cancer or colorectal cancer. In preferred embodiments, the cancer is oesophageal cancer, preferably oesophageal squamous cell carcinoma (eSCC). In some embodiments, the cancer is colorectal cancer. Examples of cancer may include primary tumours of any of the above types of cancer or metastatic tumours at alternative (non-original) sites derived from any of the above types of cancer.

It is apparentthat there have been provided in accordance with the invention products, methods, and uses, that provide for substantial advantages as set forth above. While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims.

The above aspects and embodiments are further supported by the following non-limiting examples.

EXAMPLES

Materials and Methods

For all experiments presented in this study, sample size was large enough to measure the effect size. No randomization and no blindingwere performed in this study.

TCGA

Copy number variation (Corsello et al., 2020) datasets from 96 human esophageal squamous cell carcinoma were retrieved from the DepMap portal (https://depmap.org/portal/).

Cancer cell line profiling with CCLE

The analysis was performed using 22 human esophageal squamous cell carcinoma cell lines. The transcriptomic (Nusinow et al., 2020), proteomic (Nusinow et al., 2020) and copy number variation (Corsello et al., 2020) datasets were retrieved from the DepMap portal (https://depmap.org/portal/). DeepOmicNet was used to compare the predictive power of models for CRISPR-Cas9 gene essentialities.

Histological staining and immunocytochemistry

6 age-matched human control and 6 esophagus cancer cases were obtained from Erasme Biobank. All procedures were carried out in accordance with relevant guidelines and regulations of the faculty of Medicine of the Universite Libre de Bruxelles (ULB). Paraffine-embedded tissue sections of 60 esophageal cancer cases (Human Esophageal Tissue MicroArray, cancer) were purchased from Novus bio (Cambridge, UK; NBP2-30266). These paraffine-embedded tissue sections (4 pm thick) were stained with anti-SHIP2 (NOVUS bio, H00003636-M01). The immunohistochemical labelling was performed using the ABC method (Elite). Briefly, deparaffinized tissue sections were treated with H₂O₂ to inhibit endogenous peroxidase and incubated with the blocking solution of 10% (v/v) normal horse serum in Tris Buffer Saline (TBS; 0.01 M Tris, 0.15 M NaCl, pH 7.4). After an overnight incubation with the diluted primary anti body, the sections were sequentially incubated with either horse anti-mouse or goat anti-rabbit antibodies conjugated to biotin (Vector) followed by the ABC complex (Vector). The peroxidase activity was developed using diaminobenzidine (Dako; Heverlee, Belgium) as chromogen. The slides were successively dehydrated, mounted with DPX (Sigma) and observed with a Leica DM500 microscope.

For quantitative analysis, images were taken at Nanozoomer (Hamamatsu) at a 40X objective and were quantified using Image J for optical density of SHIP2 labelling as previously reported (Ando et al., 2020). Cell culture

Human eSCC lines were obtained from DSMZ (German Collection of Microorganisms and Cell cultures Gmbh, Braunschweig, Germany) - KYSE-30, KYSE-70, KYSE-140, KYSE-150, KYSE180, KYSE-270, KYSE-410, KYSE-450, KYSE-510, KYSE-520, COLO-680N) ; and Riken BRC Cell Bank (Tsukuba, Ibaraki, Japan) - TE-4, TE-5, TE-6, TE-8, TE-9, TE-10, TE-11 , TE-14, TE-15. Cells were cultured according to the supplier’s recommendations. All lines were cultivated in RPMI 1640 medium (Gibco/Thermo Fisher Scientific, Waltham, MA, USA, 21875-034) supplemented with 10% fetal bovine serum (Gibco, 10270-106) and 1% penicillin/streptomycin (Gibco, 15070-063); except KYSE-30, KYSE-150 and KYSE-270 that were cultivated with 49% RPMI 1640 and 49% Ham’s F12 (Gibco, 11765-054) supplemented with 2% fetal bovine serum and 1% penicillin/streptomycin.

Human colorectal cancer cell lines (HCT116, DLD1 , LOVO, and HT29) were cultured according to the supplier’s recommendations.

All cells were cultured under humidified atmosphere with 5% CO₂ and 37°C.

Antibodies

The following primary antibodies were used for Western blot: polyclonal anti-SHIP2 (C-terminal Ab#1 , Muraille et al 1999), monoclonal anti-SHIP2 (Ab#2, Abeam, ab166916), for immunocytochemistry (NOVUS bio, H00003636-M01 ); anti-GAPDH (#2118), anti-AKT (#9372), anti-pAKT S473 (#9271 ) are from Cell Signaling Technology (Bioke). For immunostaining we used the following: anti-Caspase-3 (R&D Systems, AF835), anti-Ki67 (Abeam, ab15580), anti- phospho-Histone-3 (LabNed, LN2000577), anti-E-cadherin (Invitrogen, 13-1900), anti-Keratin-14 (Abeam, ab197893), anti-lntegrin-a6 (Biolegend, 313602).

Protein extract preparation and immunoblottting

Whole cell extracts were generated from cell cultures under basal conditions or after the treatments specified. Briefly, cells were lysed in buffer A [10mM Tris-HCl (pH 7.5), 150 mM KCL, 12 mM p-mercaptoethanol, 100 mM NaF, 0.5% NP-40, 0.01 mg/ml leupeptin, 0.3 mg/ml pefabloc, 2.5 pM okadoic acid, 1 mM sodium vanadate, and 2 mM EDTA] and protease inhibitors (Roche). The cells were scraped, and lysates were cleared by centrifugation at 14,000 g. The lysates were normalized based on total protein content measured using the Bradford assay (Bio-Rad). Equal amounts of protein i.e. 50-100 pg were resolved on SDS-PAGE and transferred into nitrocellulose membranes. The proteins were detected with the corresponding primary antibodies. Immunoblot analysis was conducted using enhanced chemiluminescence (ECL) reagent accordingto manufacturer’s protocol (PerkinElmer, NEL104001 EA; or Advasta K-12042). Images were obtained on a Vilber Lourmat Fusion Solo S (Vilber, Marne-la-Vallee, France) and Azure c600 (Azure Biosystems, Sierre Court, CA, USA).

Transfection of cells with SHIP2-specific siRNA eSCC human cell lines were transfected with siRNA targeting INPPL1 (HSS_105471 for silNPPL1_1 and HSS_179945 for silNPPL1_2, Invitrogen) and a non-targeting siRNA (ON- TARGETplus, D-001810-01-05, Dharmacon) using the Lipofectamine RNAi Max according to the manufacturer’s instructions.

CRISPR/Cas9

CRISPR/Cas9 technology was used for SHIP2 ablation in KYSE-410 cells (ref: PMC3969860) using the CRISPR/Cas9n plasmids. Plasmids were co-transfected with a GFP-based CRISPR reporter (Addgene #48138) using the Lipofectamine CRISPRMAX according to the manufacturer’s instructions. GFP-positive cells were subjected to FACS sorting (ref FACS sorter) by a standard protocol and collected in a 96-well plate. Cells were expanded and positive clones were screened by Western blot for SHIP2, and the targeted locus was PCR amplified [5'- GTACCACCGCGACCTGAG-3' (forward; SEQ ID NO: 5) and 5'-cttggctttctcctgggtct-3' (reverse; SEQ ID NO: 6)]. PCR products were sequenced to determine SHIP2 sequence and genotype.

Cell proliferation assay

Cell proliferation was detected with 5-ethynyl-2’-deoxyuridine (EdU) assays. Cells were seeded in glass coverslips in 24-well plates and treated accordingly. After the treatment, cells were incubated with 10 pM EdU (Invitrogen, C10635) for 1 h at 37°C. Briefly, cells were fixed with 4% formaldehyde (VWR, 11699455) for 15 min, then cells were washed in PBS and permeabilized with 0.1 % Triton in PBS for 10 min. After washing with PBS cells were incubated for 30 min with EdU reaction prepared in TBS containing 100 mM Tris-HCl, 1 mM CuSO4, 100 mM Ascorbic Acid and AF594 Picolyl azide (Invitrogen, C10339B) diluted 1 :2000. Hoechst (ref) was used as nuclear counterstain. Slides were mounted using Glycergel (Dako) supplemented with 2.5% DABCO (Sigma-Aldrich). Cells were observed on a Zeiss Axio Imager M2 fluorescence microscope with a Zeiss Axiocam 503 mono camera at 20 x. Acquired CZI images were then processed using QPath. The ratio of EdU positive cells to total cell count was taken over random fields and represented as a percentage.

Cell viability assays

The proportion of viable cells was determined using the CellTiter 96® Aqueous One solution Cell Proliferation assay (MTS assay, Promega, G3581) according to the manufacturer's instructions. Briefly, cells were seeded in 96-well plates, in quintuplets for each condition, and incubated with the respective treatments. To measure cell viability, after 72h, MTS reagent was added to each well and cells were incubated at 37 °C for 1 h. The optical density was determined at 490 nm and compared to a blank made without cells. All the experiments were performed with at least 3 biological replicates. eSCC cells xenografts and treatment by SHIP2 inhibitors

Athymic immunodeficient NOD/SCID mice were purchased from Janvier Labs (NOD.CB-17- PrKdc-scid/Rj; France). Mouse colonies were maintained in a certified animal facility in accordance with the European guidelines. All the experiments were approved bythe AnimalCare and Ethics Committee of the Universite Libre de Bruxelles and in conformity with regulatory standards (LA1230406 - projects 785N, 668N and 821 N). All mice used in this study were composed of males and females with 6 to 8-week-old. 2*10⁵ cells were suspended in Matrigel ® Basement Membrane matrix (Corning, 354234) and injected in duplicates subcutaneously in at least 3 mice per cell line and condition. Tumour appearance was monitored every week. Once mice developed tumours, mice were treated with vehicle alone (5% DMSO) and 10 mg/kg K149 (Echelon, B-0345) (herein also referred to as compound of Formula IV) or AS1949490 (herein also referred to as compound of Formula II) by intra-peritoneal injection every other day for 5 days (3 injections).

Tumour histology and immunostaining

Forthe staining on frozen sections, harvested tumours were directly embedded in O.C.T. (Tissue Tek) and then flash frozen for cryopreservation. Samples were sectioned at 6 pm sections using a M1860 cryostat (Leica Microsystems GmbH). Sections were fixed with 4% formaldehyde for 15 min and, after PBS washing, nonspecific antibody binding was blocked with 5% horse serum (HS), 1% Bovine Serum Albumin (BSA) and 0.2% Triton X-100 - blocking buffer - for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C in blocking buffer. Sections were rinsed three times in PBS and incubated with secondary antibodies during 1 h at room temperature. Nuclei were stained with Hoechst. Slides were mounted using Glycergel (Dako) supplemented with 2.5% DABCO (Sigma-Aldrich). Imaging was performed on a Zeiss Axio Imager M2 fluorescence microscope with a Zeiss Axiocam 503 mono camera. Acquired CZI images were then processed using QPath. The ratio of Ki67 or Active-Caspase3 positive epithelial cells to total epithelial cell count was taken over random fields and represented as a percentage. E-cadherin and/or a6-lntegrin staining was used to define the epithelial region within the sections. Cell treatment eSCC cells were treated with K149 (Echelon, B-0345), AS1949490 (Tocris, 3718), Volasertib (Selleckchem, S2235) (herein also referred to as compound of Formula III), GSK481364 (herein also referred to as GSK, compound of Formula V), Palbociclib (Selleckchem, S116) and BYL719 (Selleckchem, S2814) at various concentrations as described in the Results section.

CRC cells were treated with K149 (Echelon, B-0345), and Volasertib (Selleckchem, S2235) (herein also referred to as compound of Formula III), at various concentrations as described in the Results section. mRNA extraction, RNA-seq and analysis of bulk samples

RNA extraction was performed using the MicroElute Total RNA kit (Omega Biotek, R6831 -02) according to the manufacturer’s recommendations with DNase I digestion protocol on column. RNAquality was checked usinga Bioanalyzer 2100 (Agilent technologies). Indexed cDNA libraries were obtained using the Ovation Solo RNA-Seq System (NuGen) following manufacturer’s recommendations. The multiplexed libraries were loaded on a NovaSeq 6000 (Illumina) using a S2 flow cell and sequences were produced using a 200 Cycle Kit. Paired-end reads were mapped against the human reference genome hg38 using STAR software (version 2.5.3a) to generate read alignments for each sample. After transcripts assembling, gene level counts were obtained using HTSeq. Total raw counts were loaded on degust 4.1 .1 . All analyses were performed using EdgeR, TMM normalisation and “Min gene read count” set at 10. Control condition (CTRL) is defined as oesophageal cancer cell lines treated with DMSO or transfected with non-targeting siRNAs control for pharmacological inhibition and INPPL1 knockdown experiments, respectively. These biological samples were used as reference all along the paper to calculate the fold change of gene expressions. MDS plots and Volcano plots have been generated using degust 4.1.1. Heatmaps and Kmeans enrichment plots were generated using iDep 0.96 and represent values in logCPM scaled by row for the 1000 most variable genes between CTRL and cells treated with pharmacological inhibitors for 72h or transfected with siRNA for 48h.

Gene Set Enrichment Analysis (GSEA) analysis

GSEA analysis was performed using preranked gene set enrichment analysis from the fgsea package in R version 3.6.3, with “nperm=1000” and “maxSize=500”. The values of LFCwere used as the ranking metric. Gene sets were generated by taking the most up regulated genes compared to CTRL. The C2, C5, C6, Hallmarks, KEGG, and Reactome collections have been downloaded (http://bioinf.wehi.edu.au/software/MSigDB/) and the function “fgseaMultilevel” has been used. Pathways with adjusted p-value <0.05 were considered as significant. Synergy determination with SynergyFinder

To evaluate the drug combination effects between K149 and Volasertib, K149 and GSK481364, and AS1949490 and Volasertib a multi-dimensional two-drug synergy analysis was performed using the Synergyfinder 3.0 (https://synergyfinder.org). Cells were seeded in 96 well plates and treated with increasing concentrations of K149, Volasertib, GSK481364, and/or AS1949490 for 72h. At the end of the experiment MTS assay was performed to measure cell viability. The expected drug combination responses were calculated based on the zero interaction potency (ZIP) reference model with SynergyFinder 3.0 (doi.org/10.1093/nar/gkac382). Deviations between observed and expected responses with positive and negative values denote synergy a nd antagonism respectively. The synergy scores are interpreted as follows: a score inferior to -10 denotes antagonism, a score between -10 and 10 an additive effect, a score superior to 10 shows a synergistic effect.

RESULTS

INPPL1 Is Frequently Amplified in Human Esophageal Squamous Cell Carcinoma

Analysis of whole-genome sequencing data and copy number alterations (CNAs) in 96 samples of oesophageal squamous cell carcinoma (eSCC) from the Cancer Genome Atlas (TCGA) revealed frequent amplification of the 11 q 13 locus in this cancer (Fig. 1A). This recurrent CNA results in the amplification of CCND1 (11q13.3) and INPPL1 (11q13.4), which encodes the SH2 domain-containing inositol 5-phosphatase 2, known as SHIP2 (Fig. 1 B). In these eSCC samples, the amplification of INPPL1 is associated with reduced methylation of the gene and elevated expression of SHIP2 mRNA (Fig. 1C). SHIP2 functions as a phosphatidylinositol 3,4,5- trisphosphate 5-phosphatase (PI(3,4,5)P3 5-phosphatase) that could regulate the PI3K/AKT pathway (Fig. 1 D). We examined CNAs in genes encoding PI 4-phosphatases (INPP4A, INPP4B) and PI 5-phosphatases (SYNJ1 , SYNJ2, INPP5D, INPP5K, INPP5J, and OCRL) and found thatthese genes are less frequently altered than INPPL1 (Fig. 1 E). Interestingly, we observed a significant co-occurrence of CCND1 (11q13.3) and PIK3CA (3q26) amplifications in eSCC, suggesting that some CNAs associated with PI3K/AKT pathway activation are positively selected in the context of CCNDI amplification.

Immunostaining for SHIP2 revealed high expression in human oesophageal mucosa and heterogeneous expression in eSCC tumour epithelial cells (Fig. 1 F). Using tissue microarrays, we noted that while SHIP2 expression appeared higher in well-differentiated tumours compared to non-differentiated ones (Fig. 1G), there was no apparent correlation between SHIP2 expression intensity in the tumour and its stage (Fig. 1 H).

Analysis of data from the Cancer Cell Line Encyclopaedia (CCLE) in a panel of 22 different human eSCC cell lines confirmed the positive correlation between SHIP2 mRNA expression and the absolute copy number of INPPL1 (Fig. 11). Based on the INPPL1 copy number and SHIP2 mRNA expression level, three profiles were delineated: (1 ) cells lacking INPPL1 amplification with low SHIP2 mRNA expression (KYSE-30, KYSE-180, and TE-11), and (2) cells with INPPL1 amplification and high SHIP2 mRNA expression (COLO-680N, KYSE-520, and TE-14). (3) Several eSCC lines such as the KYSE-410 line had an intermediate phenotype, characterized by an intermediate SHIP2 expression as well as the absence of INPPL1 amplification. Furthermore, these data unveiled that SHIP2 mRNA is more abundant than any other PI 5-phosphatase (OCRL, INPP5B, SHIP1/2, Synaptojanin 1/2, INPP5E, INPP5J, and INPP5K) (Fig. 1J). Conversely, the SHIP2 homolog, SHIP1 (encoded by INPP5D), is virtually absent from these cells.

SHIP2 Knockdown Decreases Human Oesophagus Squamous Cell Carcinoma Cell Survival

In order to assess the impact of SHIP2 on eSCC cell growth, we employed the MTS assay to measure cell survival 72 hours after siRNA-mediated SHIP2 knockdown (KD) across various eSCC cells (Fig. 2A). Treatment with two different siRNA targeting SHIP2 (siSHIP2 and siSHIP2#2) led to a reduction KYSE-410 cell survival (Fig. 2B). The strongest impact was obtained with siSHIP2 and we focused on this in siRNA for the subsequent experiments. SHIP2 knockdown leads to a reduction in SHIP2 protein levels when compared to control siRNA (siCT), as well as a notable decrease in AKT S473 phosphorylation (Fig. 2C). To explore the role of SHIP2 in human eSCC cells in vitro, we then utilized multiple cell lines characterized by either high or low SHIP2 expression profiles based on the INPPL1 copy number and SHIP2 mRNA expression level, confirmed by Western blot (Fig. 2D). In these six eSCC lines, siRNA mediated SHIP2 KD resulted in a decrease in cell survival (Fig. 2E). To ascertain whether SHIP2 influences eSCC cell survival by modulating the PI3K/AKT pathway, we assessed AKT phosphorylation 48 hours after transfection with either control siRNA or siRNA specifically targeting SHIP2. In eSCC cell lines with varying SHIP2 expression levels, inhibition of EGF-induced pAKT S473 was clearly observed following SHIP2 KD (Fig. 2F). We further investigated into the role of SHIP2 using RNA sequencing of the eSCC lines with either a high (KYSE-520) or a low (KYSE-30) SHIP2 profile, which growth was the most decreased 48 hours post-SHIP2 KD, and compared these data with the ones obtained on KYSE-410 cells RNA sequencing (RNA-seq) (Fig. 2G, H). Gene Set Enrichment Analysis (GSEA) of RNA-seq data from KYSE-410 eSCC cells revealed a significant downregulation of transcripts associated with cell cycle-related 'G2/M checkpoint' (Fig. 2I).

Collectively, these findings suggest that SHIP2 modulates eSCC cell survival and proliferation, exhibiting variable effects on the transcriptome in vitro. Furthermore, its role in cell survival in vitro is closely tied to the regulation of AKT phosphorylation.

Pharmacological Inhibition ofSHIP2 Inhibits Human Esophagus Squamous Cell Carcinoma Cell Survival and Proliferation In Vitro and In Vivo

To assess the potential impact of SHIP2 pharmacological inhibition to decrease human eSCC cell growth in vitro, we treated our range of eSCC cell lines with compound K149 (herein also referred to as compound of Formula I), a potent SHIP1/2 inhibitor. Given the virtual absence of SHIP1 (encoded by INPP5D) in eSCC cells (Fig. 1 J), K149 can be regarded as a SHIP2-specific inhibitor in our model (Fig. 3A). Our observations indicate that a 72-hour treatment with K149 within the micromolar range led to reduced eSCC survival across six distinct cell lines (Fig. 3B). Utilizing an EdU incorporation assay upon K149 treatment, we substantiated that SHIP2 inhibition resulted in decreased cell proliferation (Fig. 3C). This effect was evident across cell lines exhibiting low (TE-11 , KYSE-180), intermediate (KYSE-410), and high (KYSE-520, TE-14, and COLO-680N) levels of SHIP2 expression. Furthermore, in these same cell lines, K149 treatment attenuated AKT phosphorylation in response to EGF stimulation for 5-10 min (Fig. 3D).

Concurrently, we conducted an in vivo experiment by transplanting eSCC cells with an intermediate SHIP2 expression level (KYSE-410) and treating them with K149 for one week (Fig. 3E). Our results demonstrated that this treatment effectively reduced tumour epithelial cell proliferation by measuring Ki67 staining and induced apoptosis (active-Casp3) (Fig. 3F,G).

To deepen our understanding of SHIP2 enzymatic activity inhibition in eSCC cells, we profiled KYSE-410 cells 48 hours after SHIP2 inhibition using K149 through RNA-seq analysis (Fig. 3H). This investigation unveiled the dysregulation of numerous transcripts upon SHIP2 pharmacological inhibition (Fig. 3I). Notably, Gene Set Enrichment Analysis (GSEA) revealed the enrichment of transcripts related to the cell cycle, including genes like TOP2A, MKI67, and E2F2 (Fig. 3I, J). Indeed, within the top 30 most significantly downregulated genes, 16 were directly associated with cell division, and in particular PLK1 (Fig. 3K).

To validate the effect of SHIP2 inhibition on the cell cycle, we replicated the experiment using AS1949490 (compound of Formula II), a specific albeit less potent SHIP2 inhibitor largely used to probe SHIP2 function by others (Figure 4A). Our findings revealed that SHIP2 inhibition with AS1949490 significantly downregulated 15 out of 16 transcripts related to cell division, consistent with those downregulated by K149 treatment (including PLK1 , Figure 4B). By comparing KYSE-410 cells treated with K149, AS1949490, and DMSO (i.e. the control condition), we determined that 40% to 50% of the transcripts deregulated by AS1949490 were also affected by K149 (Figure 4C, D). Collectively, these results underscore the potential of pharmacological SHIP2 inhibition as an innovative strategy for curtailing eSCC cell survival/proliferation both on cells in vitro and in mice in vivo.

SHIP2 Inhibition in eSCC Unravels Compensatory Mechanisms

To assess the viability of blocking SH I P2 as a potential strategy for eSCC treatment, we embarked on characterizing the outcomes of SHIP2 knockout (KO). Leveraging CRISPR/Cas9 technology, we generated four distinct KYSE-410 SHIP2 KO eSCC lines originating from individual cell colonies (Fig. 5A). Of note, SHIP2 protein expression remained undetectable in all four KO lines, while AKT phosphorylation levels only displayed a minor reduction in three out of the four lines (Fig. 5B). Morphologically, the KO lines exhibited similarities to the control line. In addition, SHIP2 KO cells grew with the same kinetic than control cells (Fig. 5C). These observations suggest an ability to compensate for the loss of SHIP2 in vitro. Seeking to identify potential adaptive mechanisms, we subjected the four KO lines and a control line (transfected with the same plasmid but devoid of guide RNA) to RNA-seq analysis (Fig. 5D). The data revealed a cluster comprising three SHIP2 KO lines (D4, G10, and H3) exhibiting distinct transcriptional profiles from the control line, while a fourth line (G4) displayed a comparable profile. Comparative analysis across these lines highlighted their individual differences (Fig. 5E). Remarkably, the three lines with the greatest divergence from the control line displayed an upregulation of another PI 5-phosphatase (INPP5J), potentially compensating for the absence of SHIP2 (Fig. 5E). Additionally, these lines exhibited downregulation of transcripts associated with the Notch pathway (HES2, HEY2) and squamous differentiation (EDAR, FLG, KRT6B, and OVOL1). Employing the K-means method, we also observed global modifications in ion transport and cell differentiation processes in these three lines (Fig. 5F). Further analysis of the G4 SHIP2 KO line, the closest match to the parental line despite SHIP2 protein loss, unveiled an upregulation of transcripts encoding aldo-keto reductases (AKR1 B10, AKR1C3 and AKR1 B15) (Fig. 5G) contrasting the downregulation observed in SHIP2 KD cells (refer to Fig. 2G). Gene Set Enrichment Analysis (GSEA) highlighted an enrichment of transcripts linked to cell division ('E2F_targets' and 'G2/M_checkpoint') and the Myc pathway (Fig. 5H). Interestingly, these changes ran counter to the direction observed in SHIP2 KD cells. These findings suggest the potential for compensatory mechanisms following SHIP2 loss, at least in vitro, and that exclusive SHIP2 inhibition (monotherapy) may prove ineffective for sustained eSCC treatment over extended periods due to the development of secondary resistance.

SHIP2 inhibition sensitizes eSCC cells to PLK1 inhibitor

In an effort to uncover vulnerabilities induced by SHIP2 pharmacological inhibition, we conducted a screening of the most prominently downregulated transcripts subsequent to SHIP2 inhibitor treatment. As elucidated in previous figures (i.e. Fig 3), SHIP2 inhibition determines the downregulation of transcripts implicated in cell division. Notably, within the roster of the ten most significantly downregulated genes following K149 treatment, we pinpointed polo-like kinase-1 (PLK1 ) as a pivotal player, known for its mastery over the cell cycle (Fig. 6A). Consistent with a direct or indirect regulation of PLK1 expression by SHIP2, we observed that small interfering RNA (siRNA)-mediated SHIP2 knockdown (KD) also results in the downregulation of PLK1 (Fig. 6B).

Volasertib (Fig. 6C; compound of Formula III), a potent PLK1 inhibitorthat showed clinical activity in a phase l/ll study in adult patients with relapsed/refractory acute myeloid leukemia, emerged as a salient candidate forexploration. This inhibitor elicited a discernible reduction in cell survival in eSCC cell lines at nanomolar concentrations (Fig. 6D). Motivated by these findings, we sought to discern whether concurrent SHIP2 and PLK1 inhibition could engender supplementary (additional) effects on in vitro cell survival. To this end, we exposed five distinct cell lines (KYSE- 520, COLO-680N, KYSE-410, TE-11, and KYSE-30) to individual doses of K149, Volasertib, or a dual application of both compounds (Fig. 6E). Impressively, the combined administration of the two drugs elicited an augmented impact on cell survival across all these lines (Fig. 6E). Particularly intriguing was the observation within TE-11 and KYSE-30 cells, where a K149 concentration insufficient to significantly attenuate cell survival was sufficient to enhance Volasertib's impact. This phenomenon, indicative of drug synergy, prompted us to quantitatively assess the synergy of K149 and Volasertib using a range of concentrations. Leveraging SynergyFinder 3.0, these analyses revealed ZIP scores consistently exceeding 10, indicative of robust synergistic interactions. Notably, areas of heightened synergy emerged (most synergistic areas), underscored by ZIP scores surpassing 20 (Fig. 6F). To challenge this result, we treated KYSE-410 cells with low concentrations of Volasertib and K149 (Fig. 6G). Strikingly, while each drug alone had virtually no impact on eSCC cell survival, the combination of the two inhibitors decreased cells survival by 25%. To determine whether K149 may sensitize eSCC cells to another cell cycle inhibitor, we examined the combination of K149 with Palbociclib, a specific inhibitor of CDK4/6. I ntrigu i ngly, our observations revealed the absence of any synergistic effects between the SHIP2 and Palbociclib, a CDK4/CDK6 inhibitor (Data not shown). This distinctive outcome underscores the unique and specific nature of the synergy observed between K149 and Volasertib.

To decipher the mechanistic underpinnings of this synergistic interplay, we focused on COLO- 680N, which exhibited heightened sensitivity to K149. Through examining pAKT S473 expression levels in cells treated with K149 and/or Volasertib, as well as BYL719 (alpelisib) — a small molecule inhibitor of PI3K p110a developed for cancer therapy and currently FDA-approved for the treatment of metastatic breast cancer — we uncovered pivotal insights (Fig. 6H). Specifically, Volasertib alone, akin to K149, lowered pAKTS473 in eSCC cells. However, the co-administration of both agents led to a near-abolition of pAKT S473, mirroring the efficacy of a PI3K inhibitor (Fig. 6H). This compelling convergence in the regulation of AKT phosphorylation underscores a potentially crucial nexus between SHIP2 and PLK1 inhibitors.

Synergistic effects of SHIP2 and PLK1 inhibition were also confirmed for other inhibitor compounds, in particular for the combinations of K149 and GSK461364, and AS1949490 and Volasertib in KYSE-410 eSCC cells using SynergyFinder 3.0 analyses as shown in Figures 7 and 8. Figure 7 shows a ZIP score of 29.6 between K149 and GSK461364, indicative of robust synergistic interaction between these two compounds as well. Figure 8A-B shows a ZIP score of 21 .07 and a Bliss score of 30.05 between AS1949490 and Volasertib also showing robust synergistic interaction between these two compounds.

SHIP2 inhibition sensitizes colorectal cancer cells to PLK1 inhibitor

The investigate whether the synergistic effect can also be observed in other cancer cell types, the effect of SHIP2 inhibition in combination with PLK1 inhibition was evaluated in colorectal cancer (CRC). To this end, 4 distinct cell lines (HCT116, DLD1 , LOVO, HT29) were exposed to individual doses of K149, Volasertib, or a dual application of both compounds for 72h (Fig. 9). Cell viability was assessed using the MTS assay and drug synergy was calculated using SynergyFinder 3.0. As shown in Fig. 9 B, the combination of K149 with Volasertib shows a synergistic effect in all four CRC cell lines as evidenced by a ZIP score above 10.

Claims

1 . A SH2 domain-containing inositol 5’-phosphatase 2 (SHIP2) inhibitor and a Polo-like kinase I (PLK1) inhibitor for use in the treatment of cancer in a subject.

2. The SHIP2 inhibitorand PLK1 inhibitorfor use accordingto claim 1 , wherein the SHIP2 inhibitor and the PLK1 inhibitor are administered to the subject simultaneously or sequentially in any order.

3. A SHIP2 inhibitor for use in the treatment of cancer in a subject, wherein the SHIP2 inhibitor is administered to the subject simultaneously or sequentially in any order with a PLK1 inhibitor.

4. A PLK1 inhibitor for use in the treatment of cancer in a subject, wherein the PLK1 inhibitor is administered to the subject simultaneously or sequentially in any orderwith a SHIP2 inhibitor.

5. A kit of parts comprising a SHIP2 inhibitor and a PLK1 inhibitor for use in the treatment of cancer in a subject.

6. The kit of parts for use according to claim 5 wherein the kit comprises a dosage form of the SHIP2 inhibitor and a dosage form of the PLK1 inhibitor which are separate and which allow either simultaneous or sequential in any order administration of both dosage forms.

7. The SHIP2 inhibitor and PLK1 inhibitor for use according to claim 1 or 2, the SHIP2 inhibitor for use according to claim 3, the PLK1 inhibitor for use according to claim 4, or the kit of parts for use according to claim 5 or 6, wherein the cancer is a solid tumour; preferably wherein the solid tumour is selected from the group consisting of oesophageal cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, brain/CNS cancer, cervical cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, endometrial cancer, pancreatic cancer, sarcoma, and melanoma.

8. The SHIP2 inhibitor and PLK1 inhibitor for use according to claim 7, or the SHIP2 inhibitor for use according to claim 7, or the PLK1 inhibitor for use according to 7, or the kit of parts for use according 7, wherein the cancer is selected from the group consisting of oesophageal cancer, colorectal cancer, and head and neck cancer.

9. The SHIP2 inhibitor and PLK1 inhibitor for use according to claim 7, or the SHIP2 inhibitor for use according to claim 7, or the PLK1 inhibitor for use according to 7, or the kit of parts for use according 7, wherein the cancer is oesophageal cancer or colorectal cancer.

10. The SHIP2 inhibitor and PLK1 inhibitor for use according to 7, the SHIP2 inhibitor for use according to 7, the PLK1 inhibitor for use according to 7, or the kit of parts for use according to 7, wherein the cancer is oesophageal cancer, preferably oesophageal squamous cell carcinoma (eSCC).

11 . The SHIP2 inhibitor and PLK1 inhibitor for use according to claim 7, or the SHIP2 inhibitor for use according to claim 7, or the PLK1 inhibitor for use according to 7, or the kit of parts for use according 7, wherein the cancer is colorectal cancer.

12. The SHIP2 inhibitor and PLK1 inhibitor for use according to any one of claims 1 , 2, 7 to 11 , the SHIP2 inhibitor for use according to any one of claims 3, 7 to 11 , the PLK1 inhibitor for use according to anyone of claims 4, 7 to 11 , or the kit of parts for use according to anyone of claims 5 to 11 , wherein the SHIP2 inhibitor is a specific inhibitor of SHIP2.

13. The SHIP2 inhibitor and PLK1 inhibitor for use according to anyone of claims 1 , 2, 7 to 12, the SHIP2 inhibitor for use according to any one of claims 3, 7 to 12, the PLK1 inhibitor for use according to anyone of claims 4, 7 to 12, or the kit of parts for use according to anyone of claims 5 to 12, wherein the PLK1 inhibitor is a specific inhibitor of PLK1 .

14. The SHIP2 inhibitor and PLK1 inhibitor for use according to anyone of claims 1 , 2, 7 to 13, the SHIP2 inhibitor for use according to any one of claims 3, 7 to 13, the PLK1 inhibitor for use according to anyone of claims 4, 7 to 13, or the kit of parts for use according to anyone of claims 5 to 13, wherein the subject is a human subject.

15. The SHIP2 inhibitor and PLK1 inhibitor for use according to anyone of claims 1 , 2, 7 to 14, the SHIP2 inhibitor for use according to any one of claims 3, 7 to 14, the PLK1 inhibitor for use according to any one of claims 4, 7 to 14, the kit of parts for use according to any one of claims 5 to 14, wherein the subject has been selected by assaying a sample from the subject for a mutation or amplification in one or more of the following genes: CCND1 , PIK3CA, INPPL1 ; preferably wherein the sample is a tumour sample.

16. A kit of parts comprising a dosage form of a SHIP2 inhibitor and a dosage form of a PLK1 inhibitor.

17. A pharmaceutical composition comprising a SHIP2 inhibitor and a PLK1 inhibitor.