WO2021202961A1

WO2021202961A1 - Methods of treating immunotherapy resistant metastatic melanoma

Info

Publication number: WO2021202961A1
Application number: PCT/US2021/025514
Authority: WO
Inventors: Ann Richmond; Rebecca SHATTUCK-BRANDT; Anna VILGELM
Original assignee: United States Represented By Department Of Veterans Affairs AS
Current assignee: United States Represented By Department Of Veterans Affairs AS
Priority date: 2020-04-02
Filing date: 2021-04-02
Publication date: 2021-10-07
Anticipated expiration: 2022-10-02

Abstract

Therapeutic methods and pharmaceutical compositions for treating immunotherapy resistant metastatic melanoma.

Description

METHODS OF TREATING IMMUNOTHERAPY RESISTANT METASTATIC MELANOMA

GOVERNMENT RIGHTS

[0001] The United States as represented by the Department of Veterans Affairs is an applicant. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0002] Methods of treating immunotherapy resistant metastatic melanoma using a Mouse double minute 2 homolog (MDM2) inhibitor or a combination of a MDM2 inhibitor, a BRAF inhibitor and a MEK inhibitor.

BACKGROUND OF THE INVENTION

[0003] p53 is a tumor suppressor and transcription factor that responds to cellular stress by activating the transcription of numerous genes involved in cell cycle arrest, apoptosis, senescence, and DNA repair. Unlike normal cells, which have infrequent cause for p53 activation, tumor cells are under constant cellular stress from various insults including hypoxia and pro-apoptotic oncogene activation. Thus, there is a strong selective advantage for inactivation of the p53 pathway in tumors, and it has been proposed that eliminating p53 function may be a prerequisite for tumor survival. In support of this notion, three groups of investigators have used mouse models to demonstrate that absence of p53 function is a continuous requirement for the maintenance of established tumors. When the investigators restored p53 function to tumors with inactivated p53, the tumors regressed.

[0004] p53 is inactivated by mutation and/or loss in 50% of solid tumors and 10% of liquid tumors.

Other key members of the p53 pathway are also genetically or epigenetically altered in cancer. MDM2, an oncoprotein, inhibits p53 function, and it is activated by gene amplification at incidence rates that are reported to be as high as 10%. MDM2, in turn, is inhibited by another tumor suppressor, pl4ARF. It has been suggested that alterations downstream of p53 may be responsible for at least partially inactivating the p53 pathway in p53^WT tumors (p53 wild type). In support of this concept, some p53^WT tumors appear to exhibit reduced apoptotic capacity, although their capacity to undergo cell cycle arrest remains intact. One cancer treatment strategy involves the use of small molecules that bind MDM2 and neutralize its interaction with p53. MDM2 inhibits p53 activity by three mechanisms: 1) acting as an E3 ubiquitin ligase to promote p53 degradation; 2) binding to and blocking the p53 transcriptional activation domain; and 3) exporting p53 from the nucleus to the cytoplasm. All three of these mechanisms would be blocked by neutralizing the MDM2-p53 interaction. In particular, this therapeutic strategy could be applied to tumors that are p53^WT, and studies with small molecule MDM2 inhibitors have yielded promising reductions in tumor growth both in vitro and in vivo. Further, in patients with p53-inactivated tumors, stabilization of wild type p53 in normal tissues by MDM2 inhibition might allow selective protection of normal tissues from mitotic poisons. As used herein, MDM2 means a human MDM2 protein and p53 means a human p53 protein. It is noted that human MDM2 can also be referred to as HDM2 or hMDM2. Several MDM2 inhibitors are in human clinical trials for the treatment of various cancers.

[0005] Although recently immunotherapy has been approved for treating metastatic melanoma, some may be resistant to immunotherapy or progress after the immunotherapy. The present invention provides a method of treating metastatic melanoma.

SUMMARY OF THE INVENTION

[0006] The present invention relates to a method of treating immunotherapy resistant metastatic melanoma comprising the step of administering to a human subject in need thereof, a therapeutically effective amount of a MDM2 inhibitor or a MDM2 inhibitor in combination with a BRAF inhibitor and a MEK inhibitor.

[0007] In one aspect, the present invention relates to a method of treating metastatic melanoma in a human subject previously treated with immunotherapy comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor. In an embodiment, the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof.

[0008] In an embodiment, the method further comprises detecting the BRAF genotype in the human subject. In an embodiment, the human subject exhibits a wild-type BRAF^⁶⁰⁰ (BRAF^) genotype.

[0009] In an embodiment, the method further comprises detecting the NRAS genotype in the human subject. In an embodiment, the human subject exhibits a wild-type NRAS ( NRAS ^WT) genotype.

[0010] In an embodiment, the method further comprises detecting the NF1 genotype in the human subject. In an embodiment, the human subject exhibits a wild-type NF1 genotype (A/F1^WT). In an embodiment, the human subject exhibits BRAF^⁷, A/RAS^and NF1^WT.

[0011] In an embodiment, the human subject exhibits a mutant NRAS. In an embodiment, the human subject exhibits a mutant NF1.

[0012] In an embodiment, the immunotherapy is an ex vivo cell therapy selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR)-engineered peripheral blood lymphocytes (PBL) and chimeric antigen receptor ((CAR)-engineered PBL).

[0013] In an embodiment, the immunotherapy is an immune checkpoint protein inhibitor therapy.

[0014] In an embodiment, the immune checkpoint protein inhibitor is an anti-PD-Ll antibody selected from the group consisting of BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.

[0015] In an embodiment, the immune checkpoint protein inhibitor is an anti-PD-1 antibody selected from group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab-rwlc, AMP-224, AMP-514, PDR001, and fragments, conjugates, biosimilars, or variants thereof.

[0016] In an embodiment, the immune checkpoint protein inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and fragments, conjugates, biosimilars, or variants thereof.

[0017] In an embodiment, wherein the immunotherapy is a T-cell engager selected from catumaxomab, FBTA05, Ertumaxomab, Ektomun, blinatumomab, solitomab, and fragments, conjugates, biosimilars, or variants thereof. [0018] In another aspect, the present invention relates to a method of treating metastatic melanoma in a human subject previously treated with immunotherapy comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, a BRAF inhibitor and a MEK inhibitor.

[0019] In an embodiment, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof.

[0020] In an embodiment, the BRAF inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and combinations thereof.

[0021] In an embodiment, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, selumetinib, pimasertib, binimetinib, and combinations thereof.

[0022] In an embodiment, the method further comprises detecting the BRAF genotype in the human subject. In an embodiment, the human subject exhibits BRAF^V¥0 mutation. In an embodiment, the human subject exhibits NRAS ^QG1 mutation.

[0023] In an embodiment, the immunotherapy is an ex vivo cell therapy selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR)-engineered peripheral blood lymphocytes (PBL) and chimeric antigen receptor ((CAR)-engineered PBL).

[0024] In an embodiment, the immunotherapy is an immune checkpoint protein inhibitor therapy. [0025] In an embodiment, the immune checkpoint protein inhibitor is an anti-PD-Ll antibody selected from the group consisting of BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.

[0026] In an embodiment, the immune checkpoint protein inhibitor is an anti-PD-1 antibody selected from group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab-rwlc, AMP-224, AMP-514, PDR001, and fragments, conjugates, biosimilars, or variants thereof.

[0027] In an embodiment, the immune checkpoint protein inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and fragments, conjugates, biosimilars, or variants thereof.

[0028] In an embodiment, the immunotherapy is a T-cell engager selected from catumaxomab, FBTA05, Ertumaxomab, Ektomun, blinatumomab, solitomab, and fragments, conjugates, biosimilars, or variants thereof.

[0029] In one embodiment, the immune checkpoint protein inhibitor is an anti-PD- L2 antibody. In one embodiment, the anti-PD- L2 antibody is rHlgM12B7A.

[0030] In an embodiment, the compound of Formula (I) is in a free form.

[0031] In an embodiment, the MDM2 inhibitor is a pharmaceutically acceptable salt of a compound of Formula (I).

[0032] In an embodiment, the compound of Formula (I) is in an amorphous form.

[0033] In an embodiment, the compound of Formula (I) is administered once daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg.

[0034] In an embodiment, the compound of Formula (I) is administered twice daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg.

[0035] In an embodiment, the human is treated with the MDM2 inhibitor for a period selected from the group consisting of about 3 days, about 5 days, about 7 days, 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, and about 56 days.

[0036] In an embodiment, the compound of Formula (I) is orally administered. [0037] In an embodiment, the MDM2 inhibitor is administered before administration of the BRAF inhibitor and MEK inhibitor.

[0038] In an embodiment, the MDM2 inhibitor is administered after administration of the BRAF inhibitor and MEK inhibitor.

[0039] In an embodiment, the MDM2 inhibitor is administered concurrently with administration of the BRAF inhibitor and MEK inhibitor.

[0040] In an embodiment, the therapeutically effective amount of the MDM2 inhibitor is 120 mg.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.

[0042] FIG. 1A illustrates patient demographics and molecular characterization of PDX tumors. A panel of 15 PDX melanoma tumors is arranged by distinct genetic phenotypes based on the mutation status. For each PDX, the patient demographics are shown including Clark's Level tumor stage and prior treatments. Prior treatments are list in the order they were received. The genetic results are based upon NextGen sequencing of the primary tumor sample or PDX P2 passage (PDX1577, 1668, 2316 and 2552, indicated by a *) using a Comprehensive Cancer Panel, and the results for 10 melanoma driver mutations are shown. STOP LOST, indicates a nonsense mutation within a stop codon, and Splice Site indicates a mutation (deletion) involving a splice site. For PDX1839 both the primary and the lung metastasis were sequenced, and the BRAF^V600E mutation was present in the metastasis but not the primary tumor. The metastatic tumor was used to generate the PDX.

[0043] FIG. IB illustrates the TP53 mutations by NGS. The specific TP53 mutations detected by NextGen sequencing are shown for each PDX. The identification of the mutations, the small nucleotide polymorphism (SNP) Effect and Impact, and the designation of germline or somatic mutation was determined by using QIAGEN NGS Data Web Analysis Web Portal. SnpEff is an open-source tool that annotates variants and predicts their effects on genes by using an interval forest approach. The variant frequency is also listed for each non-synonymous mutation (NSM). The PDX numbers listed below the horizontal line are those tumors that express the P53P72R polymorphism. The gray shading highlights those PDX tumors expressing point NSMs within the TP53 gene. NGS was performed on the patient tumor (P0) except for PDX1577, 1668, 2316, and 2552. For these four PDX, the second passage (P2) was sequenced.

[0044] FIG. 2A illustrates those PDX lines that responded to the standard therapy dabrafenib and trametinib (D+T) but not to the compound of Formula (I) (Group I), with PDX1839 as an example.

[0045] FIG. 2B illustrates those PDX lines that responded to the combination therapy (the compound of Formula (I) + dabrafenib + trametinib) synergistically (Group II), but not to the compound of Formula (I) alone or trametinib alone or standard therapy dabrafenib and trametinib (D+T), with PDX1946 as an example.

[0046] FIG. 2C illustrates those PDX lines that responded the compound of Formula (I) alone, but not to standard therapy dabrafenib and trametinib (D+T) (Group III), with PDX2316 as an example.

[0047] FIG. 2D illustrates those PDX lines that responded the compound of Formula (I) alone, but not to standard therapy dabrafenib and trametinib (D+T) (Group III), with PDX1595 and 1668 as examples, and also illustrates the compound of Formula (I) treatment increases nuclear localization of P53. IHC staining of huP53 in melanoma PDX tumors from mice treated with the vehicle or the compound of Formula (I). 20X images are shown and the scale marker is 100 pm. Tumor growth is also shown. The standard therapy for PDX1595 was trametinib and for PDX1668 the standard therapy was dabrafenib + trametinib. For FIGs. 2A, 2B and 2C, each panel in A-C includes data showing the effect of drug treatments on Tumor Growth, Final Tumor Weight, %Ki67 Staining and the Tumor Growth Statistical Analysis. Tumor volume was analyzed on the natural log scale to better meet normality assumptions and the predicted mean and standard error of tumor volume over time for each treatment group is shown. Dot plot of tumor weight (g) by treatment and % positive Ki67 by treatment (gray line: mean with standard error) are shown. A t-ratio table for pairwise comparison in tumor growth rate between treatments based on the mixed-effect model with post hoc tests is shown.

[0048] FIG. 3A illustrates a summary of statistical summary of t. ratios obtained from the statistical analysis of treatment difference comparisons of the tumor growth rate based on the tumor volume for each PDX treatment comparison. The BRAF and TP53 mutational status of each PDX is also listed. The group assignment, shown in the final column, is based on the response to the compound of Formula (I) and T+/-D treatment.

[0049] FIG. 3B illustrates the synergy analysis of the effect of the compound of Formula (I) and dabrafenib + trametinib on the PDX lines. The mean estimated tumor growth rate of 4 independent PDX Group II lines is graphed with a 95% confidence interval by treatment. Mice implanted with PDX1351, 1577, 1946, and 2552 were treated in vivo with Vehicle, the compound of Formula (I), D+T, or the compound of Formula (l)+D+T as described in FIGs. 2A-2D.

[0050] FIG. 4A illustrates synergy associated with changes in cell morphology is consistent with karyorrhexis and large vacuole formation. FI&E is shown for Vehicle and the compound of Formula (I) + D+T treated mice implanted with PDX1351, PDX1946, PDX2552 and PDX1577 (Group III). The t-ratio for the statistical difference between the vehicle or the compound of Formula (I) treatment group compared to the compound of Formula (l)+D+T treatment group is shown. 20X images are shown and the scale marker is lOOpm.

[0051] FIG. 4B illustrates alterations in protein expression associated with synergism. A volcano plot of RPPA data obtained from vehicle-treated and the compound of Formula (I) + D+T treated Group III PDX tumors samples for PDX1179, PDX1351, and PDX2252 is shown. Three tumors were analyzed for each PDX treatment.

[0052] FIG. 4C illustrates alterations in protein expression associated with synergism. A heat map of RPPA data obtained from vehicle-treated and the compound of Formula (I) + D+T treated Group III PDX tumors samples for PDX1179, PDX1351, and PDX2252 is shown. Three tumors were analyzed for each PDX treatment.

[0053] FIG. 5A illustrates the results of oncoprint cluster analysis. DNA sequence analysis was performed using NextGen sequencing. Paired targeted analysis for all 15 PDX tumors was performed using Oncoprint on the cBioPortal (http://www.cbioportal.org/) hosted by Sloan Kettering Institute. These results show the analysis of the 10 genes listed in Table 1. The inset table lists the brackets and terms used to describe the copy number variations.

[0054] FIG. 5B is a volcano plots of RPPA data obtained from vehicle-treated and the compound of Formula (I) treated Group I and II.

[0055] FIG. 5C is a heat map of RPPA data obtained from vehicle-treated and the compound of Formula (I) treated Group I and II.

[0056] FIG. 5D is a volcano plots of RPPA data obtained from vehicle-treated and the compound of Formula (I) treated Group III. [0057] FIG. 5E is a heat map of RPPA data obtained from vehicle-treated and the compound of Formula (I) treated Group III. For FIGs. 5A-5E, three tumors were analyzed for each PDX treatment. Group I and II PDX tumors were PDX1179, 1351 and 2552 and Group III PDX tumors were PDX1129,

1595, 1668 and 2316. Only those proteins with an FDR <0.05 and log2(FC)=+/- 0.4 were included in the heat map.

[0058] FIG. 6A illustrates the tumor growth of PDX1577. Tumor volume was analyzed on the natural log scale to better meet normality assumptions and the predicted mean and standard error of tumor volume over time for each treatment group is shown.

[0059] FIG. 6B illustrates the final tumor weight of PDX1577 under different treatment. Dot plot of tumor weight (g) by treatment (gray line: mean with standard error).

[0060] FIG. 6C is a T-ratio table for PDX tumors treated with Navitoclax. A t-ratio table for pairwise comparison in tumor growth rate between treatments based on the mixed-effect model with post hoc tests is shown.

[0061] FIG. 7A illustrates NGS summary. For the panel of 15 PDX melanoma tumors, which were analyzed by NextGen sequencing, the mean read depth and number of high confidence variants are listed for each PDX.

[0062] FIG. 7B illustrates the mutations and copy number variations (cnv) for ten driver genes and MDM2. All NSMs and CNV noted by NGS for the 10 driver genes and MDM2 are listed. CNV is not available for PDX0807, 1129, 2195 and 9164. The following brackets were used for labeling CNV. HOMDEL 0-0.5 copies; HETLOSS 0.51-1.5 copies; GAIN: 2.5-3.5 copies; AMP >3.5 copies (see FIG. 5A). There were no NSMs for MDM2 in any PDX tumor. The primary tumor sample (P0) was analyzed except for PDX1577, 1668, 2316 and 2552. For these 4 PDX tumors, the second passage (P2) was analyzed and these PDX tumors are indicated by an *.

[0063] FIG. 8A illustrates short tandem repeat (STR) analysis results. STR analysis on second passage (P2) PDX tumors, the originating patient tumor (P0), and when available the patient's blood, was used to confirm that the PDX was derived from the patient tumor. In all 8 PDX tumors, STR analysis at 16 different loci matched the parent tumor sample (P0) and the blood when available. Two distinct isolates from each tumor (A and B) were analyzed. The clonal variation between either the blood, patient sample or PDX tumor are noted and these variations all represented the loss of an allele. No gain of an allele was detected. [0064] FIG. 8B illustrates the validation of PDX tumors via immunohistochemistry. Patient samples (P0) and second passage PDX tumors (P2) were stained by H&E and IHC for Ki67, Melan-A, and SOX10. Either DAB or FastRed was used as the chromogen. 20X magnification is shown. All PDX's stained positive for the melanoma marker SOX10. The levels of Ki67 and Melan-A staining were similar in both the patient tumor and P2 PDX passage. Also, when comparing the H&E staining of the patient sample (P0) to the PDX (P2), it was noted that melanotic tumors (PDX1767) remained melanotic while those that were amelanotic (PDX2552, PDX 1595 and PDX 1668) remained amelanotic. These data are representative of all 13 PDX tumors. All images are at 20X magnification and the scale is 3.556 mm = 100 pm.

[0065] FIG. 9A illustrates the effect of drug treatments on estimated tumor growth for PDX1839 tumor.

[0066] FIG. 9B illustrates the effect of drug treatments on estimated tumor growth for PDX9164 tumor.

[0067] FIG. 9C illustrates the effect of drug treatments on estimated tumor growth for PDX2195 tumor.

[0068] FIG. 9D illustrates the effect of drug treatments on estimated tumor growth for PDX2252 tumor.

[0069] FIG. 9E illustrates the effect of drug treatments on estimated tumor growth for PDX1351 tumor.

[0070] FIG. 9F illustrates the effect of drug treatments on estimated tumor growth for PDX1179 tumor.

[0071] FIG. 9G illustrates the effect of drug treatments on estimated tumor growth for PDX1946 tumor.

[0072] FIG. 9H illustrates the effect of drug treatments on estimated tumor growth for PDX2552 tumor.

[0073] FIG. 91 illustrates the effect of drug treatments on estimated tumor growth for PDX1577 tumor.

[0074] FIG. 9J illustrates the effect of drug treatments on estimated tumor growth for PDX2316 tumor. [0075] FIG. 9K illustrates the effect of drug treatments on estimated tumor growth for PDX1129 tumor.

[0076] FIG. 9L illustrates the effect of drug treatments on estimated tumor growth for PDX1767 tumor.

[0077] FIG. 9M illustrates the effect of drug treatments including Navitoclax on estimated tumor growth for PDX1129 tumor.

[0078] FIG. 9N illustrates the effect of drug treatments including Navitoclax on estimated tumor growth for PDX1351 tumor.

[0079] FIG. 90 illustrates the effect of drug treatments including Navitoclax on estimated tumor growth for PDX1577 tumor.

[0080] FIG. 9P illustrates the effect of drug treatments including Navitoclax on estimated tumor growth for PDX1668 tumor. For FIGs. 9A-P, Tumor volume was analyzed on the natural log scale to better meet normality assumptions. The predicted mean and standard error of tumor volume over time for each treatment group is shown. A statistics table for pairwise comparison in tumor growth rate between treatments based on the mixed-effect model with post hoc tests is shown below each growth curve. The final tumor weight for all PDX tumors is shown. Dot plot of tumor weight (g) by treatment (gray line: mean with standard error) is shown.

[0081] FIG. 10 illustrates alterations in protein expression associated with D+T response in Group I PDX tumors. RPPA data obtained from Group I PDX tumors (PDX1839, 2195 and 2252) treated with D+T or vehicle control were analyzed and the volcano plot is shown. Three tumors for each PDX treatment were analyzed.

[0082] FIG. 11 illustrates that protein expression patterns did not predict the compound of Formula (I) responsiveness in Group III PDX tumors compared to Group I and II PDX tumors. RPPA data obtained from vehicle-treated Group I PDX tumors (PDX1839, 2195 and 2252) and Group II PDX tumors (PDX 1179, 1351, and 2552) were compared to vehicle-treated Group III PDX tumors (PDX1129, 1595, 1668 and 2316). These data were analyzed and the volcano plot is shown. Three tumors for each PDX treatment were analyzed. No significant differences in protein expression were noted.

[0083] FIG. 12 illustrates the association between mutation status and t ratio. The correlation between the BRAF^V600 mutation status ("Yes", for mutation and "No", for wild-type BRAF or mutations outside of the V600 codon) are plotted relative to the t-ratio for the difference in tumor growth rate between the compound of Formula (I) treated and vehicle treated mice.

DETAILED DESCRIPTION OF THE INVENTION

[0084] While preferred embodiments of the invention are shown and described herein, such embodiments are provided by way of example only and are not intended to otherwise limit the scope of the invention. Various alternatives to the described embodiments of the invention may be employed in practicing the invention.

[0085] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

[0086] The terms "administered in combination with" and "co-administration" as used herein, encompass administration of two or more active pharmaceutical ingredients to a subject so that both agents and/or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more agents are present.

[0087] The term "combination" or "pharmaceutical combination" is defined herein to refer to either a fixed combination in one dosage unit form, a non-fixed combination or a kit of parts for the combined administration where the therapeutic agents may be administered together, independently at the same time or separately within time intervals, which preferably allows that the combination partners show a cooperative, e.g. synergistic effect. Thus, the single compounds of the pharmaceutical combination of the present disclosure could be administered simultaneously or sequentially.

[0088] Furthermore, the pharmaceutical combination of the present disclosure may be in the form of a fixed combination or in the form of a non-fixed combination.

[0089] The term "effective amount" or "therapeutically effective amount" refers to that amount of an active pharmaceutical ingredient or combination of active pharmaceutical ingredients as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, and other factors which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, (e.g., the reduction of platelet adhesion and/or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[0090] The term "fixed combination" means that the therapeutic agents, e.g., the single compounds of the combination, are in the form of a single entity or dosage form.

[0091] The term "IC50" refers to the half maximal inhibitory concentration, i.e. inhibition of 50% of the desired activity. The term "EC50" refers to the drug concentration at which one-half the maximum response is achieved.

[0092] In an embodiment, compounds described herein include of the isomers, stereoisomers, and enantiomers thereof.

[0093] The term "non-fixed combination" means that the therapeutic agents, e.g., the single compounds of the combination, are administered to a patient as separate entities or dosage forms either simultaneously or sequentially with no specific time limits, wherein preferably such administration provides therapeutically effective levels of the two therapeutic agents in the body of the subject, e.g., a mammal or human in need thereof.

[0094] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, and absorption delaying agents. The use of such media and agents for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional media or agent is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Supplementary active ingredients can also be incorporated into the described compositions. Unless otherwise specified, or clearly indicated by the text, reference to therapeutic agents useful in the pharmaceutical combination of the present disclosure includes both the free base of the compounds, and all pharmaceutically acceptable salts of the compounds.

[0095] The term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counter ions known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In selected embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.

[0096] The terms "QD," "qd," or "q.d." means quaque die, once a day, or once daily. The terms "BID," "bid," or "b.i.d." mean bis in die, twice a day, or twice daily. The terms "TID," "tid," or "t.i.d." mean ter in die, three times a day, or three times daily. The terms "QJD," "qid," or "q.i.d." mean quaterin die, four times a day, or four times daily.

[0097] "Solvate" refers to a compound in physical association with one or more molecules of a pharmaceutically acceptable solvent.

[0098] A "therapeutic effect" as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0099] When ranges are used herein to describe, for example, physical or chemical properties such as molecular weight or chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. Use of the term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") includes those embodiments such as, for example, an embodiment of any composition of matter, method or process that "consist of" or "consist essentially of" the described features.

[0100] Compounds of the invention also include crystalline and amorphous forms, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as combinations thereof. "Crystalline form" and "polymorph" are intended to include all crystalline and amorphous forms of the compound, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms, as well as combinations thereof, unless a particular crystalline or amorphous form is referred to.

Administration of compounds

[0101] The present invention relates to pharmaceutical combinations or pharmaceutical compositions that are particularly useful as a medicine. Specifically, the combinations or compositions of the present disclosure can be applied in the treatment of immunotherapy resistant metastatic melanoma.

[0102] While nearly 60% of melanoma patients respond to treatment with an immune checkpoint inhibitor (ICI) or targeted therapy, identification of therapeutic regimes that successfully treat the 40% of melanoma patients who fail to respond or progress after therapy, remains elusive. Here the response of patient-derived xenograft tumors established from melanoma patients who progressed on previous ICI therapy to a new therapeutic regimen was evaluated: treatment with either MDM2 antagonist alone, or in combination with BRAF/MEK or MEK inhibitor, depending upon BRAF mutation status. Data show that those tumors that did not exhibit the BRAF^^OE/M _mut_ati₀n responded to MDM2 inhibition with significant growth inhibition, while those with BRAF^®®^/M mutation responded to treatment with combined MDM2, BRAF, and MEK inhibition. These data provide key insight into the potential for using MDM2 inhibitors alone or combined with BRAF and/or MEK inhibitors for the treatment of therapy- resistantmelanoma.

[0103] The American Cancer Society estimates that there will be over 96,000 new cases of melanoma in the United States in 2019, with an estimated 7,230 deaths. The World Health Organization estimates nearly 300,000 melanoma skin cancers occur globally each year with the highest rates in Australia and New Zealand. The incidence of melanoma has risen rapidly over the past 30 years, especially in the population over 50, which places melanoma as the 5¹-*¹ most common form of new cancer diagnoses. For the period between 2006-2015, the incidence rate increased by 3% per year.

[0104] Advances in metastatic melanoma treatment followed the identification and classification of melanoma driver mutations, particularly mutations in BRAF characterized in 2002. Characterization of driver mutations paved the way for new treatment strategies focused on small molecule inhibitors targeting specific proteins involved in the melanoma pathogenesis. The most common oncogenic driver mutations are in the mitogen-activated protein-kinase (MAPK) pathway where BRAF mutations occur in approximately 50% of melanomas and NRAS mutations in 15-20% of melanomas, with neither BRAF nor NRAS mutations occurring concomitantly. Activating mutations in NRAS occur at either codon 12 or codon 61, or less frequently codon 13. The mutation at codon Q61, resulting in the Q61R/K/L substitutions, disrupts the GTPase activity of RAS, resulting in a constitutively active conformation; whereas, mutations at codon G12 or G13, affect the Walker A-motif of the protein, thus decreasing its sensitivity to GTPase-accelerating proteins. The primary alteration and the most common of the BRAF mutations is V600E/K/M. This activating mutation accounts for nearly 90% of all the BRAF mutations in melanomas. Inhibitors of BRAF^^^/K/M _were developed including dabrafenib, vemurafenib, and encorafenib, and shown successfully in Phase III clinical trials. MEK kinases (MEK1 and MEK2), which function immediately downstream of BRAF, also have been studied as potential targets for inhibition, especially in combination with BRAF inhibition. Three BRAF-MEK inhibitor combinations (dabrafenib- trametinib, vemurafenib-cobimetinib, and encorafenib and binimetinib) were successful in Phase III clinical trials. Treatment of patients with wild-type BRAF has proved much more difficult, especially for those with NRAS mutations. There are currently no targeted therapies that directly target mutant NRAS; however, MEK inhibitors (trametinib and pimasertib) have been shown to have some effect in wild type BRAF and mutant NRAS melanoma.

[0105] The suboptimal response rates for targeted therapies, particularly within tumors not expressing the BRAF^OO mutation, have led to increased interest in other therapeutic options, including combination therapies targeting different pathways. The P53 tumor suppressor protein is one such option. In contrast to many other cancers, over 80% of human melanomas express TP53^wt, but P53 degradation can be enhanced through overexpression of the murine double minute (MDM) proteins, MDM2 or MDMX. Loss or mutation of CDKN2A in ~40% of melanoma tumors results in loss of function of the MDM2 inhibitor, pl4/pl9, thus increasing MDM2 mediated degradation of P53. In the past several years, several molecules have been developed to interrupt the interaction between P53 and MDM2. These include the cis-imidazolines "Nutlins" (RG7112 and RG7388), the spirooxindoles, MI- 77301 and, MK- 8242, as well as other recently developed compounds such as CGM097, H DM201, and the piperidinone, the compound of Formula (I) Interestingly, the compound of Formula (I) was shown to have antitumor activity in cell and animal models of melanoma with BRAF and KRAS mutations. These studies showed that the compound of Formula (I) treatment led to increased P53, CDKN1A, MDM2, and PUMA proteins in TP53 wild-type cells (SJSA-1, FICT116), but not in TP53-mutant FIT-29 cells. The compound of Formula (I) also has been reported to be an effective MDM2 inhibitor in glioblastoma cell lines, patient-derived stem cells (27), and a wide variety of TP53^wt but not in homozygous TP53 mutant tumor cell lines

[0106] In one example, the anti-tumor efficacy of combining the compound of Formula (I) with BRAF and MEK inhibitors using melanoma patient-derived xenograft models (PDX) was studied. A well- characterized panel of melanoma PDX tumors, which represented an array of BRAF and NRAS genotypes, was used. PDX tumors directly established from patient tumors closely resemble the clinical lesion and the response to targeted therapies. Importantly, except for PDX2316, all tumors were obtained from patients who had progressed on prior targeted and immunotherapy. When the compound of Formula (I) was tested as a single agent, only tumors that expressed wild type BRAF^®^ (6/15 or 40%) had slower growth rates in response to the compound of Formula (I) compared to vehicle treated mice. The remaining 9 PDX tumors were synergistically responsive to the compound of Formula (I) in combination with BRAF and MEK inhibition. These studies demonstrate that the compound of Formula

(I) is an effective potential agent for the treatment of melanoma tumors that are either S/?AF^wt or Pan^T (BRAF^wt, NRAS^wt, NFl^wt). Furthermore, the compound of Formula (I) is an effective agent in combination with dabrafenib and trametinib for the treatment of BRAF^OOmut tumors.

[0107] The present invention relates to a method of treating immunotherapy resistant metastatic melanoma comprising the step of administering to a human subject in need thereof, therapeutically effective amounts of a MDM2 inhibitor or a MDM2 inhibitor in combination with a BRAF inhibitor and a MEK inhibitor. In an embodiment, the human subject is previously treated with immunotherapy described herein for treating metastatic melanoma. In an embodiment, the previously treated metastatic melanoma has progressed after treated with immunotherapy described herein. In an embodiment, the human subject is previously treated with immunotherapy described herein for treating the metastatic melanoma, but the metastatic melanoma relapses or progresses or metastasizes.

[0108] In some embodiments, the immunotherapy resistant metastatic melanoma is melanoma including different types of melanoma such as superficial spreading melanoma, nodular melanoma, acral-lentiginous melanoma, lentigo maligna melanoma, amelanotic and desmoplastic melanomas, or metastatic melanoma.

[0109] In one aspect, the present invention relates to a method of treating metastatic melanoma in a human subject previously treated with immunotherapy comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor. In an embodiment, the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof.

[0110] In an embodiment, the method further comprises detecting the BRAF genotype in the human subject. In an embodiment, the human subject exhibits a wild-type BRAF^⁶⁰⁰ (BRAF^) genotype.

[0111] In an embodiment, the method further comprises detecting the NRAS genotype in the human subject. In an embodiment, the human subject exhibits a wild-type NRAS (NRAS^) genotype.

[0112] In an embodiment, the method further comprises detecting the NF1 genotype in the human subject. In an embodiment, the human subject exhibits a wild-type NF1 genotype (NFl^). In an embodiment, the human subject exhibits BRAF^, A/RAS^and NFl^. [0113] In an embodiment, the human subject exhibits a mutant NRAS. In an embodiment, the human subject exhibits a mutant NF1.

[0114] In an embodiment, the immunotherapy is an ex vivo cell therapy selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR)-engineered peripheral blood lymphocytes (PBL) and chimeric antigen receptor ((CAR)-engineered PBL).

[0115] In an embodiment, the immunotherapy is an immune checkpoint protein inhibitor therapy.

[0116] In an embodiment, the immune checkpoint protein inhibitor is an anti-PD-Ll antibody selected from the group consisting of durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.

[0117] In an embodiment, the immune checkpoint protein inhibitor is an anti-PD-1 antibody selected from group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab-rwlc, AMP-224, AMP-514, PDR001, and fragments, conjugates, biosimilars, or variants thereof.

[0118] In one embodiment, the immune checkpoint protein inhibitor is an anti-PD- L2 antibody. In one embodiment, the anti-PD- L2 antibody is rHlgM12B7A.

[0119] In an embodiment, the immune checkpoint protein inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and fragments, conjugates, biosimilars, or variants thereof.

[0120] In an embodiment, wherein the immunotherapy is a T-cell engager selected from catumaxomab, FBTA05, Ertumaxomab, Ektomun, blinatumomab, solitomab, and fragments, conjugates, biosimilars, or variants thereof.

[0121] In another aspect, the present invention relates to a method of treating metastatic melanoma in a human subject previously treated with immunotherapy comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, a BRAF inhibitor and a MEK inhibitor.

[0122] In an embodiment, the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof.

[0123] In an embodiment, the BRAF inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and combinations thereof.

[0124] In an embodiment, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, selumetinib, pimasertib, binimetinib, and combinations thereof.

[0125] In an embodiment, the method further comprises detecting the BRAF genotype in the human subject. In an embodiment, the human subject exhibits BRAF^V¥0 mutation. In an embodiment, the human subject exhibits NRAS ^QS1 mutation.

[0126] In an embodiment, the immunotherapy is an ex vivo cell therapy selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR)-engineered peripheral blood lymphocytes (PBL) and chimeric antigen receptor ((CAR)-engineered PBL).

[0127] In an embodiment, the immunotherapy is an immune checkpoint protein inhibitor therapy.

[0128] In an embodiment, the immune checkpoint protein inhibitor is an anti-PD-Ll antibody selected from the group consisting of BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.

[0129] In an embodiment, the immune checkpoint protein inhibitor is an anti-PD-1 antibody selected from group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab-rwlc, AMP-224, AMP-514, PDR001, and fragments, conjugates, biosimilars, or variants thereof. [0130] In an embodiment, the immune checkpoint protein inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and fragments, conjugates, biosimilars, or variants thereof.

[0131] In an embodiment, the immunotherapy is a T-cell engager selected from catumaxomab, FBTA05, Ertumaxomab, Ektomun, blinatumomab, solitomab, and fragments, conjugates, biosimilars, or variants thereof.

[0132] In one embodiment, the immune checkpoint protein inhibitor is an anti-PD- L2 antibody. In one embodiment, the anti-PD- L2 antibody is rHlgM12B7A.

[0133] In an embodiment, the compound of Formula (I) is in a crystalline form.

[0134] In an embodiment, the compound of Formula (I) is in a free form.

[0135] In an embodiment, the MDM2 inhibitor is a pharmaceutically acceptable salt of a compound of Formula (I).

[0136] In an embodiment, the compound of Formula (I) is in an amorphous form.

[0137] In an embodiment, the compound of Formula (I) is administered once daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg.

[0138] In an embodiment, the compound of Formula (I) is administered twice daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg.

[0139] In an embodiment, the human is treated with the MDM2 inhibitor for a period selected from the group consisting of about 7 days, 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, and about 56 days.

[0140] In an embodiment, the compound of Formula (I) is orally administered.

[0141] In an embodiment, the MDM2 inhibitor is administered before administration of the BRAF inhibitor and MEK inhibitor.

[0142] In an embodiment, the MDM2 inhibitor is administered after administration of the BRAF inhibitor and MEK inhibitor. [0143] In an embodiment, the MDM2 inhibitor is administered concurrently with administration of the B/? F inhibitor and MEK inhibitor.

[0144] In an embodiment, the therapeutically effective amount of the MDM2 inhibitor is 120 mg or more.

[0145] The combination may be administered by any route known in the art. In an exemplary embodiment, the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered by oral, intravenous, intramuscular, intraperitoneal, intravitreal, subcutaneous or transdermal means. In one embodiment, the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is administered orally.

[0146] In an exemplary embodiment, the MDM2 inhibitor is in the form of a pharmaceutically acceptable salt.

[0147] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type BRAF^W0° (BRAF^) genotype.

[0148] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type NRAS (NRAS^) genotype.

[0149] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type NF1 genotype (NF1^WT).

[0150] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the human subject exhibits BRAF ^V600 mutation.

[0151] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the human subject exhibits NRAS ⁰⁶¹ mutation.

[0152] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with pembrolizumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the human subject exhibits NF1 mutation.

[0153] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the human subject has a wild-type BRAF^W¥ (BRAF^) genotype.

[0154] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):

[0155] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):

[0156] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I):

[0157] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I):

[0158] In an embodiment, the disclosure provides a method of for treating metastatic melanoma in a human subject previously treated with nivolumab comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, dabrafenib and trametinib, wherein the MDM2 inhibitor is a compound of Formula (I):

MDM2 inhibitor

[0159] The compound of Formula (I) has the structure and name shown below.

2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-l-((S)-l-(isopropylsulfonyl)-3-methylbutan-2-yl)-3- methyl-2-oxopiperidin-3-yl) acetic acid:

[0160] The synthesis of the compound of Formula (I) is set forth in International Applications: W02011/153509 and W02014/200937; U.S. Patent No. 8,569,341; 9,593,129; 9,296,736; 9,623,018; 9,757,367; 9,801,867; 9;376;386; and 9,855,259, the disclosure of which are incorporated by reference herein in its entirety.

[0161] In an embodiment, the compound of Formula (I) is in an amorphous form. In an embodiment, the MDM2 inhibitor is the compound of Formula (I) in a crystalline form. In an embodiment, the MDM2 inhibitor is the compound of Formula (I) in a crystalline anhydrous form. In an embodiment, the MDM2 inhibitor is the compound of Formula (I) in a crystalline anhydrous form characterized by a powder X-ray diffraction pattern comprising peaks at diffraction angle 2 theta degrees at approximately 11.6, 12.4, 18.6, 19.0, 21.6 and 23.6. In an embodiment, the MDM2 inhibitor is the compound of Formula (I) in a crystalline anhydrous form having the X-ray diffraction pattern substantially shown in FIG. 1. The method of making such crystalline form was disclosed in the International Application W02014200937, the disclosure of which is incorporated herein by reference in its entirety. The compound of Formula (I) is also referred as AMG or AMG-232 or AMG 232 in the drawings.

BRAF Inhibitor

[0162] The MDM2 inhibitors of the present invention can be used in combination with MAP kinase pathway inhibitors. Examples of proteins in the MAP kinase pathway that can be inhibited and the inhibitors of such proteins used in combination with an MDM2 inhibitors are BRAF inhibitors, Pan-RAF inhibitors, and MEK inhibitors. There are three main RAF isoforms: ARAF, BRAF and CRAF. A pan-RAF inhibitor shows inhibitory activity on more than one RAF isoform. In contrast, a BRAF inhibitor exhibits more inhibitor activity (or selectivity) towards SRAFthan the other RAF proteins.

[0163] The MDM2 inhibitors of the present invention can be used in combination with BRAF inhibitors, such as those found in published PCT application WO2008/153,947. A particular compound is AMG 2112819 (also known as 2112819) (Example 56). Another particular BRAF inhibitor that can be used in the combinations of the present invention is dabrafenib. Another BRAF inhibitor that can be used in the combinations of the present invention is vemurafenib.

Encorafenib

[0164] In an embodiment, the BRAF inhibitor is encorafenib. Encorafenib has the chemical structure and name shown as:

[0165] methyl N-[(2S)-l-[[4-[3-[5-chloro-2-fluoro-3-(methanesulfonamido)phenyl]-l-propan-2- ylpyrazol-4-yl]pyrimidin-2-yl]amino]propan-2-yl]carbamate (CAS No. : 1269440-17-6)

Vemurafenib

[0166] In an embodiment, the BRAF inhibitor is vemurafenib. Vemurafenib has the chemical structure and name shown as:

N-[3-[5-(4-chlorophenyl)-lH-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]propane-l- sulfonamide (CAS No.: 918504-65-1)

Dabrafenib

[0167] In an embodiment, the BRAF inhibitor is dabrafenib. Dabrafenib has the chemical structure and name shown as:

[0168] N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-l,3-thiazol-4-yl]-2-fluorophenyl]-2,6- difluorobenzenesulfonamide (CAS No.: 1195765-45-7)

Sorafenib

[0169] In an embodiment, the BRAF inhibitor is sorafenib. Sorafenib has the chemical structure and name shown as:

4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methylpyridine-2-carboxamide (CAS No.: 284461-73-0)

MEK Inhibitor

The MDM2 inhibitors of the present invention can be used in combination with MEK inhibitors, such as those found in published PCT application W02002/006213. A particular compound is N-(((2R)-2,3- dihydroxypropyl)oxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide, also known as AMG 1009089 or 1009089, (Example 39).

[0170] The MDM2 inhibitors of the present invention can be used in combination with MEK inhibitors. Particular MEK inhibitors that can be used in the combinations of the present invention include PD0325901, trametinib, pimasertib, MEK162, TAK-733, GDC-0973 and AZD8330. A particular MEK inhibitor that can be used along with MDM2 inhibitor in the combinations of the present invention is trametinib (also called AMG 2712849 or 2712849). Another particular MEK inhibitor is N-(((2R)-2,3- dihydroxypropyl)oxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide, also known as AMG 1009089, 1009089 or PD0325901.

Trametinib

[0171] In an embodiment, the MEK inhibitor is trametinib. Trametinib has the chemical structure and name shown as:

[0172] N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3- d]pyrimidin-l-yl]phenyl]acetamide (CAS No.: 871700-17-3)

Cobimetinib

[0173] In an embodiment, the MEK inhibitor is cobimetinib. Cobimetinib has the chemical structure and name shown as:

[3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3-hydroxy-3-[(2S)-piperidin-2-yl]azetidin-l- yljmethanone (CAS No.: 934660-93-2)

Selumetinib

[0174] In an embodiment, the MEK inhibitor is selumetinib. Selumetinib has the chemical structure and name shown as:

[0175] 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5- carboxamide (CAS No.: 606143-52-6)

Pimasertib

[0176] In an embodiment, the MEK inhibitor is pimasertib. Pimasertib has the chemical structure and name shown as:

[0177] N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4-carboxamide (CAS No.: 1236699-92-5)

Binimetinib

[0178] In an embodiment, the MEK inhibitor is binimetinib. Binimetinib has the chemical structure and name shown as:

[0179] 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5- carboxamide (CAS No.: 606143-89-9)

Immunotherapy

[0180] In some embodiments, the immunotherapy described herein refers to an immune checkpoint immunotherapy wherein an immune checkpoint protein inhibitor is administered to a subject in need thereof. The immune checkpoint protein inhibitor is an agent that modulates a target selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, LAG 3, B7-H3, B7-H4, KIR, 0X40, IDO-1, IDO-2, CEACAM1, INFR5F4, BTLA, OX40L, and TIM3 or combinations thereof.

[0181] In some embodiments, the immunotherapy is a T-cell engager.

[0182] In some embodiments, the immune checkpoint protein inhibitor is a PD-1 inhibitor selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab.

[0183] In some embodiments, the immune checkpoint protein inhibitor is a CTLA-4 inhibitor selected from the group consisting of ipilimumab and tremelimumab.

[0184] In some embodiments, the immune checkpoint protein inhibitor comprises a PD-1 immune checkpoint protein inhibitor and a CTLA-4 immune checkpoint protein inhibitor.

[0185] In some embodiments, the immune checkpoint protein inhibitor is a PD-L1 inhibitor selected from the group consisting of BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.

[0186] In an embodiment, the immune checkpoint protein inhibitor is an anti-PD-L2 antibody. In one embodiment, the anti-PD- L2 antibody is rHlgM12B7A.

PD-1 Inhibitors

[0187] The PD-1 inhibitor may be any PD-1 inhibitor or PD-1 blocker known in the art. In particular, it is one of the PD-1 inhibitors or blockers described in more detail in the following paragraphs. The terms "inhibitor" and "blocker" are used interchangeably herein in reference to PD-1 inhibitors. For avoidance of doubt, references herein to a PD-1 inhibitor that is an antibody may refer to a compound or antigen binding fragments, variants, conjugates, or biosimilars thereof. For avoidance of doubt, references herein to a PD-1 inhibitor may also refer to a compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug thereof.

[0188] In some embodiments, the compositions and methods described include a PD-1 inhibitor that binds human PD-1 with a KD of about 100 pM or lower, binds human PD-1 with a KD of about 90 pM or lower, binds human PD-1 with a KD of about 80 pM or lower, binds human PD-1 with a KD of about 70 pM or lower, binds human PD-1 with a KD of about 60 pM or lower, binds human PD-1 with a KD of about 50 pM or lower, binds human PD-1 with a KD of about 40 pM or lower, or binds human PD-1 with a KD of about 30 pM or lower.

[0189] In some embodiments, the compositions and methods described include a PD-1 inhibitor that binds to human PD-1 with a k_aSsoc of about 7.5 ^c 10⁵ l/M-s or faster, binds to human PD-1 with a k_aSsoc of about 7.5 x 10⁵ l/M-s or faster, binds to human PD-1 with a k_assoc of about 8 ^c 10⁵ l/M-s or faster, binds to human PD-1 with a k_ass0c of about 8.5 ^c 10⁵ l/M-s or faster, binds to human PD-1 with a k_ass0c of about 9 ^c 10⁵ l/M-s or faster, binds to human PD-1 with a k_ass0c of about 9.5 ^c 10⁵ l/M-s or faster, or binds to human PD-1 with a k_ass0c of about 1 ^c 10^s l/M-s or faster.

[0190] In some embodiments, the compositions and methods described include a PD-1 inhibitor that binds to human PD-1 with a k_dissoc of about 2 ^c 10⁵ I/s or slower, binds to human PD-1 with a k_dissoc of about 2.1 x 10⁵ I/s or slower, binds to human PD-1 with a k_dissoc of about 2.2 ^c 10⁵ I/s or slower, binds to human PD-1 with a k_dissoc of about 2.3 x 10⁵ I/s or slower, binds to human PD-1 with a k_dissoc of about 2.4 x 10⁵ I/s or slower, binds to human PD-1 with a k_dissoc of about 2.5 x 10⁵ I/s or slower, binds to human PD-1 with a k_dissoc of about 2.6 x 10⁵ I/s or slower or binds to human PD-1 with a k_dissoc of about 2.7 x 10⁵ I/s or slower, binds to human PD-1 with a k_dissoc of about 2.8 x 10⁵ I/s or slower, binds to human PD-1 with a k_dissoc of about 2.9 x 10⁵ I/s or slower, or binds to human PD-1 with a k_dissoc of about 3 x 10⁵ I/s or slower.

[0191] In some embodiments, the compositions and methods described include a PD-1 inhibitor that blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 10 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 9 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 8 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 7 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 6 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower, blocks or inhibits binding of human PD-LI or human PD- L2 to human PD-1 with an IC50 of about 3 nM or lower, blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 2 nM or lower, or blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC₅₀ of about 1 nM or lower. [0192] In an embodiment, an anti-PD-1 antibody comprises nivolumab (Bristol-Myers Squibb) or antigen-binding fragments, conjugates, or variants thereof. Nivolumab is referred to as 5C4 in International Patent Publication No. WO 2006/121168. Nivolumab is assigned CAS registry number 946414-94-4 and is also known to those of ordinary skill in the art as BMS-936558, MDX-1106 or ONO- 4538. Nivolumab is a fully human lgG4 antibody blocking the PD-1 receptor.

[0193] In an embodiment, the anti-PD-1 antibody is an antibody disclosed and/or prepared according to U.S. Patent No. 8,008,449 or U.S. Patent Application Publication No. 2009/0217401 or 2013/0133091, the disclosures of which are specifically incorporated by reference herein. For example, in an embodiment, the monoclonal antibody includes 5C4 (referred to herein as nivolumab), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in U.S. Patent No. 8,008,449, the disclosures of which are hereby incorporated by reference. The PD-1 antibodies 17D8, 2D3, 4H1, 5C4, and 4A11, are all directed against human PD-1, bind specifically to PD-1 and do not bind to other members of the CD28 family. The sequences and CDR regions for these antibodies are provided in U.S. Patent No. 8,008,449, in particular FIG. 1 through FIG. 12; all of which are incorporated by reference herein in their entireties.

[0194] In another embodiment, the anti-PD-1 antibody comprises pembrolizumab, which is commercially available from Merck, or antigen-binding fragments, conjugates, or variants thereof. Pembrolizumab is referred to as h409AI I in International Patent Publication No. WO 2008/156712, U.S. Patent No. 8,354,509 and U.S. Patent Application Publication No. 2010/0266617, 2013/0108651 and 2013/0109843. Pembrolizumab has an immunoglobulin G4, anti-(human protein PDCD1 (programmed cell death 1)) (human-Mus musculus monoclonal heavy chain), disulfide with human-Mus musculus monoclonal light chain, dimer structure. The structure of pembrolizumab may also be described as immunoglobulin G4, anti-(human programmed cell death 1); humanized mouse monoclonal [228-L- proline(FllO-S>P)]y4 heavy chain (134-218')-disulfide with humanized mouse monoclonal k light chain dimer (226-226":229-229")-bisdisulfide. Pembrolizumab is assigned CAS registry number 1374853-91-4 and is also known as lambrolizumab, MK-3475, and SCFI-900475.

[0195] In an embodiment, the anti-PD-1 antibody is an antibody disclosed in U.S. Patent No. 8,354,509 or U.S. Patent Application Publication No. 2010/0266617, 2013/0108651, 2013/0109843, the disclosures of which are specifically incorporated by reference herein. [0196] In an embodiment, the anti-PD-1 antibody is pidilizumab, which is also known as CT-011 (CureTech Ltd.), and which is disclosed in U.S. Patent No. 8,686,119 B2, the disclosures of which are specifically incorporated by reference herein.

[0197] In another embodiment, anti-PD-1 antibodies and other PD-1 inhibitors include those described in U.S. Patent No. 8,287,856, 8,580,247, and 8,168,757 and U.S. Patent Application Publication No. 2009/0028857, 2010/0285013, 2013/0022600 and 2011/0008369, the teachings of which are hereby incorporated by reference. In another embodiment, antibodies that compete with any of these antibodies for binding to PD-1 are also included. In another embodiment, the anti-PD-1 antibody is an antibody disclosed in U.S. Patent No. 8,735,553, the disclosures of which are incorporated herein by reference.

[0198] The PD-1 inhibitor may also be a small molecule or peptide, or a peptide derivative, such as those described in U.S. Patent No . 8,907,053; 9,096,642; and 9,044,442 and U.S. Patent Application Publication No. 2015/0087581; 1,2,4 oxadiazole compounds and derivatives such as those described in U.S. Patent Application Publication No. 2015/0073024; cyclic peptidomimetic compounds and derivatives such as those described in U.S. Patent Application Publication No. 2015/0073042; cyclic compounds and derivatives such as those described in U.S. Patent Application Publication No. 2015/0125491; 1,3,4 oxadiazole and 1,3,4 thiadiazole compounds and derivatives such as those described in International Patent Application Publication No. WO 2015/033301; peptide-based compounds and derivatives such as those described in International Patent Application Publication No. WO 2015/036927 and WO 2015/04490, or a macrocyclic peptide-based compounds and derivatives such as those described in U.S. Patent Application Publication No. 2014/0294898; the disclosures of each of which are hereby incorporated by reference in their entireties.

[0199] In an embodiment, the PD-1 inhibitor is selected from group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, PDR001, AUNP-12 and combinations thereof. In an embodiment the PD-1 inhibitor is nivolumab. In an embodiment the PD-1 inhibitor is pembrolizumab. In an embodiment the PD-1 inhibitor is Pidilizumab. In an embodiment the PD-1 inhibitor is AMP-224.

PD-L1 and PD-L2 Inhibitors

[0200] The PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2 inhibitor or blocker known in the art. In particular, it is one of the PD-L1 or PD-L2 inhibitors or blockers described in more detail in the following paragraphs. The terms "inhibitor" and "blocker" are used interchangeably herein in reference to PD-L1 and PD-L2 inhibitors. For avoidance of doubt, references herein to a PD-L1 or PD-L2 inhibitor that is an antibody may refer to a compound or antigen-binding fragments, variants, conjugates, or biosimilars thereof. For avoidance of doubt, references herein to a PD-L1 or PD-L2 inhibitor may refer to a compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug thereof.

[0201] In an embodiment, the anti-PD-Ll antibody is durvalumab, which is also known as MEDI4736 (Medimmune) or antigen-binding fragments, conjugates, or variants thereof. In an embodiment, the anti-PD-Ll antibody is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013/0034559, the disclosures of which are specifically incorporated by reference herein. The clinical efficacy of durvalumab (MEDI4736) has been described in: Page, Ann. Rev. Med., 2014, 65, 185-202; Brahmer, J. Clin. Oncol. 2014, 32, 5s (supplement, abstract 8021); and McDermott, Cancer Treatment Rev., 2014, 40, 1056-64.

[0202] In an embodiment, the anti-PD-Ll antibody is atezolizumab, also known as MPDL3280A or RG7446 (Genentech) or antigen-binding fragments, conjugates, or variants thereof. In an embodiment, the anti-PD-Ll antibody is an antibody disclosed in U.S. Patent No. 8,217,149, the disclosure of which is specifically incorporated by reference herein. In an embodiment, the anti-PD-Ll antibody is an antibody disclosed in U.S. Patent Application Publication No. 2010/0203056, 2013/0045200, 2013/0045201, 2013/0045202 or 2014/0065135, the disclosures of which are specifically incorporated by reference herein.

[0203] In an embodiment, the anti-PD-Ll antibody is avelumab, also known as MSB0010718C (Merck KGaA/EMD Serono) or antigen-binding fragments, conjugates, or variants thereof. In an embodiment, the anti-PD-Ll antibody is an antibody disclosed in U.S. Patent Application Publication No.

2014/0341917, the disclosure of which is specifically incorporated by reference herein.

[0204] In an embodiment, the anti-PD-Ll antibody is MDX-1105, also known as BMS-935559, which is disclosed in U.S. Patent No. 7,943,743, the disclosures of which are specifically incorporated by reference herein. In an embodiment, the anti-PD-Ll antibody is selected from the anti-PD-Ll antibodies disclosed in U.S. Patent No. 7,943,743 which is specifically incorporated by reference herein.

[0205] In an embodiment, the anti-PD-Ll antibody is a commercially-available monoclonal antibody, such as INVIVOMAB anti-m-PD-Ll clone 10F.9G2 (BioXCell). A number of commercially-available anti-PD- Ll antibodies are known to one of ordinary skill in the art. [0206] In an embodiment, the anti-PD-L2 antibody is a commercially-available monoclonal antibody, such as BIOLEGEND 24F.10C12 Mouse lgG2a, k isotype (Biolegend), anti-PD-L2 antibody (Sigma-Aldrich), or other commercially-available anti-PD-L2 antibodies known to one of ordinary skill in the art.

[0207] In an embodiment, the PD-L1 inhibitor is an anti-PD-Ll antibody. In an embodiment, the PD-L1 inhibitor is selected from the group consisting of Atezolizumab, Avelumab, Durvalumab, BMS-936559 and combinations thereof. In an embodiment, the anti-PD-Ll inhibitor is durvalumab (MEDI4736). In an embodiment, the anti-PD-Ll inhibitor is BMS-936559 (also known as MDX-1105-01). In an embodiment, the anti-PD-Ll inhibitor is Atezolizumab. In an embodiment, the anti-PD-Ll inhibitor is Avelumab.

[0208] In an embodiment, the immunotherapy is a PD-L2 inhibitor. In an embodiment, the PD-L2 inhibitor is an anti-PD-L2 antibody. In one embodiment, the anti-PD- L2 antibody is rHlgM12B7A.

CTLA-4 Inhibitors

[0209] In some embodiments, the at least one immune checkpoint protein inhibitor is an inhibitor of CTLA-4. In some embodiments, the at least one immune checkpoint protein inhibitor is an antibody against CTLA-4. In some embodiments, the at least one immune checkpoint protein inhibitor is a monoclonal antibody against CTLA-4. In other or additional embodiments, the at least one immune checkpoint protein inhibitor is a human or humanized antibody against CTLA-4. In one embodiment, the anti-CTLA-4 antibody blocks the binding of CTLA-4 to CD80 (B7-1) and/or CD86 (B7-2) expressed on antigen presenting cells. Exemplary antibodies against CTLA-4 include: Bristol Meyers Squibb's anti- CTLA-4 antibody ipilimumab (also known as Yervoy™, MDX-010, BMS-734016 and MDX-101); anti-CTLA4 Antibody, clone 9H10 from Millipore; Pfizer's tremelimumab (CP-675,206, ticilimumab); and anti-CTLA4 antibody clone BNI3 from Abeam.

[0210] In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibody disclosed in any of the following patent publications (which is incorporated by reference in its entirety): WO 2001014424; WO 2004035607; US2005/0201994; EP 1212422; WO 2003086459; WO 2012120125; WO 2000037504; WO 2009100140; WO 200609649; WO 2005092380; WO 2007123737; WO 2006029219; W020100979597; W0200612168; and WO1997020574. Additional CTLA-4 antibodies are described in U.S. Patent No. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; in PCT Publication No. WO 01/14424 and WO 00/37504; and in U.S. Publication No. 2002/0039581 and 2002/086014; and/or U.S. Patent No. 5,977,318, 6,682,736, 7, 109,003 and 7,132,281, incorporated herein by reference). [0211] In some embodiments, the anti-CTLA-4 antibody is an, for example, those disclosed in: WO 98/42752; U.S. Patent No. 6,682,736 and 6,207,156; Hurwitz, "CTLA-4 blockade synergizes with tumor- derived granulocyte-macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma," Proc. Natl. Acad. Sci. USA, 95: 10067-10071 (1998); Camacho, "Phase 1 clinical trial of anti- CTLA4 human monoclonal antibody CP-675,206 in patients (pts) with advanced solid malignancies," J. Clin. Oncol., 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr, "Realization of the Therapeutic Potential of CTLA-4 Blockade in Low-Dose Chemotherapy-treated Tumor-bearing Mice," Cancer Res., 58:5301-5304 (1998).

[0212] In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand as disclosed in WO 1996040915. In other embodiments the CTLA-4 inhibitor may be B7-like peptides or nucleic acid molecules disclosed in U.S. Patent No. 6,630,575.

T-cell Engager

[0213] In an embodiment, the immunotherapy is a T- cell engager. In some embodiments, the T cell engager is selected from an antigen binding domain or ligand that binds to (e.g., and in some embodiments activates) one or more of CD3, TCRa, TCRp, TCRy, TCRC, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In other embodiments, the T cell engager is selected from an antigen binding domain or ligand that binds to and does not activate one or more of CD3, TCRa, TCRp, TCRy, TCRC, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In some embodiments, the T cell engager binds to CD3.

[0214] In some embodiments, the T cell engager is selected from the group consisting of: Catumaxomab mAb (anti CD3X anti-EpCAM), FBTA05 / Lymphomun (CD3X anti-anti-CD20), duly cable Eritrea mAb (Ertumaxomab) (anti-CD3 X anti-HER2 / neu), Ektomun (anti-CD3 X anti-GD2), Bona spit mAb (blinatumomab) and B. thuringiensis FIG mAb (solitomab).

Pharmaceutical Compositions for Oral Administration

[0215] In selected embodiments, the invention provides a pharmaceutical composition for oral administration a combination comprising a MDM2 inhibitor and a pharmaceutical excipient suitable for oral administration. In an embodiment, the MDM2 inhibitor is a compound of Formula (I) or a pharmaceutically acceptable salt thereof. [0216] In selected embodiments, the invention provides a pharmaceutical composition for oral administration a combination comprising a MDM2 inhibitor, a BRAF inhibitor, and a MEK inhibitor, and a pharmaceutical excipient suitable for oral administration. In an embodiment, the MDM2 inhibitor is a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In an embodiment, the BRAF inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and combinations thereof. In an embodiment, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, selumetinib, pimasertib, binimetinib, and combinations thereof.

[0217] In selected embodiments, the pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption. Pharmaceutical compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such dosage forms can be prepared by any of the methods, but all methods include the step of bringing the active ingredient(s) into association with the carrier, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient(s) with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0218] The invention further encompasses anhydrous pharmaceutical compositions and dosage forms since water can facilitate the degradation of some compounds. For example, water may be added (e.g., 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms of the invention which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.

[0219] The pharmaceutical compositions may further comprise a carrier. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.

Dosages and Dosing Regimens

[0220] The amount of a MDM2 inhibitor, a BRAF inhibitor, or a MEK inhibitor administered will be independently dependent on the human being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compounds and the discretion of the prescribing physician. However, an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, such as about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, such as about 0.05 to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect - e.g., by dividing such larger doses into several small doses for administration throughout the day.

[0221] In some embodiments, the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered in multiple doses for treating immunotherapy resistant metastatic melanoma. In an embodiment, dosing may be once, twice, three times, four times, five times, six times, or more than six times per day. In some embodiments, the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, once every other day, once weekly, twice weekly, three times weekly, four times weekly, biweekly, or monthly.

[0222] Administration of the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor may independently continue as long as necessary. In some embodiments, the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more days. In some embodiments, the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered for about 3 days, 5 days, 7 days, 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days. In some embodiments, the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered for more than about 6, 10, 14, 28 days, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months or one year.

[0223] In some embodiments, an effective dosage of the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 10 mg to about 200 mg, about 20 mg to about 150 mg, about 30 mg to about 120 mg, about 10 mg to about 90 mg, about 20 mg to about 80 mg, about 30 mg to about 70 mg, about 40 mg to about 60 mg, about 45 mg to about 55 mg, about 48 mg to about 52 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about 105 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 202 mg.

[0224] In some embodiments, a MDM2 inhibitor or a pharmaceutically acceptable salt thereof is administered at a dosage of 10 to 500 mg BID, including a dosage of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg BID.

[0225] In some embodiments, a MDM2 inhibitor or a pharmaceutically acceptable salt thereof is administered at a dosage of 10 to 500 mg QD, including a dosage of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg QD. [0226] An effective amount of the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including buccal, sublingual, and transdermal routes, by intra-arterial injection, intravenously, parenterally, intramuscularly, intravitreal, subcutaneously or orally.

[0227] In some embodiments, the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject intermittently, known as intermittent administration. By "intermittent administration", it is meant a period of administration of a therapeutically effective dose, followed by a time period of discontinuance, which is then followed by another administration period and so on. In each administration period, the dosing frequency can be independently selected from three times daily, twice daily, daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly or monthly.

[0228] By "period of discontinuance" or "discontinuance period" or "rest period", it is meant to the length of time when discontinuing of the administration of the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor. The time period of discontinuance may be longer or shorter than the administration period or the same as the administration period. For example, where the administration period comprises three times daily, twice daily, daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, seven times weekly or monthly dosing, the discontinuance period may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, one month, two months, three months, four months or more days.

[0229] In an embodiment, the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a human subject in need thereof for treating immunotherapy resistant metastatic melanoma for a first administration period, then followed by a discontinuance period, then followed by a second administration period, and so on. In an embodiment, the immunotherapy resistant metastatic melanoma is metastatic melanoma. In an embodiment, the first administration period, the second administration period, and the discontinuance period are independently selected from the group consisting of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, one month, two months, three months, four months and more days, in which the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject three times daily, twice daily, daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly or monthly. In an embodiment, the first administration period is at same length as the second administration period. In an embodiment, the first administration period is shorter than the second administration period. In an embodiment, the first administration period is longer than the second administration period. In an embodiment, the first administration period and the second administration period are about three weeks, in which the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject daily; and the discontinuance is about two weeks. In an embodiment, the first administration period and the second administration period are about three weeks, in which the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject weekly; and the discontinuance is about two weeks. In an embodiment, the first administration period and the second administration period are about four weeks, in which the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject daily; and the discontinuance is about two weeks. In an embodiment, the first administration period and the second administration period are about four weeks, in which the MDM2 inhibitor, the BRAF inhibitor, or the MEK inhibitor is independently administered to a subject weekly; and the discontinuance is about two weeks. In an embodiment, the MDM2 inhibitor is the compound of Formula (I). In an embodiment, the BRAF inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and combinations thereof. In an embodiment, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, selumetinib, pimasertib, binimetinib, and combinations thereof.

[0230] In an embodiment, the MDM2 inhibitor is administered to a human intermittently; while the BRAF inhibitor and the MEK inhibitor are administered to a human non-intermittently. In an embodiment, while the BRAF inhibitor and the MEK inhibitor are administered to a human intermittently; while the MDM2 inhibitor is administered to a human non-intermittently. In an embodiment, the MDM2 inhibitor, the BRAF inhibitor, and the MEK inhibitor are administered to a human intermittently. In an embodiment, the MDM2 inhibitor, the BRAF inhibitor, and the MEK inhibitor are administered to a human non-intermittently.

Examples

[0231] The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein. Abbreviations

[0232] D, dabrafenib (Tafinlar), V600B-Raf mutant Inhibitor; G, GM-CSF (Oncovax); I, interferon; Ipi, ipilimumab (Yervoy), monoclonal Ab CTLA-4; N, nivolumab (Opdivo), monoclonal Ab PD-1; Pern, pembrolizumab (Keytruda), lgG4 isotype antibody PD-1; S, sunitinib (Sutent), multi-targeted receptor tyrosine kinase (RTK) inhibitor; S+T, sorafenib (Nexavar) + tivantinib, tyrosine-protein kinase inhibitor, MET inhibitor; T, trametinib (Mekinist), MEK1/2 inhibitor; V, vemurafenib (Zelboraf), B-Raf inhibitor.

Example 1: Immunotherapy Resistant Metastatic Melanomas Respond to HDM2 Inhibition as a Single Agent or in Combination with BRAF/ MEK inhibition

[0233] Animal studies were approved by the Vanderbilt IACUC. NSG mice (NOD.Cg-Prkdc^scid Il2_rg^tmiw^ji|s_zj) were purchased from Jackson Labs and Athymic Nude mice (Hsd: Athymic Nude-Foxnlnu) were purchased from Envigo. PDX tumors were established and propagated as described previously (Vilgelm, MDM2 and aurora kinase A inhibitors synergize to block melanoma growth by driving apoptosis and immune clearance of tumor cells. Cancer Res. 2015, 75:181-93). The mice were all 3-6 month-old females weighing 20-26 grams the compound of Formula (I) and navitoclax were prepared in 5% DMSO and 95% corn oil and administered five days a week by oral gavage in a total volume of 100- 200uL based upon the weight of the mouse. Corn oil with 5% DMSO was used as the vehicle control. Dabrafenib and trametinib were prepared in 0.5% hydroxypropyl methylcellulose. At least 5 mice per treatment group were used, with each mouse bearing 2 tumors. Mouse body weight was measured daily for gavage dosing and recorded twice a week and tumor measurements were taken twice a week with micro-calipers. Tumor volume was estimated as 0.5 x length x width x depth. Treatment began when tumors reached 50-100mm³ volume on average and continued until tumors reached 15 mm in diameter or became perforated. At the end of the experiment, the final weight of the tumors was recorded and tumors were either flash-frozen for RPPA analysis or fixed in 10% buffered formalin for paraffin embedding. PDX models 1577, 1668, 1767, 1595, 2316 and 2252 were provided to the Patient- Derived Models Repository at NCI-Frederick. All models were established according to the recommended Minimal Information Standard (Meehan, PDX-MI: Minimal information for patient- derived tumor xenograft models. Cancer Res. 2017, 77:e62).

[0234] DNA from patient or PDX tumor (PDX1577, 1668, 2316, and 2552) and peripheral blood from the PDX donor were submitted to the VANTAGE core for QC and DNAseq using Fluman Comprehensive Cancer Panel (Qjagen). Identification of tumor-associated somatic mutations and copy number alterations was performed using QIAGEN NGS Data Analysis Web Portal. A color-coded map of genetic alterations in PDXs was constructed using OncoPrinter available through cBioPortal (Gao, Integrative Analysis of Complex Cancer Genomics and Clinical Profiles Using the cBioPortal. Sci Signal. 2013, 6:pll; Cerami, The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012, 2:401-4).

[0235] DNA extraction from tumor tissue was achieved using standard phenol: chloroform extraction methods and ethanol precipitation or using the DNeasy Blood and Tissue Kit. For a subset of tumors with high melanin content, the DNA was further purified using the Qjagen DNeasy PowerClean Pro Cleanup K Kit. DNA from matching blood samples, when available, was extracted using the QIAamp DNA Mini and Blood Mini Kit. DNA from matched patient blood, when available, P0 (patient's original tumor), and P2A and P2B xenograft tumors were subject to STR Profiling, at the Interdisciplinary Center for Biotechnology Research at the University of Florida, leveraging the PowerPlex 16 FIS System (Promega). PDX specimens were considered a match to the patient specimen when one allele from each 16 STR locus was present in the P0 specimen. The amelogenin alleles for all of the specimens match the patient sex that was reported.

[0236] Immunohistochemical (IHC) analyses were performed by Vanderbilt's Translational Pathology Shared Resource (TPSR). Slides were placed on the Leica Bond Max IHC Stainer. All steps besides dehydration, clearing and cover-slipping were performed on the Bond Max. Slides were deparaffinized and heat-induced antigen retrieval was performed on the Bond Max using their Epitope Retrieval 2 solution for 30 minutes. The sections were incubated with Ready-to-Use antibody as indicated below. The Bond Refine Polymer detection system was used for visualization. Slides were then dehydrated, cleared and coverslipped. I HC slides were scanned at the Digital Pathology Shared resource. The automated quantification of the percentages of the KI67-positive cells was performed by Leica Biosystems' Digital Image Flub, using the software available with the Leica SCN400 Slide Scanner. The antibodies used were as follows: anti-p53 (Leica Biosystems) for 30 minutes; anti-MelanA (Leica) for 15 minutes; anti-Ki67 (StatLab) for 30 minutes; anti-SOX-10 (Cell Marque) for one hour.

[0237] Flash-frozen tumor tissue was processed in RIPA buffer using a Precellys Homogenizer. Lysates were submitted to MD Anderson Cancer Center RPPA core for analysis with a 382 antibody panel. For each condition, three independent tumors were analyzed from three PDX tumors, resulting in 9 samples analyzed for each treatment. The only exception to this was for the compound of Formula (I) + dabrafenib +Trametinb treatment of PDX1351 where only two independent tumors were analyzed. RPPA data was analyzed after a log² transformation. All comparisons were performed based on a mixed- effect model to take into account the correlation structure with the measured data from the sample PDX. Using model-based (least-square) means, the average adjusted difference (log²FC: fold change in a log2 scale) between treatments (or groups) was estimated and compared using the Wald test.

Bonferroni correction was used to adjust for multiple testing. Three tumors were analyzed using RPPA for each PDX treatment. Changes in protein expression were considered to be significant (shown in red) based upon two criteria, the significance of the change (false discovery rate (FDR) < 0.05) and the degree of change (when log²(FC)=+/- 0.4). A heatmap was used for the visualization of proteins with significantly different expression across treatments. Hierarchical clustering analysis using Ward's distance was used to measure the dissimilarity between two clusters of observations.

[0238] For a comparison of more than two groups, one-way analysis of variance (ANOVA) with pairwise t test comparison was used. For in vivo experiments, tumor volume or tumor weight were analyzed on the natural log scale to better meet the normality assumption. The mixed-effects model with posthoc tests was used to compare the difference in tumor growth or tumor weight between treatments. P-value was adjusted using the Bonferroni method. With pairwise comparison, the p-value was adjusted using the Bonferroni method to correct for inflated Type I error (the higher the change for a false positive). For each PDX, initial statistical and data analysis was based on all treatment groups, which included three experimental agents tested individually and in combination compared to vehicle controls, but data analyses are presented based upon the statistical analysis for the subset of experimental agents. For synergy analysis, a mixed-effect model with individual effect and interactive effect was used to assess treatment differences in tumor volume over days. Each observation was classified according to the PDX. Tukey's adjusted p-value was used for multiple comparisons. All tests of statistical significance were two-sided. Unless otherwise noted in the manuscript, the word "significant" was used to represent statistical significance (p<0.05). *** for p<0.001, ** for p<0.01, * for p<0.05. Analyses were performed using R version 3.4.3.

[0239] Melanoma patient-derived xenografts (PDX) were established from melanoma tumors obtained from patients who were referred for surgical resection or biopsy. From a larger set of PDX tumors, 15 PDXs (FIG. 1A) were chosen for this study to approximately represent the range of mutations found in melanoma tumors. The patient population was 47% male and 53% female and represented ages from 29 to 77. The tumors were staged according to the American Joint Committee on Cancer (AJCC) (Flartman, A Review in Detection, Staging, and Management. Flematol Oncol Clin North Am [Internet] Elsevier Inc; 2019, 33:25-38) and the majority were Stage IV or Stage V; however, the panel did include two patients with locally advanced stage III melanoma (PDX2316 and 2552). The prior systemic therapies are listing in FIG. 1A, in the order in which they were received. Four patients were not treated systemically before resection (PDX2195, 2252, 1668, and 2316). The remaining 10 patients had tumor progression after targeted therapy or immunotherapy specifically the two patients with BRAF^V600 mutations treated with dabrafenib and trametinib (D+T) had tumor progression after therapy.

[0240] Except for patient 2316, all tumors were screened by either SNaPshot analysis or in the case of patient 2552 by Onkosight. SNaPshot is designed to screen for 43 somatic mutations in 6 genes (BRAF, NRAS, mast/stem cell growth factor receptor KIT, guanine nucleotide-binding protein q polypeptide (GNAQ), Guanine nucleotide-binding protein subunit alpha-11 (GNA11), and Catenin beta-1 (CTNNB1)( Lovly, Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials. PLoS One. 2012, 7). SNaPshot analysis indicated that three tumors (1129, 1668 and 1767) were BRAFwt, NRASwt, and NFlwt. To further understand the genetic make-up of the PDX tumors utilized in this study, either the original patient tumor or for PDX1577, 1668, 2316 and 2552, an early (second) PDX passage were analyzed by Next-Generation Sequencing (NGS) using a comprehensive cancer panel that targets exonic regions of genes with known associations to cancer. In all cases, the mutations noted in the patients' tumors by SNaPshot and Foundation One sequencing were confirmed by NGS and NGS results are shown in FIG. 1 and FIGs. 7A-B.

[0241] NGS did provide additional genetic information relevant to this study. The UV carcinogenic etiology of melanoma results in melanoma having the highest prevalence of somatic mutation across cancer types (Alexandrov, Signatures of mutational processes in human cancer. Nature. 2013, 500:415- 21). NGS of the patient and PDX tumors revealed a wide variation in the number of mutations. PDX1767 had the fewest non-synonymous mutations (NSMs), with 295 high confidence variants at a mean read depth in the target region of 83, while PDX1668 had the most NSMs with 2,352 high confidence variants at a mean read depth of 199 (FIGs. 7A-B). Neither of these tumors had the canonical mutations in BRAF and NRAS. For two of the tumors where no mutations were identified by SNaPshot (1129 and 1668),

NGS identified additional mutations in driver genes. For PDX1668, NGS identified a loss of the stop codon in NF1, and two mutations in BRAF, S147N, and N140T, with unknown significance. Further analysis of PDX1129 by NGS identified two mutations in BRAF, D549N and K483T. While BRAF mutations that are distinct from the V600 mutations are of unknown significance, the D549N mutation has also been identified in the NZM41 cell line (Stones, Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors. 2013, 4:1-6). In addition, there are reports of the D549N mutation in melanoma, colorectal and lung carcinomas and hairy cell leukemia (Boyd, High resolution melting analysis for detection of BRAF exon 15 mutations in hairy cell leukemia and other lymphoid malignancies. Br J Haematol. 2011, 155:609-12; Boursault, Tumor Homogeneity between Primary and Metastatic Sites for BRAF Status in Metastatic Melanoma Determined by Immunohistochemical and Molecular Testing. PLoS One. 2013, 8; Mufti, A Case Series of Two Patients Presenting With Pericardial Effusion as First Manifestation of Non-Small Cell Lung Cancer With BRAF Mutation and Expression of PD- L1 . World J Oncol. 2018, 9:56-61.). PDX2316 also was retrospectively analyzed by NGS and was found to lack mutations in BRAF and NRAS, although mutations in NF1, CDKN2A, MTOR, and KIT were detected (FIG. 1). The tumors used in this study closely mimic the expected distribution of mutations within the driver mutation genes BRAF, NRAS, and NF1, with 53% exhibiting mutated BRAF^V600 and 13% exhibiting mutated NRAS^QS1 mutations. Furthermore, PDX2316 was BRAF^wt, NRAS^wt and PDX1767 was BRAF^wt,

N RAS^wt, and NFl^wt.

[0242] Given the high number of variants in the melanoma tumors, the cBioPortal for Cancer Genomics was used to identify the 10 most frequently mutated genes in melanoma. The data set included 1414 patient/case sets for melanoma, including acral, desmoplastic and lentigo maligna but not uveal melanoma. The distribution of occurrence of mutations in these top 10 genes is shown in FIG. 1A and FIG. 7B and the hotspot mutations are listed, where a hot spot in the cBioPortal is defined as, "this mutated amino acid was identified as a recurrent hotspot (statistically significant) and a 3D clustered hotspot in a population-scale cohort of tumor samples of various cancer types using methodology based in part on Chang, Nat Biotechnol, 2016 and Gao, Genome Medicine, 2017 (Fredriksson, Systematic analysis of noncoding somatic mutations and gene expression alterations across 14 tumor types. Nat Genet. 2014, 46; Chang, Identifying recurrent mutations in cancer reveals widespread lineage diversity and mutational specificity. Nat Biotechnol. Nature Publishing Group; 2016, 34:155-63). Close attention was paid to the mutations and copy number variations (CNV) within the TP53 locus (FIG. IB and FIG. 7B). By far the most prevalent TP53 variant was the P72R polymorphism, which was present in all the PDXs, except PDX9164. Three PDX tumors, PDX9164, 2252, and 0807, had nonsynomonous mutations (NSM) in the TP53 locus. Both PDX9164 and PDX2252 had mutations resulting in early translation termination and PDX2252 also had a decreased TP53 copy number of 0.94 (CNV could not be determined for PDX9164). PDX0807 had two mutations, an insertion at codon 138 (A-ADG) and an NSM at codon T140 (T-S). The mutations in PDX0807 occurred at a low frequency of 26-27%. In addition to these NSMs, PDXs 1668, 1839, and 1946 had mutations within splice sites. While the exact nature and frequency of these mutations in not reported in the NGS data, these three PDX tumors also had a loss of heterozygosity in the TP53 locus of 1.46, 1.38 and 1.08 respectively. All mutations and alterations in copy number within the 10 focus genes and MDM2, regardless of snpEff scores, for each PDX are listed in FIG. 7B. Interestingly, only copy number alterations were noted for MDM2.

[0243] Eight PDX tumors were characterized by Short Tandem Repeat (STR) analysis to confirm that the resulting PDX tumors were derived from the patient sample (FIG. 8A). DNA samples were extracted from the primary tumor specimen, the patient's blood when available, and two independent second passage isolates from the established PDX for PDX samples 9164, 0807, 1577, 1595, 1668, 1767, 2316, and 2552. A total of 16 STR loci (D351358, TH01, D18551, Penta E, D55828, D135317, D75820, D165539, CSF1P0, Dmelogenin, vWA, D851179, TPOX, FGA) were co-amplified in each sample. The STR profiles demonstrate that each analyzed PDX was derived from the primary specimen. As previously reported (Ben-David, Patient-derived xenografts undergo mouse-specific tumor evolution. Nat Genet. Nature Publishing Group; 2017, 49: 1567-75), mouse PDX tumors do undergo genetic evolution, and in accordance with this, there were changes noted between the STR profiles of the human tumor sample and the PDX, with occasional loss of an allele, consistent with clonal selection.

[0244] For each PDX tumor, pathology and melanoma IHC markers were confirmed relative to the patient diagnosis by a clinical pathologist. Representative results are shown for PDXs 2552, 1767, 1668 and 1595 (FIG. 8B). The markers used for melanocytic differentiation were Melan-A/Mart 1 and SOX10. Also, patient tumors (P0) and the second PDX passage (P2) were stained by H&E and for Ki-67, a proliferation marker used to determine the mitotic rate. All melanoma tumors and resulting PDXs were positive for SOX10 (Willis, SOX10: A useful marker for identifying metastatic melanoma in sentinel lymph nodes. Appl Immunohistochem Mol Morphol. 2015, 23:109-1247) while a subset was positive for Melan-A (Berset, Expression of Melan-a / Mart-1 Antigen As a Prognostic Factor in. Int J Cancer. 2001, 77:73-7) consistent with prior results. In addition to the correlation between SOX10 and Melan-A expression between the tumor sample and PDX P2 passage, a correlation with melanin expression was found, as shown for PDX1767. PDX tumors derived from tumors with high expression of melanin also had high expression of melanin in the PDX. Likewise, if a tumor did not express detectable melanin, the PDX did not either, as shown for PDX2552 and PDX1668. PDX1595 had a low but detectable level of melanin as did the patient tumor. All tumors and derived PDXs stained positive for Ki-67. [0245] To evaluate the effects of the compound of Formula (I) on the melanoma PDX panel, 50 mg/kg the compound of Formula (I), comparable to a human dose of 250mg/kg, was administered by oral gavage to the mice once the PDX tumor reached a volume of 50-100mm³. Mice were treated 5- days/week and treatment was continued until one or more tumors reached the endpoint size limit defined by the IACUC protocol (1.5 cm diameter). The compound of Formula (I) response to an FDA approved therapy was compared to, either dabrafenib (30 mg/kg) and trametinib (1 mg/kg) (D+T) or trametinib (T) alone (for NRAS mutant tumors) administered by oral gavage 5-days/week. For those mice harboring mutated BRAF^V600 tumors, dabrafenib and trametinib (D+T) were administered in vivo either with or without 50 mg/kg the compound of Formula (I). The response to all drugs was compared to the appropriate vehicle control group. Toxicity was not detected with any treatment group based on as AST and ALT levels, weight loss or morbidity; however, at higher doses of the compound of Formula (I) (100 mg/kg) significant morbidity was noted (data not shown). Representative data from these studies for PDX1839, PDX1946, PDX2316, PDX1668, and PDX1595 are shown in FIG. 2. (Data from all the PDX tumors are shown in FIGs. 9A-P). For each drug study, the tumor growth rate was statistically determined, the final tumor weight was measured, and FFPE sections were stained and quantified to measure proliferation based on Ki67 expression. To facilitate comparisons between PDX tumors and the responses to therapy, a summary of t-ratios for treatment group comparisons across 15 PDX tumors is shown in FIGs. 2 and 3A. The t-ratio is a ratio of the difference in tumor slope between control and a treatment group relative to its standard error, calculated by estimated marginal means based on the mixed effect model. For each PDX, response to a drug was based on a positive t-ratio greater than 2.6. The lowest positive t-ratio considered was 2.598 for the compound of Formula (I) treatment of PDX0807, which correlated with an adjusted p-value of 0.063. Any treatment which resulted in a decreased tumor growth compared to vehicle control is indicated by a positive t-ratio. Likewise, any treatment which resulted in increased tumor growth compared to vehicle control is indicated by a negative t-ratio.

[0246] The response to drug treatment could be classified into three distinct categories. FIG. 2A, with PDX1839 as an example, represents those PDX tumors that responded to the standard therapy (D+T) but not to the compound of Formula (I). This group of PDX tumors is referred to as Group I. In fact, in the case of PDX1839 in vivo administration of the compound of Formula (I) caused a slight, albeit insignificant, increase in growth (Bonferroni adjusted (adj.) p=0.138) (FIG. 2A). In contrast, D+T, an FDA approved therapy for BRAF^V600 mutated melanoma, did decrease the growth rate of the Group I PDX tumors (t-ratio=5.4741, adj. p <0.001). The combination of the compound of Formula (I) with D+T resulted in a further reduction in the growth rate compared to the compound of Formula (I) alone treatment (t-ratio=9.00, adj. p<0.001), but not compared to D+T standard therapy (t-ratio=1.58, adj. p<0.7). A decrease in tumor size was also observed when comparing the response to the compound of Formula (I) alone versus the compound of Formula (I) + D+T (adj. p = 0.014). Also shown in FIG. 2, Ki67 levels were not affected by in vivo administration of either the compound of Formula (I) or D+T (adj. p=0.714 and 0.184 respectively); however, a significant decrease in Ki67 staining compared to the vehicle control was seen with the combination therapy of the compound of Formula (I) and D+T (adj. p<0.001). A significant decrease was also observed when comparing the combination therapy (the compound of Formula (I) + D+T) to the compound of Formula (I) alone treatment (adj. p< 0.001) but not when compared to D+T therapy (adj. p=0.999). In summary, in Group I PDX tumors, D+T has a predominant effect on decreasing melanoma PDX tumor growth while the compound of Formula (I) therapy does not inhibit tumor growth in Group I PDX tumors.

[0247] FIG. 2B, with PDX1946 as an example, represents those PDX lines that did not respond to either the compound of Formula (I) or the standard therapy alone but responded synergistically to the combination of the compound of Formula (I) and the standard therapy (Group II). Group II was comprised of 5 PDX lines. Four of the five lines exhibited BRAF ^V600 mutation and one line, PDX 1179, had a nras ^QS1 mutation. In the case of PDX1179, mice were treated only with trametinib (T). As shown in FIG. 2B, neither the compound of Formula (I) or the standard therapy (D+T or T) resulted in a significant decrease in either growth rate (adj. p=0.999) or final tumor size. Flowever, in vivo administration of the compound of Formula (I) in combination with the standard therapy, resulted in a significant decrease in the tumor growth rate compared to the vehicle control (t. ratio=3.75, adj. p=0.002).

[0248] The second group of tumors shown in FIG. 2B includes those PDX tumors that did not respond to either the compound of Formula (I) or the standard therapy alone but responded synergistically to the combination of the compound of Formula (I) and the standard therapy (Group II). Group II was comprised of 6 PDX tumors and PDX1946 is representative of the response to therapy for Group II tumors. Five of the six lines exhibited BRAF^V600 mutations. The sixth line, PDX 1179, had an NRAS^QS1 mutation and a decreased BRAF copy number to 1.36 (see FIG. 7B) and was treated with trametinib but not dabrafenib. As shown in FIG. 2B, neither the compound of Formula (I) or the standard therapy (D+T or T) resulted in a significant decrease in either growth rate (adj. p=0.999) or final tumor size. Flowever, in vivo administration of the compound of Formula (I) in combination with the standard therapy, resulted in a significant decrease in the tumor growth rate compared to the vehicle control (t-ratio=3.75, adj. p=0.002) and a significant decrease in Ki67 positive cells (FIG. 2B and FIGs. 9A-P).

[0249] The third group of tumors, PDX tumors 0807, 1129, 1595, 1668, 1767, and 2316, did respond to the compound of Formula (I) alone with growth inhibition (Group III) (FIG. 2C and D). Two of the lines, PDX2316 and PDX1767, were BRAF^wt and NRAS^wt so mice were treated only with the compound of Formula (I). One line, PDX1595, expressed an NRAS^Q61R mutation, so the mice were treated with either the compound of Formula (I), trametinib, or the combination. The final three lines, PDXs 0807, 1129 and 1668, had BRAF mutations that are not in the V600 position (FIG. 1 and FIG. 3). The mice carrying these three PDX tumors were treated with the compound of Formula (I) and D+T alone and PDX0807 and 1129 were treated with the combination. PDX2316, PDX1668, and PDX1595 are shown as examples of the three different genetic subgroups within the group that responded to the compound of Formula (I) [FIG. 2C (PDX2316) and D (PDX1595 and PDX1668)]. For PDX2316, PDX1668, and PDX1595, the compound of Formula (I) treatment resulted in a significant decrease in tumor growth rate (adj. p<0.001 for PDX2316 and 1595 and adj. p=0.018 for PDX1668), and as shown for PDX2316, the compound of Formula (I) alone also decreased the final tumor weight (adj. p=0.042) and the level of Ki-67 staining (adj. p<0.001). When the compound of Formula (I) was used in combination with the standard therapy, as shown in FIG. 2D for PDX1595, the standard therapy alone did not affect the growth rate (adj. p=0.999), except for PDX0807 and there was no further decrease in growth rate comparing the rate with the compound of Formula (I) to the combination therapy (adj. p=0.999). In summary, in Group III PDX tumors, the compound of Formula (I) has a predominant effect on tumor growth inhibition that is not enhanced by D+T therapy.

[0250] The compound of Formula (I) previously has been shown to increase P53 protein levels in cell lines and mouse xenografts (Canon, The MDM2 Inhibitor AMG 232 Demonstrates Robust Antitumor Efficacy and Potentiates the Activity of p53-lnducing Cytotoxic Agents. Mol Cancer Ther. 2015, 14:649- 58; Werner, Small Molecule Inhibition of MDM2-p53 Interaction Augments Radiation Response in Fluman Tumors. Mol Cancer Ther. 2015, 14:1994-2003). To determine whether the compound of Formula (I) was affecting P53 expression and localization in the melanoma PDX models, the P53 protein level was monitored by IHC in two Group III PDX tumors that had been treated in vivo with the compound of Formula (I) (FIG. 2D). For both PDX1595 and PDX1668, the P53 protein level was very low in tumors from the vehicle-treated mice but the compound of Formula (I) treatment resulted in a substantial increase in P53 protein and nuclear localization. In contrast, neither dabrafenib or trametinib altered P53 levels or localization.

[0251] As exemplified in FIG. 2 and FIG. 3, the 15 PDXs studied here were classified into three subgroups: Group I PDXs did not respond to the compound of Formula (I), responded to D+T, and treatment with the combination of D+T and the compound of Formula (I) resulted in a small augmentation of tumor growth inhibition. Group II PDXs did not respond to either the compound of Formula (I) alone or D+T alone but responded to the combination of the compound of Formula (I) and D+T (AMG+D+T). Group III PDXs responded to the compound of Formula (I) monotherapy, as indicated by the gray shading. A larger t-ratio indicates that there was a larger difference between the treatment group and the comparison group. Interestingly, PDX 1767 was the only PDX with MDM2 amplification, and this PDX had the largest t-ratio when comparing the effect of the compound of Formula (I) to vehicle treatment (t-ratio=5.182). This supplements prior findings in glioblastoma cell lines, where lines with MDM2 amplification were the most sensitive to the compound of Formula (I) (Her, Potent effect of the MDM2 inhibitor AMG232 on suppression of glioblastoma stem cells. Cell Death Dis. 2018, 9:1-12).

[0252] In the Group II PDX tumors, the range of t-ratios for the combined treatment with the compound of Formula (I) and D+T was 3.028 to 8.106, which is suggestive of a synergistic relationship between the compound of Formula (I) and D+T treatments. The synergistic effect was defined as an interaction between the compound of Formula (I) and D+T treatments that causes the total effect of the drugs to be greater than the sum of the individual effects of each drug. This analysis did not include data from PDX1179, since this NRAS^Q61H tumor, was treated only with trametinib, in contrast to the other Group II PDX tumors, which were treated with dabrafenib and trametinib. The analysis results showed a significant interaction effect of the compound of Formula (I) and D+T for four independent PDX tumors over time (p=0.004) using the mixed-effect model. With model-based means, tumor growth rate with 95% confidence intervals for each treatment was estimated (FIG. 3B). The multiple comparison graph showed that the tumor growth rate in the combination group (AMG+D+T) was significantly reduced compared to either the compound of Formula (I) group (Tukey's adjusted p<0.001) or the D+T group (Tukey's adjusted p<0.001). There was no difference in the tumor growth rate between the compound of Formula (I) alone and D+T groups (Tukey's adjusted p=0.696). In other words, the Group II PDX tumors did not respond to the compound of Formula (I) or D+T alone, but when administered together there was synergistic inhibition of tumor growth. The power of this analysis is based on analyzing treatment effectiveness for multiple independent PDX tumors (FIG. 3). [0253] Interestingly, analysis of H&E staining of those PDX tumors which had the highest t-ratios when comparing the effect of the compound of Formula (I) treatment to the effect of the compound of Formula (I) + D+T revealed morphologic differences after the compound of Formula (I) and D+T therapy (FIG. 4). H&E staining of tumors isolated from vehicle-treated and D+T treated cells revealed that the tumor cells were tightly packed with minimal stroma, uniform in size and appearance, and had large nuclei with a high mitotic rate and a high Ki67 expression. In contrast, PDX tumors exhibiting a synergistic growth-inhibitory response to the compound of Formula (I) and D+T were characterized by mostly karyorrhectic nuclei with vacuolar changes around the karyorrhectic debris. This change in histology was only present in PDX tumors where the t-ratio comparing the effect of the compound of Formula (I) treatment to the effect of the compound of Formula (I) combined with D+T (or T for PDX1179) was greater than 2.6 (PDX1351 and 1946).

[0254] To identify changes in protein expression associated with the synergistic response to the compound of Formula (I) and D+T, RPPA analysis of protein and phosphoprotein expression was conducted (FIG. 4B). Unexpectedly, protein markers for different modalities of cell death, including bcl- 2-like protein 4 (BAX), B-cell lymphoma 2 (BCL2), p53 upregulated modulator of apoptosis (PUMA), Poly (ADP-ribose) polymerase (PARP), Beclin, janus kinase (JAK), induced myeloid leukemia cell differentiation protein (MCL1), and the Caspase, signal transducer and activator of transcription (ST AT), and heat shock proteins (HSP) families of proteins, while present in the antibody panel, were notably absent from the list of positive hits when comparing vehicle-treated PDX tumors to the compound of Formula (I) +D +T treated tumors. In contrast, a decreased expression hypoxia-inducible factor 1 subunit alpha (HIF-lot), monocarboxylate transporter 4 (MCT4), phosphorylated N-myc downstream-regulated gene-1 (pNDRGl), and phosphorylated mitogen-activated protein kinase (pMAPK) were noted in the compound of Formula (I) + D +T treatment group as compared to vehicle-treated tumors. Lactate dehydrogenase A (LDFIA) was also decreased, although the fold change was slight and below the cutoff of log2(FC)=+/- 0.4. The decreased levels of phosphorylated MAPK kinase were expected since these tumors were treated with dabrafenib and trametinib, both of which regulate the phosphorylation of MAPK. In contrast, in response to the combination of the compound of Formula (I) and D+T, the observed decreased expression of H IF-l-ot, MCT-4, LDFIA, and decreased phosphorylation of NDRG was unexpected and indicates treatment mediated decreased rates of glycolysis (Zheng, Energy metabolism of cancer: Glycolysis versus oxidative phosphorylation (review). Oncol Lett. 2012, 4:1151-7; Warburg,

On the Origin of Cancer Cells. Science (80), 1956; 123:3090314; Tran, Rubric Ass. 2016, 32:177-93). The change in expression of HIF-1-a, pNDRG and LDHA was only seen in PDX tumors treated with the combination of the compound of Formula (I) and D+T (Group II: PDX1179, 1351, and 2252). Changes in MCT-4 expression were also observed when the compound of Formula (I) was used as a single agent (FIG. 5D and E). Furthermore, Group I PDX tumors 1839, 2195, and 2252 responded to D+T as a single agent, and exhibited decreased phosphorylation of MAPK, but did not exhibit altered expression of H IF- 1-a, MCT-4, LDFIA nor was there a decrease in phosphorylation of NDRG in response to D+T without the compound of Formula (I) (FIG. 10). These data suggest that the synergistic action of the compound of Formula (I) and D+T result in metabolic reprogramming of the tumor cells.

[0255] To identify potential biomarkers for the compound of Formula (I) response, both the genetic mutation and copy number variation data obtained by NGS, and gene expression changes obtained by RPPA analysis (FIG. 5) were examined. Analysis and comparison of basal (vehicle-treated) protein and phosphoprotein expression in PDX tumors that were either resistant or responsive to the compound of Formula (I) did not identify any significant proteins or phosphoproteins (FDR >0.05) that could be used as predictors of the compound of Formula (I) responsiveness, as shown in the Volcano Plot comparing vehicle-treated samples from PDX tumors in Groups I and II compared to those in Group III (FIGs. 10 and 11). While protein expression patterns were not a predictor of the compound of Formula (I) responsiveness, the BRAF mutation status was a strong predictor of the compound of Formula (I) responsiveness.

[0256] The Oncoprint Cluster Analysis shown in FIG. 5 represents the analysis with the 10 most relevant genetic alterations, as discussed in FIG. 1. No further insights were gained by including all 300 genes analyzed by NGS (data not shown). This analysis indicates that the mutational status of BRAF is highly correlated with responsiveness to the compound of Formula (I). Of the 9 PDX tumors that were resistant to the compound of Formula (I) (Groups I and II), all but one had a V600 mutation in BRAF (FIG. 1)·

[0257] In contrast to the high prevalence of V600 BRAF mutations in the PDX tumors that were resistant to the compound of Formula (I), all of the PDX tumors that responded to the compound of Formula (I) lacked the BRAF^V600 mutation, although other mutations were noted in the BRAF gene. PDX1129 had two coding changes, D594N and K483T and PDX1668 and PDX1595 had missense mutations, of unknown significance. For PDX1668 the two non-synonymous BRAF mutations were S147N and N140T and for PDX1595 the BRAF mutation was P334S. While there are reports of the D549N mutation in melanoma, colorectal and lung carcinomas and hairy cell leukemia, none of the other mutations have been reported and there are no data on the biological significance of these mutations (Boyd, High resolution melting analysis for detection of BRAF exon 15 mutations in hairy cell leukemia and other lymphoid malignancies. Br J Haematol. 2011, 155: 609-12; Boursault, Tumor Homogeneity between Primary and Metastatic Sites for BRAF Status in Metastatic Melanoma Determined by Immunohistochemical and Molecular Testing. PLoS One. 2013, 8; Mufti, Case Series of Two Patients Presenting With Pericardial Effusion as First Manifestation of Non-Small Cell Lung Cancer With BRAF Mutation and Expression Of PD-L1. World J Oncol. 2018, 9:56-61). There was also amplification of the BRAF gene but these occurred in equal percentages in both the PDX tumors that were resistant to the compound of Formula (I) (3/9 or 33.3%) and those that were sensitive (2/6 or 33.3%). Statistical analysis of the correlation between the BRAF^V600 mutation and the in vivo effectiveness of the compound of Formula (I) in decreasing tumor growth, as measured by the t-ratio was analyzed using the Wilcoxon rank sums test and Pearson's product-moment correlation. The Point-Biserial correlation coefficient (a special case of Person's correlation coefficient) was -0.7938, with a Bonferroni adjusted p-value of <0.001. A dot plot showing the correlation is shown in FIG. 12.

[0258] RPPA analysis indicated the compound of Formula (I) monotherapy of Group III PDX tumors (1129, 1595, 1668 and 2316) altered the levels of six proteins in the compound of Formula (I) responsive tumors based on the limits of an FDR < 0.5 and log2(FC)>+/- 0.4. The P53-inducible P21 protein (CDKN1A) exhibited the most significant increase in expression in response to the compound of Formula (I) treatment. The other notable protein induced by the compound of Formula (I) was the receptor tyrosine kinase ephrin type-A receptor 2, (EPHA2) the erythropoietin-producing hepatocellular receptor A2, which has been reported to play a critical role in oncogenic signaling in many types of solid tumors (Zhou, Emerging and diverse functions of the EphA2 noncanonical pathway in cancer progression. Biol Pharm Bull. 2017, 40:1616-24). There were also decreases in the cell cycle regulatory protein Cyclin B1 (CCNB1), PLK1 which belongs to the CDC5/Polo subfamily, and forkhead box protein Ml (FoxMl) in response to the compound of Formula (I) (Stark, Analyzing the G2 / M Checkpoint. 280:51-82; Down, Watson RJ. Binding of FoxMl to G2/M gene promoters is dependent upon B-Myb. Biochim Biophys Acta - Gene Regul Mech. 2012, 1819:855-62). All three proteins are involved in the regulation of M-phase of the cell cycle (Liao, Regulation of the master regulator FOXM1 in cancer. Cell Commun Signal. 2018, 16:1-1555; Murakami, Regulation of yeast forkhead transcription factors and FoxMl by cyclin- dependent and polo-like kinases. Cell Cycle. 2010, 9:3233-42). (FIG. 5 D&E). Finally, the monocarboxylate transporter 4 (MCT4), which releases lactate from glycolytic tumors (Draoui, Lactate shuttles at a glance: From physiological paradigms to anti-cancer treatments. DMM Dis Model Mech. 2011, 4:727-32) was decreased by the compound of Formula (I) treatment.

[0259] Interestingly, RPPA analysis of PDX tumors that did not respond to the compound of Formula (I) treatment had a similar increase in p21 expression even though the growth of these tumors was not decreased by the compound of Formula (I) treatment (FIG. 5 B&C). Thus, in the compound of Formula (I) resistant tumors, the increase in P21 was not accompanied by significant changes in the downstream pathways regulated by MDM2 including cell cycle regulatory proteins or proteins involved in the regulation of apoptosis (El-Deiry WS, p21(WAFl) mediates cell-cycle inhibition, relevant to cancer suppression and therapy. Cancer Res. 2016, 76:5189-91). Since all these PDX tumors have activating mutations in the MEK/ERK pathway, these data suggest that activation of BRAF or NRAS interferes with the pathways associated with the growth inhibitory effects of the compound of Formula (I).

[0260] Detailed analysis of the TP53 status in the panel of 15 melanoma PDX tumors revealed 6 PDXs had either a non-synonymous mutation in the TP53 gene and/or a loss of heterozygosity (FIG. 1A, B and FIG. 7B). As shown in FIG. 3A, the PDX tumors with alterations in TP53 status still responded to the compound of Formula (I) either as a single agent (PDX0807 and PDX1668) or in combination with D+T (PDX2252 and PDX1946), but the response was reduced compared to the TP53^wt tumors and was at the lower limits of significance. For example, PDX0807 and PDX1668, both of which responded to the compound of Formula (I) as a single agent had t-ratios of 2.598 and 2.829, respectively, while the TP53wt PDX tumors in Group III, PDX 1595, 2316, 1129 and 1767, had t-ratios greater than 3.5 (FIG. 3).

[0261] BRAF^V600 mutant melanoma tumors often develop resistance to MAPK inhibitors like dabrafenib and trametinib. It was postulated that D+T resistant tumors might respond to a Bcl-2 inhibitor to block tumor growth and proliferation through an alternate pathway. Indeed, it has been demonstrated that combining the MDM2 inhibitor, nutlin RG7388, with the BCL-2 inhibitor, ABT-199, provides better therapeutic efficacy than either drug alone in acute myeloid leukemia (AML) (Pan, Synthetic Lethality of Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic Efficacy. Cancer Cell, Elsevier Inc.; 2017, 32:748-760. e6). To determine if similar effects occur in melanoma, two BRAF^mut and two BRAF^wt tumors were treated with navitoclax, a BCL2 inhibitor, either as a single agent or in combination with the compound of Formula (I). The summary data for navitoclax treatment are shown in FIG. 6C. Navitoclax slightly enhanced tumor growth for two PDX tumors (PDX1351 and 1577), as indicated by the negative t-ratio. While navitoclax was not effective as a single agent in any PDX tumor, navitoclax in combination with the compound of Formula (I) in BRAF^V600E (PDX1351 and 1577) tumors was more effective than either drug alone and resulted in tumor regression in three PDX models, as shown by example for PDX1577 (FIG. 6). Interestingly, the growth of only the mutant BRAF^V600E tumors was inhibited by the combination of navitoclax and the compound of Formula (I). In those tumors, that were BRAF^V600wt (PDX1129 and PDX1668), the effects of the compound of Formula (I) and navitoclax were not more effective than the compound of Formula (I) alone as indicated by a t-ratio of less than 2.6 when comparing the compound of Formula (I) + navitoclax treatment groups to the compound of Formula (I) alone. Similar effects were noted in the weights of the final tumors.

[0262] While recent therapeutic advances for metastatic melanoma have significantly increased the overall survival of melanoma patients, a subset of patients does not respond to immunotherapy or targeted therapies. In this study, a panel of melanoma PDX tumors was observed that therapy with the compound of Formula (I), either singularly or in combination with dabrafenib and trametinib treatment, effectively decreased tumor growth in 100% of the tumors tested. Similar results were obtained in a small Phase 1/2 clinical trial testing AMG232 with trametinib and/or dabrafenib, where the compound of Formula (I) was found to reduce tumor size in 73% of the patients with metastatic cutaneous melanoma expressing BRAF nonV600-mutant, with 13% (2/15) showing a greater than 30% reduction in size according to the RECIST criteria (Eisenhauer, New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1) European J. Cancer (2009) 45:228-247). Furthermore, 100% (6/6) of patients treated with the compound of Formula (I) in combination with trametinib or dabrafenib had a reduction in tumor size, with 67% (4/6) patients having a decrease in tumor size of greater than 30%.

The dose-escalation design for the compound of Formula (I) in the Phase I/ll trial (NCT02110355) included treatment for seven days of each 3-week cycle (7/21) at 120, 240, or 480 mg. Based on this, the maximally tolerated dose was 180 mg in combination with tremetanib or tremetanib + dabrafenib. The incidence of adverse effects was notable, albeit acceptable, with 1 patient having a grade 3 adverse event and all remaining patients have either a G1 or G2 adverse event (Moschos, Phase 1 study of the p53-MDM2 inhibitor AMG 232 combined with trametinib plus dabrafenib or trametinib in patients (Pts) with TP53 wild type (TP53WT) metastatic cutaneous melanoma (MCM) J. Clin. Oncol. 2017, 2575). Similarly, based on the calculations described by Nair and Jacob (Nair, J Basic Clin Pharm. 2016, 7(2):27- 31), the human equivalent dose for 50 mg/kg the compound of Formula (I) used in mice is 250 mg the compound of Formula (I) for an average 62 kg individual.

[0263] In the study, tumors responding to the compound of Formula (I) and dabrafenib and trametinib could be classified into three separate groups. Group I, consisted of three BRAF^V600E mutant PDX tumors, which responded to dabrafenib and trametinib but not to the compound of Formula (I) as a single agent. In combination, these agents further inhibited tumor growth. Group II, consisted of BRAF^V600E/M and one NRAS^Q61H mutant tumors that did not respond to either the compound of Formula (I) or dabrafenib and trametinib alone, but when used in combination, the inhibition of tumor growth was demonstrated to be statistically significant and synergistic. Finally, in Group III, the BRAF^V600wt PDX tumors responded to the compound of Formula (I) alone with significant inhibition of tumor growth. The mutational status of BRAF was an effective predictor of the compound of Formula (I) monotherapy response. In the PDX panel, only those tumors that were BRAF^V600wt were responsive to the compound of Formula (I) alone, while, except for PDX1179, tumors with BRAF^V600 mutations were resistant to the compound of Formula (I), but responded synergistically to the compound of Formula (I) combined with dabrafenib and trametinib.

[0264] As seen with the compound of Formula (I), in both the RG7388 sensitive and resistant cell lines, the protein level of P21 is abundantly induced. Through a series of elegant experiments using RG7388 in combination with a Bcl-2 inhibitor, ABT-199 (venetoclax), they demonstrated that P53 plays a pivotal role in regulating the Ras/Raf/MEK/ERK signaling cascade (Pan, Synthetic Lethality of Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic Efficacy. Cancer Cell, Elsevier Inc.; 2017, 32:748-760.e6). While Pan did not explore the mechanistic role of P53 in regulating the MAPK pathway, others have identified P53-regulated transcription of four phosphatases, wild-type p53-induced phosphatase 1 (Wipl), mitogen-activated protein kinase phosphatase 1 (MKP1), phosphatase of activated cells 1 also known as dual specificity phosphatase 2 (PAC1/DUSP2), and DUSP5, that negatively regulate MAPK signaling (Gen, The functional interactions between the p53 and MAPK signaling pathways. Cancer Biol Ther. 2004, 3:156-61). While further experiments are needed, it was proposed that in tumors with BRAF^V600 mutations, activation of P53 through MDM2 inhibition will not inhibit the MAPK pathway; therefore, those tumors with BRAF^V600 mutations are resistant to the effects of the compound of Formula (I) and other MDM2 inhibitors. However, in the presence of dabrafenib and trametinib, two inhibitors of the MAPK pathway, inhibition of the constitutively activated MAPK pathway reverses the resistance to MDM2 inhibition, resulting in a decrease in MAPK T202/Y204 phosphorylation. The MAPK pathway is also a key regulator of the transcription factor Myc through both phosphorylation and transcription control. While the RPPA panel did not include p-Myc or N-Myc, the N-Myc downstream-regulated gene NDRG-1 was decreased by the compound of Formula (I) and D+T treatment. Additionally, Myc has been shown to stabilize HIF-Ia (Doe, Myc posttranscriptionally induces HIF1 protein and target gene expression in normal and cancer cells. Cancer Res. 2012, 72:949-57) , which explains the decrease in HIF-Ia in the compound of Formula (I) and D+T treated tumors in the studies described here. Therefore, the studies suggest that the treatment of melanoma PDX tumors expressing the activating BRAF^V600 mutation with the combination of dabrafenib, trametinib and the compound of Formula (I), modulates the underlying "triad" of proteins, MYC, H IF-1, and P53. These three proteins play a key role in the regulation of the Warburg phenomenon (Yeung, Roles of p53, MYC and H IF-1 in regulating glycolysis-The seventh hallmark of cancer. Cell Mol Life Sci. 2008, 65:3981-99; Li, Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy, Immunity, Elsevier Inc.; 2018, 49:178-193. e7; Melotte, The N-myc downstream regulated gene (NDRG) family: Diverse functions, multiple applications. FASEB J. 2010, 24:4153-66; Gordan, HIF and c-Myc: Sibling Rivals for Control of Cancer Cell Metabolism and Proliferation. Cancer Cell. 2007, 12:108-13) and are key regulators of the glycolytic switch in tumors (Yu, The glycolytic switch in tumors: How many players are involved? J Cancer. 2017, 8:3430-40).

[0265] It has also been reported that Bcl-2 inhibitors, like dabrafenib and trametinib, can overcome the resistance to the compound of Formula (I). Preclinical studies have shown that mice implanted with OCL-AML3 leukemic cells that are resistant to both the MDM2 inhibitor, RG7388, and the Bcl-2 inhibitor, ABT-199, exhibited enhanced overall survival with the two agents combined. Similarly, it was found in melanoma, treatment of BRAF^V600 mutant PDX tumors PDX1351 and 1577, which are resistant to the compound of Formula (I), the compound of Formula (I) combined with navitoclax significantly inhibited tumor growth when neither agent was effective alone. These data suggest that, as in AML, Bcl2 inhibition can overcome the resistance to MDM2 inhibitors in tumors in which the MAPK pathway is constitutively activated. It is interesting to note that in the PDX tumors treated with both combinations, the compound of Formula (I) and navitoclax and the compound of Formula (I) and dabrafenib and trametinib, that the combination of the compound of Formula (I) with navitoclax was more effective and not only inhibited tumor growth but caused tumor regression (see clinicaltrials.gov/ct2/show/NCT02110355).

[0266] While the combination of the compound of Formula (I) plus dabrafenib and trametinib in BRAF^VS00mut tumors resulted predominantly in a decreased expression of key proteins involved in the regulation of glycolysis, treatment with the compound of Formula (I) of BRAF^V600WT tumors resulted in a decrease in expression of a triad of proteins regulating the G2/M checkpoint in the cell cycle. The G2/M DNA damage checkpoint serves to prevent the cell from entering mitosis (M-phase) with genomic DNA damage and the key regulators of this process are FoxMl, PLK1, and Cyclin Bl, all of which were decreased by the compound of Formula (I) treatment. FoxMl expression is increased in a variety of solid tumors, including melanoma, and inhibition of FoxMl leads to a decrease in cell proliferation and migration, metastasis and angiogenesis. Furthermore, analysis of the TGCA database illustrated that high levels of FoxMl are related to poor prognosis in most solid tumors (Liao, Regulation of the master regulator FOXM1 in cancer. Cell Commun Signal. 2018, 16:1-15). P53, both directly and indirectly through p21, has been reported to decrease FoxMl expression (Kurinna, P53 Regulates a Mitotic Transcription Program and Determines Ploidy in Normal Mouse Liver. Hepatology. 2013, 57:2004-13, Barsotti, Pro-proliferative FoxMl is a target of p53-mediated repression. Oncogene. Nature Publishing Group; 2009, 5:4295-305). Subsequently, FoxMl induces the expression of the Cdkl activators, cyclin B and Cdc25 (Laoukili, FoxMl is required for execution of the mitotic programme and chromosome stability. Nat Cell Biol. 2005, 7:126-36), so a decrease in FoxMl expression would result in a decrease in cyclin B, as seen in the RPPA data. Cdc25 was also statistically decreased in the RPPA analysis, but the fold decrease was slight and log2(FC) was not greater than the cutoff -0.4. Interestingly, cyclin B has been proposed to be a critical target of FoxMl at the G2/M transition (Murakami, Regulation of yeast forkhead transcription factors and FoxMl by cyclin-dependent and polo-like kinases. Cell Cycle. 2010, 9:3233-42). Polo-like kinases (PLK) also help to regulate the cell cycle, mainly at the G2/M checkpoint and FoxMl and PLKs exist in a positive feedback loop where transcriptionally activated FoxMl controls the expression of PLKs while PLK1 binds and phosphorylates FoxMl and activates FoxMl as a transcription factor (Z, Malureanu, Plkl-dependent phosphorylation of FoxMl regulates a transcriptional programme required for mitotic progression. Nat Cell Biol. 2008, 10:1076-82). Therefore, the compound of Formula (I), through the P53 dependent inhibition of FoxMl expression, has a direct effect in regulating the G2/M checkpoint.

[0267] It is important to note that the tumors used in this study were very heterogeneous and there was no PDX tumor which was purely BRAF mutant and there was only one line which was a complete TP53 mutant (PDX9164). Of the PDX tumors with a BRAF^V600 mutation, the PDX with the highest mutation frequency was PDX1577 with a BRAF^V600 mutation frequency of 0.951 and the lowest was PDX1351 with a BRAF^V600 mutation frequency of 0.316. There was an average BRAF^V600 mutation frequency of 0.555. Similar heterogeneity was seen with TP53. In this study, 4 PDX tumors with mutations in the TP53 gene were included. Only one of the lines, PDX9164 had a TP53 mutation frequency of 1 at codon 192 (Q192*) and this mutation has a FATHMM pathogenic score of 0.93 suggesting it is highly pathogenic. The other mutations occurred at lower frequencies, 0.265 for PDX0807 (A138ADG and T140S); 0.607 for PDX 2252 (R196*); and 0.484 and 0.235 for PDX1839 (R306* and Y236H). This heterogeneity complicates the interpretation of the effectiveness of the compound of Formula (I) in mutant versus wild type TP53 tumors. It is interesting to note, however, that while the compound of Formula (I) was somewhat effective in melanoma PDX tumors with mutant TP53, the effect was at the lower limit of significance. Furthermore, TP53mut, BRAF^V600 mutant, PDX9164, while it did not respond to the compound of Formula (I) monotherapy, did respond synergistically to the compound of Formula (I) plus dabrafenib and trametinib. Similar results were noted in a Phase 1 trial of RG7112, a member of the Nutlin family of MDM2 inhibitors, in leukemia. In this study, 3 of 19 patients with TP53 mutations showed evidence of response to RG7112 as demonstrated by a decrease peripheral blast count and one patient was reported to have stable disease for more than 2 years (Andreeff,

Results of the phase i trial of RG7112, a small-molecule MDM2 antagonist in leukemia. Clin Cancer Res. 2016, 22:868-76).

[0268] It has been demonstrated here that 60% of melanoma PDX tumors have an inherent resistance to the compound of Formula (I) and this correlates with the incidence of BRAF^V600 mutations. In this study, of the eight BRAF^V600 mutant PDX tumors treated with dabrafenib and trametinib, five (62.5%) were resistant to dabrafenib and trametinib. Two of the five resistant PDX tumors came from patients previously treated with dabrafenib and trametinib who had progressed on treatment. In these two MAPKi-resistant PDX tumors, the resistance is undoubtedly acquired. The study of MAPKi-resistant melanoma has been extensively studied and the mechanism of resistance is complex and multifactorial. The causative factors that contribute to MAPKi-resistance can be broadly classified into three categories: mutational events and non-mutational events, which are tumor inherent and lead to either MAPK pathway reactivation or activation of a parallel signaling pathway, and changes in the surrounding microenvironment. Based on the pleiotropic nature of drug resistance it is interesting, although not surprising, the two PDX tumors previously treated with D+T remained resistant. It is also interesting that none of the BRAF^V600E mutant tumors that responded to D+T were derived from patients who had prior exposure to D+T.

[0269] Overall, the results demonstrate the efficacy of the MDM2 antagonist as a single agent or as a part of a therapeutic combination with MAPK pathway-targeting agents in preclinical melanoma models. It was previously shown in preclinical studies that melanoma tumors respond to CDK4/6 inhibitors but the addition of an MDM2 antagonist results in significantly increased inhibition of tumor growth (Vilgelm, MDM2 antagonists overcome intrinsic resistance to CDK4/6 inhibition by inducing p21. Sci Transl Med. 2019, 11). Notably, tumors that acquired resistance to the current standard of care approaches such as immunotherapy and BRAF/MEK targeted therapy, are responsive to the MDM2 antagonist given alone or as a part of combination treatment. These findings support the clinical development of MDM2 antagonists as a second-line treatment for metastatic melanoma.

Claims

CLAIMS We claim:

1. A method of treating metastatic melanoma in a human subject previously treated with immunotherapy comprising administering to the human subject a therapeutically effective amount of a MDM2 inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):

(I) or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, further comprising detecting the BRAF genotype in the human subject.

3. The method of claim 2, comprising administering the MDM2 inhibitor to a human subject with a wild-type BRAF^⁶⁰⁰ (BRAF^) genotype.

4. The method of claim 1, further comprising detecting the NRAS genotype in the human subject.

5. The method of claim 4, comprising administering the MDM2 inhibitor to a human subject with a wild-type NRAS (NRAS^) genotype.

6. The method of claim 1, further comprising detecting the NF1 genotype in the human subject.

7. The method of claim 1, comprising administering the MDM2 inhibitor to a human subject with a wild-type NF1 genotype (NFl^).

8. The method of claim 1, wherein the human subject has a genotype selected from the group consisting of BRAF^, A/RAS^and NFl^.

9. The method of claim 3, wherein the human subject has a mutant NRAS.

10. The method of claim 3, wherein the human subject has a mutant NF1.

11. The method of claim 1, wherein the immunotherapy is an ex vivo cell therapy selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR)-engineered peripheral blood lymphocytes (PBL) and chimeric antigen receptor ((CAR)-engineered PBL).

12. The method of claim 1, wherein the immunotherapy is an immune checkpoint protein inhibitor therapy.

13. The method of claim 12, wherein the immune checkpoint protein inhibitor is an anti-PD-Ll antibody selected from the group consisting of BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.

14. The method of claim 12, wherein the immune checkpoint protein inhibitor is an anti-PD-1 antibody selected from group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab-rwlc, AMP-224, AMP-514, PDR001, and fragments, conjugates, biosimilars, or variants thereof.

15. The method of claim 12, wherein the immune checkpoint protein inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and fragments, conjugates, biosimilars, or variants thereof.

16. The method of claim 1, wherein the immunotherapy is a T-cell engager selected from catumaxomab, FBTA05, Ertumaxomab, Ektomun, blinatumomab, solitomab, and fragments, conjugates, biosimilars, or variants thereof.

17. A method of treating metastatic melanoma in a human subject previously treated with immunotherapy comprising administering to the human subject a combination of a therapeutically effective amount of a MDM2 inhibitor, a BRAF inhibitor and a MEK inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof.

18. The method of claim 17, wherein the BRAF inhibitor is selected from the group consisting of encorafenib, vemurafenib, dabrafenib, sorafenib, and combinations thereof.

19. The method of claim 17, wherein the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, selumetinib, pimasertib, binimetinib, and combinations thereof.

20. The method of claim 17, further comprising detecting the BRAF genotype in the human subject.

21. The method of claim 20, wherein the human subject exhibits SfiAF ^V60° mutation.

22. The method of claim 17, wherein the human subject exhibits NRAS ⁰⁶¹ mutation.

23. The method of claim 17, wherein the immunotherapy is an ex vivo cell therapy selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR)-engineered peripheral blood lymphocytes (PBL) and chimeric antigen receptor ((CAR)-engineered PBL).

24. The method of claim 17, wherein the immunotherapy is an immune checkpoint protein inhibitor therapy.

25. The method of claim 24, wherein the immune checkpoint protein inhibitor is an anti-PD-Ll antibody selected from the group consisting of BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, and fragments, conjugates, biosimilars, or variants thereof.

26. The method of claim 24, wherein the immune checkpoint protein inhibitor is an anti-PD-1 antibody selected from group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab-rwlc, AMP-224, AMP-514, PDR001, and fragments, conjugates, biosimilars, or variants thereof.

27. The method of claim 24, wherein the immune checkpoint protein inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and fragments, conjugates, biosimilars, or variants thereof.

28. The method of claim 17, wherein the immunology is a T-cell engager selected from catumaxomab, FBTA05, Ertumaxomab, Ektomun, blinatumomab, solitomab, and fragments, conjugates, biosimilars, or variants thereof.