WO2018202910A1

WO2018202910A1 - Combination of antibiotic and bcl-2 inhibitor and uses thereof

Info

Publication number: WO2018202910A1
Application number: PCT/EP2018/061694
Authority: WO
Inventors: Micol RAVA'; Aleco D'ANDREA; Bruno Amati
Original assignee: Fondazione Istituto Italiano di Tecnologia; Istituto Europeo di Oncologia SRL IEO
Current assignee: Fondazione Istituto Italiano di Tecnologia; Istituto Europeo di Oncologia SRL IEO
Priority date: 2017-05-05
Filing date: 2018-05-07
Publication date: 2018-11-08
Anticipated expiration: 2019-11-05

Abstract

The present invention relates to the therapeutic use of a combination of at least one mitochondrially-targeted antibiotic and at least one inhibitor of BCL-2 for use in the treatment of MYC/BCL2 double positive B-cell lymphomas and relative compositions.

Description

Combination of antibiotic and BCL-2 inhibitor and uses thereof

Technical field

The present invention relates to the therapeutic use of a combination of at least one mitochondrially- targeted antibiotic and at least one inhibitor of BCL-2 for use in the treatment of MYC/BCL2 double positive B-cell lymphomas and relative compositions.

Background art

High-grade B cell lymphomas with MYC and BCL2 or BCL6 translocations, hereby referred to as double-hit lymphomas (DHL), constitute a subset of diffuse large B cell lymphoma (DLBCL) that was recently re-classified as a separate entity (1-4). Additional cases of DLBCL, commonly referred to as double-expressors, show positivity for the MYC and BCL2 proteins in the absence of the corresponding translocations. In both groups, activation of MYC and BCL2 correlates with poor prognosis in the face of current front-line treatments, including combined immuno- and chemotherapy (for example rituximab and CHOP, or R-CHOP) (1-7), calling for the development of new therapeutic regimens. A most promising prospect in this regard is the emergence of selective BCL2 inhibitors, such as venetoclax (also called ABT- 199) (3, 8-10).

The synergy between MYC and BCL2 in lymphomagenesis was originally demonstrated in transgenic mice (11, 12) and is explained by the ability of BCL2 to block the pro-apoptotic activity of MYC, while leaving intact its proliferative potential (13-15). On this basis, the inventors reasoned that compounds that exacerbate MYC-induced apoptosis would be likely to cooperate with venetoclax in killing DHL cells. A candidate compound was tigecycline (16, 17), a broad-spectrum anti-bacterial agent with documented cytotoxicity against diverse cancer cell types, most likely owing to its inhibitory effect on mitochondrial translation (18) (Fig. 15A).

Genes encoding components of the mitochondrial ribosome were coordinately activated during lymphomagenesis (19) and were critical for tumor maintenance ϊη Εμ-myc transgenic mice (16) (Fig. 15B). In line with these genetic data, pharmacological inhibition of the mitochondrial ribosome with tigecycline was synthetic lethal with MYC activation, impaired tumor cell survival in vitro, and extended lifespan in lymphoma-bearing mice (16, 17) (Fig. 15A, B).

Therefore, there is still the need for a therapeutic regime effective in the treatment MYC/BCL2 double positive B-cell lymphomas. Here, the inventors present preclinical data showing that a BCL2 inhibitor (preferably venetoclax) and a mitochondrially targeted antibiotic (preferably tigecycline) synergize in the treatment of MYC/BCL2 double-hit lymphomas, allowing tumor eradication in xenografted mice.

Summary of the invention

High-grade B cell lymphomas with concurrent activation of the MYC and BCL2 oncogenes, also known as double-hit lymphomas (DHL), show dismal prognosis with current therapies. MYC activation sensitizes cells to inhibition of mitochondrial translation by a tetracycline derivative antibiotic, tigecycline, and treatment with this compound provides a therapeutic window in a mouse model of MYC-driven lymphoma. The inventors now addressed the utility of a mitochondrially- targeted antibiotic fortreatment of DHL. BCL2 activation in mouse Εμ-myc lymphomas antagonized tigecycline-induced cell death, which was specifically restored by combined treatment with the BCL2 inhibitor venetoclax. In line with these findings, tigecycline and two related antibiotics, tetracycline and doxycycline, synergized with venetoclax in killing human MYC/BCL2 DHL cells. Treatment of mice engrafted with either DHL cell lines or a patient-derived xenograft (PDX) revealed strong anti-tumoral effects of the tigecycline/venetoclax combination, including long-term tumor eradication with one of the cell lines. This drug combination also had the potential to cooperate with rituximab, a component of current front-line regimens. Venetoclax and tigecycline are currently in the clinic with distinct indications: the inventors' preclinical results warrant the repurposing of these drugs for combinatorial treatment of DHL.

Then, the present invention provides a combination of at least one mitochondrially-targeted antibiotic and at least one inhibitor of BCL-2 for use in the treatment of a MYC/BCL2 double positive B-cell lymphoma.

Preferably, the at least one mitochondrially-targeted antibiotic is selected from the group consisting of: erythromycin and derivatives thereof, tetracycline and derivatives thereof, glycylcycline and derivatives thereof, an anti-parasitic drug and derivatives thereof, and chloramphenicol and derivatives thereof.

Still preferably, the erythromycin derivative is selected from the group consisting of: Azithromycin, Carbomycin, Cethromycin, Clarithromycin, Dirithromycin, Mitemcinal, Oleandomycin, flurithromycin, Roxithromycin, Spiramycin, Telithromycin and Tylosin. Still preferably, the tetracycline and/or glycylcycline derivative is selected from the group consisting of: Tigecycline, Tetracycline, Doxycycline, Chlortetracycline, Oxytetracycline, Demeclocycline, Lymecycline, Meclocycline, Methacycline, Minocycline and Rolitetracycline. Still preferably, the anti-parasitic drug is pyrvinium pamoate.

It is an object of the invention a combination of at least one mitochondrially-targeted antibiotic and at least one inhibitor of BCL-2 comprising more than one mitochondrially-targeted antibiotic, said more than one mitochondrially-targeted antibiotic optionally belonging to the same class of antibiotics or to different classes of antibiotics.

Preferably, the mitochondrially-targeted antibiotic is administered intravenously.

More preferably, the mitochondrially-targeted antibiotic is administered every two days.

Preferably, the inhibitor of BCL-2 is selected from the group consisting of: ABT-737, ABT-263, ABT-199 (Venetoclax).

Still preferably, the inhibitor of BCL-2 is administered for at least one cycle of 5 days.

In a preferred embodiment, the mitochondrially-targeted antibiotic is Tigecycline. In a preferred embodiment, the mitochondrially-targeted antibiotic is doxycycline. In a preferred embodiment, the mitochondrially-targeted antibiotic is tetracycline.

In a preferred embodiment, the inhibitor of BCL-2 is ABT-199.

In a preferred embodiment, the mitochondrially-targeted antibiotic is Tigecycline and the inhibitor of BCL-2 is ABT-199. In another preferred embodiment, the mitochondrially-targeted antibiotic is doxycycline and the inhibitor of BCL-2 is ABT-199. In another preferred embodiment, the mitochondrially-targeted antibiotic is tetracycline and the inhibitor of BCL-2 is ABT-199.

Preferably, the MYC/BCL2 double positive B-cell lymphoma is a double-hit lymphoma or a double- expressor lymphoma.

In a preferred embodiment, the combination as defined above further comprises at least one additional therapeutic agent.

Preferably, the additional therapeutic agent is selected from the group consisting of: an anti-CD 20 antibody, an anti-CD22 antibody, an anti-VEGF antibody, an anti-CD52 antibody, Cyclophosphamide, Doxorubicin (Hydroxydaunomycin), Vincristine (Oncovin ®), Prednisolone and a combination thereof.

Preferably, the anti-CD 20 antibody is Rituximab. Preferably, the anti-CD22 antibody is Epratuzumab. Preferably, the anti-VEGF antibody is Bevacizumab. Preferably, the anti-CD52 antibody is Alemtuzumab. In a preferred embodiment, the additional therapeutic agent is rituximab. In another preferred embodiment, the mitochondrially-targeted antibiotic is Tigecycline and the inhibitor of BCL-2 is ABT-199.

Still preferably, the mitochondrially-targeted antibiotic and the inhibitor of BCL-2 and optionally the least one additional therapeutic agent are administered simultaneously or sequentially.

The present invention also provides a pharmaceutical composition comprising at least one mitochondrially-targeted antibiotic as defined above, at least one inhibitor of BCL-2 as defined above, a pharmaceutically acceptable vehicle and optionally at least a further therapeutic agent as defined above for use in the treatment of MYC/BCL2 double positive B-cell lymphomas. Preferably, the MYC/BCL2 double positive B-cell lymphoma is a double-hit lymphoma or a double-expressor lymphoma.

In another embodiment, the present invention provides a kit comprising at least one mitochondrially- targeted antibiotic as defined above, at least one inhibitor of BCL-2 as defined above and optionally at least a further therapeutic agent as defined above for use in the treatment of MYC/BCL2 double positive B-cell lymphomas. Preferably, the MYC/BCL2 double positive B-cell lymphoma is a double-hit lymphoma or a double-expressor lymphoma.

The present invention particularly pertains to a combination of at least one mitochondrially-targeted antibiotic, at least one inhibitor of BCL-2, and optionally at least one additional therapeutic agent useful for separate, simultaneous or sequential administration to a subject in need thereof for treating or preventing a MYC/BCL2 double positive B-cell lymphoma. The present invention also pertains to said combination for use in the preparation of a pharmaceutical composition or medicament for the treatment or prevention of a proliferative disease in a subject in need thereof. In these embodiments of the present invention, said combination is used for the treatment or prevention of a proliferative disease comprising administering to the subject a combination therapy, comprising an effective amount of at least one mitochondrially-targeted antibiotic, an effective amount of at least one inhibitor of BCL-2 and optionally an effective amount additional therapeutic agent. Preferably, the mitochondrially-targeted antibiotic, the inhibitor of BCL-2, and the optional additional therapeutic agent (hereinafter also collectively referred to as "the components" or "the components of the combination") are administered at therapeutically effective dosages which, when combined, provide a beneficial effect. The administration may be separate (e.g. in a chronologically staggered manner, especially a sequence-specific manner), simultaneous or sequential. The present invention further provides a kit, i.e. a commercial package, comprising as therapeutic agents a mitochondrially-targeted antibiotic, an inhibitor of BCL-2 and optionally at least one additional therapeutic agent, together with instructions for simultaneous, separate or sequential administration thereof for use in the delay of progression or treatment of a MYC/BCL2 double positive B-cell lymphoma.

The term "combination", as used herein, may define for example a fixed combination in one dosage unit form for simultaneous administration or a kit of parts for the combined administration where the mitochondrially-targeted antibiotic, the inhibitor of BCL-2 and the optional additional therapeutic agent may be administered independently at the same time (separate administration) or separately within time intervals (sequential administration) that allow that the combination partners show a cooperative, e.g., synergistic, effect.

In particular, in the case that the components are not in the same dosage unit form, e.g. they are in a kit of parts, they can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e., simultaneously or at different time points. The parts of the kit of parts can then e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the mitochondrially-targeted antibiotic to the the inhibitor of BCL-2 and to the optional additional therapeutic agent can be varied, e.g., in order to cope with the needs of a patient sub-population to be treated or the needs of the single patient. Therapeutically effective amounts of the mitochondrially-targeted antibiotic and the inhibitor of BCL-2 and at least one additional therapeutic agent depend on the recipient of the treatment, the disorder being treated and the severity thereof, the composition containing the compound, the time of administration, the route of administration, the duration of treatment, the compound potency, its rate of clearance and whether or not another drug is co -administered. The amount of the mitochondrially-targeted antibiotic and the inhibitor of BCL-2 used to make a composition to be administered daily to a patient in a single dose or in divided doses is from about 0.03 to about 200 mg/kg body weight. Single dose compositions contain these amounts or a combination of submultiples thereof.

Preferably, for the mitochondrially-targeted antibiotic derivative an initial dose of up to 1000 mg may be used, preferably up to 500 mg, followed by up to 250 mg every 12 hours. Intravenous infusion of the mitochondrially-targeted antibiotic derivative may last approximately 30 to 60 minutes and may occur every 12 hrs for days 1 -5, and every 21 days for 2 cycles. The initial dose may be twice as much as the maintenance dose.

The components may be each independently administered, for example, bucally, ophthalmically, orally, osmotically, parenterally (intramuscularly, intraperitoneally intrasternally, intravenously, subcutaneously), rectally, topically, transdermally or vaginally.

The term "combined administration" or "co-administration" as used herein encompasses the administration of the selected therapeutic agents to a single patient, and is intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

In the present invention, a "mitochondrially-targeted antibiotic" means an antibiotic acting through inhibition of mitochondrial biogenesis and/or mitochondrial translation and includes for instance the erythromycins, the tetracyclines, the glycylcyclines, anti-parasitic drugs, and chloramphenicol. In particular, the antibiotic acting through inhibition of mitochondrial translation is a tetracycline. Methods to assess inhibition of mitochondrial biogenesis and/or mitochondrial translation are known in the art. For instance, to assess inhibition of mitochondrial biogenesis and/or mitochondrial translation surrogate markers for WB, IHC may be used as described in D 'Andrea et al, Oncotarget 2016 Nov 8;7(45):72415-72430 and in Skrtic M, et al Cancer Cell 201 1 Nov 15;20(5):674-88. D'Andrea et al, Oncotarget 2016 Nov 8;7(45):72415-72430 refer to analysis of mitochondrial membrane with electronic microscope and the study of OCR (Oxygen consumption rate). Skrtic et al, 2011 use [3H]-leucine incorporation or S-^5 -pulse label as described in Sasarman F, et al. Methods Mol Biol 2012;837:207-17.

For measuring inhibition of mitochondrial biogenesis, it is also possible to use the methods as described in Agnello et al, Cytotechnology 2008 Mar;56(3):145-9 explaining the use of Mitotracker Green (MTG) and Orange (MTO), and Confocal Laser-Scanning Microscope (CLSM), to measure in vivo the mitochondrial mass and activity.

The term "derivative" as used herein refers to any known synthetic, semi-synthetic or natural derivative of a parent agent. In a preferred embodiment, the term "derivative" refers to any agent that can be prepared, at least theoretically, from a parent agent. Preferably, the derivative is structurally related to the parent agent. In the present invention, said parent agent is preferably a mitochondrially- targeted antibiotic or an inhibitor of BCL-2 or an additional therapeutic agent as defined herein.

Semi-synthetic or synthetic derivatives of erythromycin have played an important role in antimicrobial chemotherapy. First generation derivatives such as 2'-esters and acid-addition salts significantly improved the chemical stability and oral bioavailability of erythromycin. A second generation of erythronolide -modified derivatives: roxithromycin, clarithromycin, azithromycin, dirithromycin and fiurithromycin, have been synthesized and have exhibited significant improvements in pharmacokinetic and/or microbiological features. Erythromycin derivatives according to the present invention may be for instance first generation derivatives, such as 2'-esters and acid-addition salts, or second generation erythronolide-modified derivatives, such as roxithromycin, clarithromycin, azithromycin, dirithromycin and fiurithromycin.

Tetracycline derivatives (or "tetracyclines") are a family of broad-spectrum antibiotics. They are a subclass of polyketides. The modifications of naturally occurring tetracyclines, such as chlortetracycline and tetracycline, and the synthesis of novel compounds within the tetracycline family have generated many compounds. Semi-synthetic derivatives of tetracycline include for instance doxycycline and minocycline. The tetracyclines exert their antibiotic effect primarily by binding to the bacterial ribosome and halting protein synthesis. Bacterial ribosomes have high- affinity binding site located on the 30S subunit and multiple low-affinity binding sites located at the 30S and 50S subunits. Upon binding the ribosome, the tetracyclines allosterically inhibit binding of the amino acyl-tR A at the acceptor site (A-site) and protein synthesis ceases.

The expression "tetracycline derivative" as used herein preferably refers to any known synthetic, semi-synthetic or natural derivative of tetracycline. Preferably, the tetracycline derivative according to the present invention belongs to a subclass of polyketides. Preferably, the expression "tetracycline derivative" as used herein means any agent that can be prepared, at least theoretically, from tetracycline. Tetracycline derivatives according to the present invention may exert their effect by binding to the bacterial ribosome and halting protein synthesis, for instance by binding to the bacterial ribosome, preferably at a high-affinity binding site located on the 30S subunit or at a low- affinity binding site located at the 30S or 50S subunit, allosterically inhibiting binding of the amino acyl-tRNA at the acceptor site (A-site) and ceasing protein synthesis.

Glycylcycline derivatives (or "glycylcyclines") are semi-synthetic or synthetic derivatives of any tetracycline as defined above which comprise a glycyl moiety, preferably attached to the 9-position of the tetracycline ring.

The expression "glycylcycline derivative" as used herein preferably refers to any known synthetic, semi-synthetic or natural derivative of glycylcycline. Preferably, the expression "glycylcycline derivative" as used herein means any agent that can be prepared, at least theoretically, from glycylcycline or from a tetracycline or from a tetracycline derivative. In a particularly preferred embodiment, the expression "glycylcycline derivative" refers to an agent that can be prepared, at least theoretically, from glycylcycline or from a tetracycline or from a tetracycline derivative and that comprises a glycyl moiety.

In the present invention, an "anti-parasitic drug" refers anthelmintic drugs, vermifuges, vermicides and the like, which preferably expel parasites from the body by stunning or killing them without causing significant damage to the host. Such anti-parasitic drugs include for instance: Benzimidazoles, Albendazole, Mebendazole, Thiabendazole, Fenbendazole, Triclabendazole, Flubendazole, Abamectin, Diethylcarbamazine, Ivermectin, Suramin, Pyrantel pamoate, Levamisole, Salicylanilides, Niclosamide, Nitazoxanide, Oxyclozanide, Praziquantel, Octadepsipeptides (e.g.: Emodepside), Aminoacetonitrile derivatives e.g., Monepantel, Spiroindoles (e.g., derquantel), Pelletierine sulphate and Artemisinin.

In the present invention, an "anti-parasitic drug" also refers to any known agent useful for preventing and/or treating any parasitic infestation or disease, including for instance malaria, toxoplasmosis, trypanosomiasis, Chagas disease, leishmaniasis, schistosomiasis, amebiasis, giardiasis, clonorchiasis, fasciolopsiasis, lymphatic filariasis, onchocerciasis, thricomoniasis and cestodiasis. A person of ordinary skill in the art would be able to discem which combinations of agents would be useful based on the particular characteristics of the drugs involved.

In particular, existing therapies for malaria include, but are not limited to cloroquine, proguanil, mefloquine, quinine, pyrimethamine-sulphadoxine, doxocycline, berberine, halofantrine, primaquine, atovaquone, pyrimethamine -dapsone, artemisinin and quinhaosu.

Existing therapies for leishmaniasis include, but are not limited to meglumine antimonite, sodium stibogluconate and amphotericin B.

Existing therapies for schistosomiasis include but are not limited to praziquantel and oxamniquine. Preferably, the mitochondrially-targeted antibiotic is administered intravenously. Preferably the mitochondrially-targeted antibiotic derivative is administered every two days. The duration of treatment with the mitochondrially-targeted antibiotic derivative may be 5 to 15 days.

Preferably, the "inhibitor of BCL-2" refers to a potent and highly selective BH3 mimetic antagonist of BCL2 that blocks the anti-apoptotic activity of BCL-2. The BCL2 family proteins share several conserved "BH" domains termed BH1, BH2, BH3 and BH4, as in the inhibitors (BCL-2, BCL-XL, BCL-2 MCL-1 , BFL/A1 , BCL-B), instead the activators (BIM, BID, PUMA) and the sensitizers (BAD, BMF, NOXA) possess only the BH3 domain, and hence are often referred to as "BH3-only" proteins. BH3 mimetics can occupy the inhibitors (anti-apoptotic proteins), preventing them from binding the activators and can induce apoptosis through the intrinsic apoptosis pathway. Preferred BH3 mimetic antagonists are as described in C. Billard, Mol. Cancer Ther., 2013, 9, 1691 -700, incorporated by reference. Examples of BH3 mimetic antagonists according to the present invention include: ABT-199 (also known as venetoclax), ABT-737 and ABT-263 (also known as navitoclax). Preferred inhibitor of BCL-2 is as described in WO 201 1/149492, incorporated by reference.

Preferably, the inhibitor of BCL-2 is administered for at least one cycle of 5 days.

The inhibitor of BCL-2 may be used according to the following dosage/schedule: dosages of 150— 1200 mg given once on days 3 or 7 followed by once daily; continuous daily dosing of 200-900 mg; dosages beginning at 20 mg titrated weekly to 200-600 mg.; 50 to 600 mg daily; Dosing schedules may be as follows: 3, 7, and 28 days/cycle in each 28-day cycle in the dose-escalation portion of the study (see S. Cang et al., Journal of Hematology & Oncology (2015), 8 :129 incorporated by reference).

The mitochondrially-targeted antibiotic, the inhibitor of BCL-2 and/or the additional therapeutic agent may also be present in the combination of the invention as prodrugs, isomers, salts, or solvates. The term "derivative" as used herein comprises prodrugs, isomers, salts, or solvates of the components of the composition.

A prodrug may be a pharmacologically inactive derivative of a biologically active substance (the "parent drug" or "parent molecule") that requires transformation within the body in order to release the active drug, and that has improved delivery properties over the parent drug molecule. The transformation in vivo may be, for example, as the result of some metabolic process, such as chemical or enzymatic hydrolysis of a carboxylic, phosphoric or sulphate ester, or reduction or oxidation of a susceptible functionality. In the present invention such prodrugs may be functional derivatives of the mitochondrially-targeted antibiotic, the inhibitor of BCL-2 and/or the additional therapeutic agent which are readily convertible in vivo into the required agent. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.

The components of the combination of the present invention may exist in different isomeric forms, all of which may be used in the combination and are thus encompassed within the scope of the present invention. The components of the combination of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E.L. Eliel and S.H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 11 19-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, all such stereoisomers being included in the present invention. In addition, the components of the combination disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention.

The components may be included in the combination as a free base, as well as a pharmaceutically acceptable salt or stereoisomer thereof, all of which are encompassed within the scope of the present invention. Some of the components are the protonated salts of amines. In particular, components containing one or more N atoms may be protonated on any one, some or all of the N atoms. The term "free base" refers to the amine compounds in non-salt form. The encompassed pharmaceutically acceptable salts not only include the salts exemplified for the specific agents described herein, but also all the typical pharmaceutically acceptable salts of the free form of the respective component. The free form of the specific salt component described may be isolated using techniques known in the art. For example, the free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate. The free forms may differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise pharmaceutically equivalent to their respective free forms for purposes of the invention.

The pharmaceutically acceptable salts of the components of the instant combination can be synthesized from the individual components which contain a basic or acidic moiety by conventional chemical methods. Generally, the salts of the basic components are prepared either by ion exchange chromatography or by reaction of the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, the salts of the acidic components are formed by reactions with the appropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the components of the composition of the invention include the conventional non-toxic salts of the components as formed by reaction of a basic instant compound with an inorganic or organic acid. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like. More particularly, pharmaceutically acceptable salts of this invention are the tartrate, trifiuoroacetate or the chloride salts.

When the component is acidic, suitable "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N^-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described above, and other typical pharmaceutically acceptable salts is more fully described by Berg et ai, "Pharmaceutical Salts," J. Pharm. ScL, 1977:66:1-19.

The components of the combination of the present invention are potentially internal salts or zwitterions, since under physiological conditions a deprotonated acidic moiety in the compound, such as a carboxyl group, may be anionic, and this electronic charge might then be balanced off internally against the cationic charge of a protonated or alkylated basic moiety, such as a quaternary nitrogen atom.

Still another embodiment pertains to compositions for treating diseases during which are expressed anti-apoptotic Bcl-2 proteins, said compositions comprising an excipient and a therapeutically effective mitochondrially-targeted antibiotic, preferably a tetracycline derivative, and a therapeutically effective amount of the inhibitor of BCL-2 and a therapeutically effective amount of one additional therapeutic agent or more than one additional therapeutic agent.

In the present invention the mitochondrially-targeted antibiotic and the inhibitor of BCL-2 may be administered in a single composition/formulation or as separate compositions. Similarly, the at least one additional therapeutic agent may be administered in a single composition/formulation (with the mitochondrially-targeted antibiotic and/or the inhibitor of BCL-2) or as a separate composition. The mitochondrially-targeted antibiotic and the inhibitor of BCL-2 and at least one additional therapeutic agent may be administered each independently with or without an excipient. Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof.

Excipients for preparation of compositions comprising the mitochondrially-targeted antibiotic and the inhibitor of BCL-2 and at least one additional therapeutic agent to be administered orally in solid dosage form include, for example, agar, alginic acid, aluminum hydroxide, benzyl alcohol, benzyl benzoate, 1 ,3-butylene glycol, carbomers, castor oil, cellulose, cellulose acetate, cocoa butter, corn starch, corn oil, cottonseed oil, cross-povidone, diglycerides, ethanol, ethyl cellulose, ethyl laureate, ethyl oleate, fatty acid esters, gelatin, germ oil, glucose, glycerol, groundnut oil, hydroxypropylmethyl cellulose, isopropanol, isotonic saline, lactose, magnesium hydroxide, magnesium stearate, malt, mannitol, monoglycerides, olive oil, peanut oil, potassium phosphate salts, potato starch, povidone, propylene glycol, Ringer's solution, saffiower oil, sesame oil, sodium carboxymethyl cellulose, sodium phosphate salts, sodium lauryl sulfate, sodium sorbitol, soybean oil, stearic acids, stearyl fumarate, sucrose, surfactants, talc, tragacanth, tetrahydrofurfuryl alcohol, triglycerides, water, and mixtures thereof. Excipients for preparation of compositions comprising the mitochondrially-targeted antibiotic and the inhibitor of BCL-2 and at least one additional therapeutic agent to be administered ophthalmically or orally in liquid dosage forms include, for example, 1,3- butylene glycol, castor oil, corn oil, cottonseed oil, ethanol, fatty acid esters of sorbitan, germ oil, groundnut oil, glycerol, isopropanol, olive oil, polyethylene glycols, propylene glycol, sesame oil, water and mixtures thereof. Excipients for preparation of compositions comprising the mitochondrially-targeted antibiotic and the inhibitor of BCL-2 and at least one additional therapeutic agent to be administered osmotically include, for example, chlorofiuorohydrocarbons, ethanol, water and mixtures thereof.

Excipients for preparation of compositions comprising the mitochondrially-targeted antibiotic derivative and the inhibitor of BCL-2 and at least one additional therapeutic agent to be administered parenterally include, for example, 1 ,3-butanediol, castor oil, corn oil, cottonseed oil, dextrose, germ oil, groundnut oil, liposomes, oleic acid, olive oil, peanut oil, Ringer's solution, saffiower oil, sesame oil, soybean oil, U.S. P. or isotonic sodium chloride solution, water and mixtures thereof. Excipients for preparation of compositions of this invention to be administered rectally or vaginally include, for example, cocoa butter, polyethylene glycol, wax and mixtures thereof.

As used herein, the expression "MYC/BCL2 double positive B-cell lymphoma" refers to a diffuse large B cell lymphoma (DLBCL) and comprises double-hit lymphomas (DHL) and double-expressor lymphomas. The expression "double -hit lymphoma" refers to a high-grade B cell lymphoma with MYC and BCL2 translocations. The expression "double-expressor lymphoma" refers to a DLBCL that shows positivity for the MYC and BCL2 proteins in the absence of the corresponding translacations.

As used herein, the expression "additional therapeutic agent" refers to any agent that is useful in the prevention and/or treatment of a MYC/BCL-2 double positive B-cell lymphoma and includes for example: alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, other apoptosis promoters (for example, Bcl- xL, Bcl-w and Bfi-1) inhibitors, activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic response modifiers, cyclin-dependent kinase (CDK) inhibitors, cell cycle inhibitors, cyclooxygenase-2 (COX- 2) inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapies, immunologicals, inhibitors of inhibitors of apoptosis proteins (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microR A's, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs ( SAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, pololike kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteosome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids, plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, and the like, and the combination of one or more of these agents. The combination of the mitochondrially-targeted antibiotic and the inhibitor of BCL-2 are expected to be useful when used with any ofthese agents. BiTE antibodies are bi-specific antibodies that direct T-cells to attackcancer cells by simultaneously binding the two cells. The T-cell then attacks the target cancer cell.

Examples of BiTE antibodies include adecatumumab (Micromet MT201), blinatumomab (Micromet MT 103) and the like. Without being limited by theory, one of the mechanisms by which T-cells elicit apoptosis of the target cancer cell is by exocytosis of cytolytic granule components, which include perforin and granzyme B. In this regard, Bcl-2 has been shown to attenuate the induction of apoptosis by both perforin and granzyme B. These data suggest that inhibition of Bcl-2 could enhance the cytotoxic effects elicited by T-cells when targeted to cancer cells (V.R. Sutton, D.L. Vaux and J.A. Trapani, J. of Immunology 1997, 158 (12), 5783).

SiRNAs are molecules having endogenous R A bases or chemically modified nucleotides. The modifications do not abolish cellular activity, but rather impart increased stability and/or increased cellular potency. Examples of chemical modifications include phosphorothioate groups, 2'- deoxynucleotide, 2'-OCH3-containing ribonucleotides, 2'-F- ribonucleotides, 2'-methoxyethyl ribonucleotides, combinations thereof and the like. The siR A can have varying lengths (e.g., 10- 200 bps) and structures (e.g., hairpins, single/double strands, bulges, nicks/gaps, mismatches) and are processed in cells to provide active gene silencing. A double-stranded siRNA (dsRNA) can have the same number of nucleotides on each strand (blunt ends) or asymmetric ends (overhangs). The overhang of 1 -2 nucleotides can be present on the sense and/or the antisense strand, as well as present on the 5'- and/ or the 3 '-ends of a given strand. For example, siR As targeting Mcl-1 have been shown to enhance the activity of ABT-263, (i.e., N-(4-(4-((2-(4-chlorophenyl)-5 ,5 -dimethyl- 1 - cyclohex- 1 -en- 1 -yl)methyl)piperazin- 1 -yl)benzoyl)-4-((( 1 R)-3 -(morpholin-4-y 1)- 1 - ((phenylsulfanyl)methyl)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide) or ABT- 737 (i.e., N-(4-(4-((4'-chloro(l,r-biphenyl)-2-yl)methyl)piperazin-l-yl)benzoyl)-4- ((( 1 R)-3 - (dimethylamino)- 1 -((phenylsulfanyl)methyl)propyl)amino)-3 - nitrobenzenesulfonamide) in multiple tumor cell lines (Tse et. al, Cancer Research 2008, 68(9), 3421 and references therein).

Multivalent binding proteins are binding proteins comprising two or more antigen binding sites. Multivalent binding proteins are engineered to have the three or more antigen binding sites and are generally not naturally occurring antibodies. The term "multispecific binding protein" means a binding protein capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins are tetravalent or multivalent binding proteins binding proteins comprising two or more antigen binding sites. Such DVDs may be monospecific (i.e., capable of binding one antigen) or multispecific (i.e., capable of binding two or more antigens). DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to as DVD Ig's. Each half of a DVD Ig comprises a heavy chain DVD polypeptide, a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site. Preferred further combinations include the mitochondrially-targeted antibiotic and the inhibitor of BCL-2 and the combination of CHOP (Cyclophosphamide, doxorubicin (Hydroxydaunomycin), vincristine (Oncovin ®) and Prednisolone) or R-CHOP (the combination of CHOP with the anti- CD20 monoclonal antibody Rituximab).

Alkylating agents include altretamine, AMD-473, AP-5280, apaziquone, bendamustine, brostallicin, busulfan, carboquone, carmustine (BCNU), chlorambucil, CLORETAZINE®(laromustine, VNP 40101M), cyclophosphamide, decarbazine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine (CCNU), mafosfamide, melphalan, mitobronitol, mitolactol, nimustine, nitrogen mustard N-oxide, ranimustine, temozolomide, thiotepa, TREANDA®(bendamustine), treosulfan, rofosfamide and the like.

Angiogenesis inhibitors include endothelial-specific receptor tyrosine kinase (Tie -2) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, insulin growth factor-2 receptor (IGFR-2) inhibitors, matrix metalloproteinase-2 (MMP-2) inhibitors, matrix metalloproteinase-9 (MMP-9) inhibitors, platelet-derived growth factor receptor (PDGFR) inhibitors, thrombospondin analogs, vascular endothelial growth factor receptor tyrosine kinase (VEGFR) inhibitors and the like.

Antimetabolites include ALIMTA® (pemetrexed disodium, LY231514, MTA), 5-azacitidine, XELODA® (capecitabine), carmofur, LEUSTAT®(cladribine), clofarabine, cytarabine, cytarabine ocfosfate, cytosine arabinoside, decitabine, deferoxamine, doxifiuridine, efiornithine, EICAR (5- ethynyl-l-P -D-ribofuranosylimidazole-4- carboxamide), enocitabine, ethnylcytidine, fiudarabine, 5- fluorouracil alone or in combination with leucovorin, GEMZAR® (gemcitabine), hydroxyurea, ALKERAN® (melphalan), mercaptopurine, 6-mercaptopurine riboside, methotrexate, mycophenolic acid, nelarabine, nolatrexed, ocfosfate, pelitrexol, pentostatin, raltitrexed, Ribavirin, triapine, trimetrexate, S-1 , tiazofurin, tegafur, TS-1, vidarabine, UFT and the like. Antivirals include ritonavir, hydroxychloroquine and the like.

Aurora kinase inhibitors include ABT-348, AZD-1 152, MLN-8054, VX-680, Aurora A-specific kinase inhibitors, Aurora B-specific kinase inhibitors and pan- Aurora kinase inhibitors and the like. Bcl-2 protein inhibitors include AT-101 ((-)gossypol), GENASENSE® (G3139 or oblimersen (Bcl- 2 -targeting antisense oligonucleotide)), IPI-194, IPI-565, N-(4-(4-((4'- chloro( 1 , 1 '-biphenyl)-2- yl)methyl)piperazin- 1 -yl)benzoyl)-4-((( 1 R)-3 -(dimethylamino)- 1 ((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide) (ABT-737), N-(4-(4-((2- (4- chlorophenyl)-5 ,5 -dimethyl- 1 -cyclohex- 1 -en- 1 -yl)methyl)piperazin- 1 -yl)benzoyl)-4- ((( 1 R)- 3 -(morpholin-4-yl)- 1 -((phenylsulfanyl)methy l)propyl)amino)-3

((trifluoromethyl)sulfonyl)benzenesulfonamide (ABT-263), GX-070 (obatoclax) and the like. Bcr-Abl kinase inhibitors include DASATINIB® (BMS-354825), GLEEVEC® (imatinib) and the like.

CDK inhibitors include AZD-5438, BMI-1040, BMS-032, BMS-387, CVT-2584, flavopyridol, GPC-286199, MCS-5A, PD0332991, PHA-690509, seliciclib (CYC-202, R-roscovitine), ZK- 304709 and the like.

COX-2 inhibitors include ABT-963 , ARCOXI A® (etoricoxib), BEXTRA® (valdecoxib), BMS347070, CELEBREX® (celecoxib), COX-189 (lumiracoxib), CT-3, DERAMAXX® (deracoxib), JTE-522, 4-methyl-2-(3 ,4-dimethylphenyl)-l-(4- sulfamoylphenyl-lH- pyrrole), MK-663 (etoricoxib), NS-398, parecoxib, RS-57067,

SC-58125, SD-8381 , SVT-2016, S-2474, T-614, VIOXX® (rofecoxib) and the like. EGFR inhibitors include ABX-EGF, anti-EGFR immuno liposomes, EGF-vaccine, EMD-7200, ERBrrUX® (cetuximab), HR3, IgA antibodies, IRESSA® (gefitinib), TARCEVA ® (erlotinib or OSI-774), TP-38, EGFR fusion protein, TYKERB ® (lapatinib) and the like.

ErbB2 receptor inhibitors include CP-724-714, CI- 1033 (canertinib), HERCEPTIN® (trastuzumab), TYKERB® (lapatinib), OMNrTARG® (2C4, petuzumab), TAK- 165, GW-572016 (ionafarnib), GW-282974, EKB-569, PI- 166, dHER2 (HER2 vaccine), APC-8024 (HER-2 vaccine), anti- HER/2neu bispecific antibody, B7.her2IgG3, AS HER2 trifunctional bispecfic antibodies, mAB AR- 209, mAB 2B-1 and the like. Histone deacetylase inhibitors include depsipeptide, LAQ-824, MS- 275, trapoxin, suberoylanilide hydroxamic acid (SAHA), TSA, valproic acid and the like.

HSP-90 inhibitors include 17-AAG-nab, 17-AAG, CNF-101, CNF-1010, CNF-2024, 17-DMAG, geldanamycin, IPI-504, KOS-953, MYCOGRAB® (human recombinant antibody to HSP-90), NCS- 683664, PU24FC1 , PU-3, radicicol, SNX-21 12, STA-9090 VER49009 and the like.

Inhibitors of inhibitors of apoptosis proteins include HGS1029, GDC-0145, GDC- 0152, LCL-161, LBW-242 and the like.

Antibody drug conjugates include anti-CD22-MC-MMAF, anti-CD22-MC-MMAE, anti-CD22- MCC-DM1, CR-011-vcMMAE, PSMA-ADC, MEDI-547, SGN-19Am SGN-35, SGN-75 and the like Activators of death receptor pathway include TRAIL, antibodies or other agents that target TRAIL or death receptors (e.g., DR4 and DR5) such as Apomab, conatumumab, ETR2-ST01 , GDC0145 (lexatumumab), HGS-1029, LBY-135, PRO-1762 and trastuzumab. Kinesin inhibitors include Eg5 inhibitors such as AZD4877, ARRY-520; CENPE inhibitors such as GSK923295A and the like.

JAK-2 inhibitors include CEP-701 (lesaurtinib), XL019 and INCBO 18424 and the like. MEK inhibitors include ARRY-142886, ARRY-438162 PD-325901, PD-98059 and the like.

mTOR inhibitors include AP-23573, CCI-779, everolimus, RAD-001 , rapamycin, temsirolimus, ATP-competitive TORC1/TORC2 inhibitors, including PI-103, PP242, PP30, Torin 1 and the like. Non-steroidal anti-inflammatory drugs include AMIGESIC® (salsalate), DOLOBID® (difiunisal), MOTRIN® (ibuprofen), ORUDIS® (ketoprofen), RELAFEN® (nabumetone), FELDENE® (piroxicam), ibuprofen cream, ALEVE® (naproxen) and NAPROSYN® (naproxen), VOLTAREN® (diclofenac), INDOCIN® (indomethacin), CLINORIL® (sulindac), TOLECTIN® (tolmetin), LODINE® (etodolac), TORADOL® (ketorolac), DAYPRO® (oxaprozin) and the like. PDGFR inhibitors include C-451 , CP-673, CP-868596 and the like.

Platinum chemotherapeutics include cisplatin, ELOXATIN® (oxaliplatin) eptaplatin, lobaplatin, nedaplatin, PARAPLATIN® (carboplatin), satraplatin, picoplatin and the like. Polo-like kinase inhibitors include BI-2536 and the like. Phosphoinositide-3 kinase (PI3K) inhibitors include wortmannin, LY294002, XL- 147, CAL-120, ONC-21, AEZS-127, ETP-45658, PX-866, GDC- 0941 , BGT226, BEZ235, XL765 and the like.Thrombospondin analogs include ABT-510, ABT-567, ABT-898, TSP-1 and the like. VEGFR inhibitors include AVASTIN® (bevacizumab), ABT-869, AEE-788, ANGIOZYME™ (a ribozyme that inhibits angiogenesis (Ribozyme Pharmaceuticals (Boulder, CO.) and Chiron, (Emeryville, CA)) , axitinib (AG- 13736), AZD-2171 , CP-547,632, IM- 862, MACUGEN (pegaptamib), NEXAVAR® (sorafenib, BAY43-9006), pazopanib (GW-786034), vatalanib (PTK-787, ZK-222584), SUTENT® (sunitinib, SU- 11248), VEGF trap, ZACTEVIA™ (vandetanib, ZD-6474) and the like. Antibiotics include intercalating antibiotics aclarubicin, actinomycin D, amrubicin, annamycin, adriamycin, BLENOXANE® (bleomycin), daunorubicin, CAELYX® or MYOCET® (liposomal doxorubicin), elsamitrucin, epirbucin, glarbuicin, ZAVEDOS® (idarubicin), mitomycin C, nemorubicin, neocarzinostatin, peplomycin, pirarubicin, rebeccamycin, stimalamer, streptozocin, VALSTAR® (valrubicin), zinostatin and the like. Topoisomerase inhibitors include aclarubicin, 9-aminocamptothecin, amonafide, amsacrine, becatecarin, belotecan, BN-80915, CAMPTOSAR® (irinotecan hydrochloride), camptothecin, CARDIOXANE® (dexrazoxine), difiomotecan, edotecarin, ELLENCE® or PHARMORUBICIN® (epirubicin), etoposide, exatecan, 10-hydroxycamptothecin, gimatecan, lurtotecan, mitoxantrone, orathecin, pirarbucin, pixantrone, rubitecan, sobuzoxane, SN-38, tafiuposide, topotecan and the like. Antibodies include AVASTIN® (bevacizumab), CD40-specific antibodies, chTNT- 1/B, denosumab, ERBITUX® (cetuximab), HUMAX-CD4® (zanolimumab), IGFlR-speciflc antibodies, lintuzumab, PANOREX® (edrecolomab), RENCAREX® (WX G250), RITUXAN® (rituximab), ticilimumab, trastuzimab, CD20 antibodies types I and II and the like. Hormonal therapies include ARIMIDEX®(anastrozole), AROMASIN®(exemestane), arzoxifene, CASODEX®(bicalutamide), CETROTIDE®(cetrorelix), degarelix, deslorelin, DESOPAN®(trilostane), dexamethasone, DROGENIL®(flutamide), EVISTA®(raloxifene), AFEMA™ (fadrozole),

FARESTON®(toremifene), FASLODEX®(fulvestrant), FEMARA®(letrozole), formestane, glucocorticoids, HECTOROL®(doxercalciferol), RENAGEL®(sevelamer carbonate), lasofoxifene, leuprolide acetate, MEGACE®(megesterol), MIFEPREX®(mifepristone), NILANDRON™ (nilutamide), NOLVADEX®(tamoxifen citrate), PLENAXIS™ (abarelix), prednisone, PROPECIA®(finasteride), rilostane, SUPREFACT®(buserelin), TRELSTAR®(luteinizing hormone releasing hormone (LHRH)), VANTAS®(Histrelin implant), VETORYL®(trilostane or modrastane), ZOLADEX® (fosrelin, goserelin) and the like.

Deltoids and retinoids include seocalcitol (EB1089, CB1093), lexacalcitrol (KH1060), fenretinide, PA RETIN® (aliretinoin), ATRAGEN® (liposomal tretinoin), TARGRETIN®(bexarotene), LGD-1550 and the like.

PARP inhibitors include ABT-888 (veliparib), olaparib, KU-59436, AZD-2281, AG- 014699, BSI- 201, BGP-15, INO-1001, ONO-2231 and the like.

Plant alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, vinorelbine and the like. Proteasome inhibitors include VELCADE®(bortezomib), MG132, NPI-0052, PR-171 and the like.

Examples of immunologicals include interferons and other immune-enhancing agents. Interferons include interferon alpha, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon gamma- la, ACTEVIMU E® (interferon gamma- lb) or interferon gamma-nl, combinations thereof and the like. Other agents include ALFAFERONE®,(IFN-a), BAM- 002 (oxidized glutathione), BEROMU ®(tasonermin), BEXXAR®(tositumomab), CAMPATH®(alemtuzumab), CTLA4 (cytotoxic lymphocyte antigen 4), decarbazine, denileukin, epratuzumab, GRANOCYTE®(lenograstim), lentinan, leukocyte alpha interferon, imiquimod, MDX-010 (anti- CTLA-4), melanoma vaccine, mitumomab, molgramostim, MYLOTARG™ (gemtuzumab ozogamicin), NEUPOGEN®(filgrastim), OncoVAC-CL, OVAREX®(oregovomab), pemtumomab (Y-muHMFGl), PROVENGE®(sipuleucel-T), sargaramostim, sizofilan, teceleukin, THERACYS®(Bacillus Calmette- Guerin), ubenimex, VIRULIZIN®(immunotherapeutic, Lorus Pharmaceuticals), Z-100 (Specific Substance of Maruyama (SSM)), WF- 10 (Tetrachlorodecaoxide (TCDO)), PROLEUKTN®(aldesleukin), ZADAXIN®(thymalfasin), ZENAPAX®(daclizumab), ZEVALIN®(90Y-Ibritumomab tiuxetan) and the like.

Biological response modifiers are agents that modify defense mechanisms of living organisms or biological responses, such as survival, growth or differentiation of tissue cells to direct them to have anti-tumor activity and include krestin, lentinan, sizofiran, picibanil PF- 3512676 (CpG-8954), ubenimex and the like.

Pyrimidine analogs include cytarabine (ara C or Arabinoside C), cytosine arabinoside, doxifiuridine, FLUDARA®(fludarabine), 5-FU (5-fluorouracil), fioxuridine, GEMZAR®(gemcitabine), TOMUDEX®(ratitrexed), TROXATYL™ (triacetyluridine troxacitabine) and the like.

Purine analogs include LANVIS®(thioguanine) and PURI-NETHOL® (mercaptopurine). Antimitotic agents include batabulin, epothilone D (KOS-862), N-(2-((4- hydroxyphenyl)amino)pyridin-3 -yl)-4-methoxybenzenesulfonamide, ixabepilone (BMS 247550), paclitaxel, TAXOTERE®(docetaxel), PNU100940 (109881), patupilone, XRP-9881 (larotaxel), vinflunine, ZK-EPO (synthetic epothilone) and the like.

Ubiquitin ligase inhibitors include MDM2 inhibitors, such as nutlins, NEDD8 inhibitors such as MLN4924 and the like. The combination of this invention can also be used as radiosensitizers that enhance the efficacy of radiotherapy. Examples of radiotherapy include external beam radiotherapy, teletherapy, brachytherapy and sealed, unsealed source radiotherapy and the like.

Additionally, Tigecycline and the inhibitor of BCL-2 maybe combined with other chemotherapeutic agents such as ABRAXANE™ (ABI-007), ABT-100 (farnesyl transferase inhibitor), ADVEXIN®(Ad5CMV-p53 vaccine), ALTOCOR®or MEVACOR®(lovastatin), AMPLIGEN®(poly Lpoly C12U, a synthetic R A), APTOSYN®(exisulind), AREDIA®(pamidronic acid), arglabin, L-asparaginase, atamestane (l-methyl-3,17-dione-androsta- 1,4- diene), AVAGE®(tazarotene), AVE-8062 (combreastatin derivative) BEC2 (mitumomab), cachectin or cachexin (tumor necrosis factor), canvaxin (vaccine), CEAVAC®(cancer vaccine), CELEUK®(celmoleukin), CEPLENE®(histamine dihydrochloride), CERVARIX®(human papillomavirus vaccine), CHOP® (C:CYTOXAN®(cyclophosphamide);H:ADRIAMYCIN®(hydroxy doxorubicin); O: Vincristine (ONCOVIN®); P: prednisone), CYPAT™ (cyproterone acetate), combrestatin A4P, DAB(389)EGF (catalytic and translocation domains of diphtheria toxin fused via a His- Ala linker to human epidermal growth factor) or TransMID-107R™ (diphtheria toxins), dacarbazine, dactinomycin, 5,6- dimethylxanthenone-4-acetic acid (DMXAA), eniluracil, EVIZON™ (squalamine lactate), DIMERICINE®(T4N5 liposome lotion), discodermolide, DX-8951f (exatecan mesylate), enzastaurin, EPO906 (epithilone B), GARDASIL®(quadrivalent human papillomavirus (Types 6, 11 , 16, 18) recombinant vaccine), GASTRIMMU E®, GENASENSE®, GMK (ganglioside conjugate vaccine), GVAX®(prostate cancer vaccine), halofuginone, histerelin, hydroxycarbamide, ibandronic acid, IGN-101, IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox), IL-13- pseudomonas exotoxin, interferon-a, interferon-γ, JUNO VAN™ or MEPACT™ (mifamurtide), lonafarnib, 5, 10-methylenetetrahydro folate, miltefosine (hexadecylphosphocholine), NEOVASTAT®(AE-941 ), NEUTREXIN®(trimetrexate glucuronate), NIPENT®(pentostatin), ONCONASE®(a ribonuclease enzyme), ONCOPHAGE®(melanoma vaccine treatment), ONCOVAX®(IL-2 Vaccine), ORATHECIN™ (rubitecan), OSIDEM®(antibody-based cell drug), OVAREX®MAb (murine monoclonal antibody), paclitaxel, PANDIMEX™ (aglycone saponins from ginseng comprising 20(S)protopanaxadiol (aPPD) and 20(S)protopanaxatriol (aPPT)), panitumumab, PANVAC®-VF (investigational cancer vaccine), pegaspargase, PEG Interferon A, phenoxodiol, procarbazine, rebimastat, REMOVAB®(catumaxomab),

REVLIMID®(lenalidomide), RSR13 (efaproxiral), SOMATULINE®LA (lanreotide), SORIATANE®(acitretin), staurosporine (Streptomyces staurospores), talabostat (PT100), TARGRETIN®(bexarotene), TAXOPREXIN®(DHA-paclitaxel), TELCYTA®(canfosfamide, TLK286), temilifene, TEMODAR®(temozolomide), tesmilifene, thalidomide, THERATOPE®(STn- KLH), thymitaq (2-amino-3,4-dihydro-6-methyl-4-oxo-5-(4- pyridylthio)quinazoline dihydrochloride), TNFERADE™ (adenovector: DNA carrier containing the gene for tumor necrosis factor-a), TRACLEER®or ZAVESCA®(bosentan), tretinoin (Retin-A), tetrandrine, TRISENOX®(arsenic trioxide), VIRULIZIN®, ukrain (derivative of alkaloids from the greater celandine plant), vitaxin (anti-alphavbeta3 antibody), XCYTRIN®(motexafm gadolinium), XINLAY™ (atrasentan), XYOTAX™ (paclitaxel poliglumex), YONDELIS®(trabectedin), ZD- 6126, ZINECARD®(dexrazoxane), ZOMETA®(zolendronic acid), zorubicin and the like.

In the present invention the term "effective amount" shall mean an amount which achieves a desired effect or therapeutic effect as such effect is understood by those of ordinary skill in the art.

The term "pharmaceutical composition/formulation" is defined herein to refer to a mixture or solution containing at least one therapeutic agent to be administered to a subject, e.g., a mammal or human, in order to prevent or treat a particular disease or condition affecting the mammal. The term "pharmaceutically acceptable" is defined herein to refer to those compounds, materials, compositions and/or dosage forms, which are, within the scope of sound medical judgment, suitable for contact with the tissues a subject, e.g., a mammal or human, without excessive toxicity, irritation allergic response and other problem complications commensurate with a reasonable benefit / risk ratio. Pharmaceutical compositions containing the mitochondrially-targeted antibiotic, the inhibitor of BCL-2 and optionally at least one additional therapeutic agent of the present invention may be manufactured by processes well known in the art, e.g., using a variety of well-known mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The compositions may be formulated in conjunction with one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Parenteral routes are preferred in many aspects of the invention. The pharmaceutical compositions can be chosen on the basis of the treatment requirements. Such compositions are prepared by blending and are suitably adapted to oral or parenteral administration, and as such can be administered in the form of tablets, capsules, oral preparations, powders, granules, pills, injectable, or infusible liquid solutions, suspensions, or suppositories.

Tablets and capsules for oral administration are normally presented in unit dose form and contain conventional excipients such as binders, fillers (including cellulose, mannitol, lactose), diluents, tableting agents, lubricants (including magnesium stearate), detergents, disintegrants (e.g. polyvinylpyrrolidone and starch derivatives such as sodium glycolate starch), coloring agents, flavoring agents, and wetting agents (for example sodium lauryl sulfate).

The oral solid compositions can be prepared by conventional methods of blending, filling or tableting. The blending operation can be repeated to distribute the active principle throughout compositions containing large quantities of fillers. Such operations are conventional.

Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as a dry product for reconstitution with water or with a suitable vehicle before use. Such liquid preparations can contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel, or hydrogenated edible fats; emulsifying agents, such as lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which can include edible oils), such as almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, such as methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired, conventional flavoring or coloring agents. Oral formulations also include conventional slow-release formulations such as enterically coated tablets or granules.

Pharmaceutical preparation for administration by inhalation can be delivered from an insufflator or a nebulizer pressurized pack.

For parenteral administration fluid unit dosages can be prepared, containing the combination or the components of the combination and a sterile vehicle. The combination or the components of the combination can be either suspended or dissolved, depending on the vehicle and concentration. The parenteral solutions are normally prepared by dissolving the combination or the components of the combination in a vehicle, sterilising by filtration, filling suitable vials and sealing. Advantageously, adjuvants such as local anaesthetics, preservatives and buffering agents can also be dissolved in the vehicle. To increase the stability, the composition can be frozen after having filled the vials and removed the water under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the combination or the components of the combination can be suspended in the vehicle instead of being dissolved and sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the combination or the components of the combination of the invention.

For buccal or sublingual administration, the compositions may be tablets, lozenges, pastilles, or gel. The combination or the components of the combination can be pharmaceutically formulated as suppositories or retention enemas, e.g. containing conventional suppositories bases such as cocoa butter, polyethylene glycol, or other glycerides, for a rectal administration.

Another means of administering the combination or the components of the combination of the invention regards topical treatment. Topical formulations can contain for example ointments, creams, lotions, gels, solutions, pastes and/or can contain liposomes, micelles and/or microspheres. Examples of ointments include oleaginous ointments such as vegetable oils, animal fats, semisolid hydrocarbons, emulsifiable ointments such as hydroxystearin sulfate, anhydrous lanolin, hydrophilic petrolatum, cetyl alcohol, glycerol monostearate, stearic acid, water soluble ointments containing polyethylene glycols of various molecular weights. Creams, as known to formulation experts, are viscous liquids or semisolid emulsions, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase generally contains petrolatum and an alcohol such as cetyl or stearic alcohol. Formulations suitable for topical administration to the eye also include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.

A further method of administering the combination or the components of the combination of the invention regards transdermal delivery. Typical transdermal formulations comprise conventional aqueous and non-aqueous vectors, such as creams, oils, lotions or pastes or can be in the form of membranes or medicated patches.

A reference for the formulations is the book by Remington ("Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins, 2000).

The term "treating" or "treatment" as used herein comprises a treatment relieving, reducing or alleviating at least one symptom in a subject or effecting a delay of progression of a disease. For example, treatment can be the diminishment of one or several symptoms of a disorder or complete eradication of a disorder. Within the meaning of the present invention, the term "treat" also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.

It is contemplated that the treatment will be given for one or more cycles until the desired clinical and biological result is obtained. The exact amount, frequency and period of administration of the combination of the present invention will vary, of course, depending upon the sex, age and medical condition of the patient as well as the severity and type of the disease as determined by the attending clinician.

Still further aspects include combining the therapy described herein with other anticancer therapies, such as radiotherapy, for synergistic or additive benefit.

The schedule of treatment with the combinations can foresee that mitochondrially-targeted antibiotic is administered concomitantly, before and/or after the inhibitor of BCL-2 identified above and optionally before and/or after the at least one additional agent as above indicated.

The present invention will be illustrated by means of non-limiting examples in reference to the following figures.

Fig. 1. Blockade of tigecycline-induced cell death by BCL2 in Εμ-myc lymphomas and its restoration by venetoclax. Two mouse Εμ-myc lymphoma cell lines, one wild-type (p53-wt) and one null (p53-null) for the Trp53 locus were infected with a recombinant retrovirus encoding human BCL2 or with the corresponding empty vector (EV). After selection, the infected cells were treated with tigecycline and/or venetoclax at the indicated concentrations. Total and viable cell counts were determined by Trypan blue exclusion 48 hours after addition of the drugs to the culture medium. (A) The viability of cells expressing BCL2 or empty vector is presented as the % of live relative to total cells (live + dead) in each culture. (B) The proliferative index of cells expressing BCL2 or empty vector is presented as the increase in viable cell counts over 48 hours, normalized to the corresponding increase in parallel untreated cultures (4 population doublings for the p53-wt cells with either EV or BCL2; 3.5 and 2 doublings for the p53-null cells with EV and BCL2, respectively). (C) Cell viability of ^ i-myc/BCL2 lymphomas in the presence or absence of venetoclax. (D) Cell viability of ^ i-myc/BCL2 lymphomas in the presence or absence of tigecycline and/or venetoclax. All data points represent the average ± s.d. from 3 independent experiments.

Fig. 2. Induction of cell death by tigecycline and venetoclax in human MYC/BCL2 double-hit lymphomas. (A and B) MYC and BCL2 mR As were quantified by RT-PCR with normalization to the RPLPO mR A (n = 3 technical replicates for each group) (A) or immunoblotting (B) in the indicated cell lines (Karpas-442, SU-DHL-6, DOHH-2, SU-DHL-4, OCI-LY8, OCI-LY7). The expression data shown here are consistent with the known status of MYC or BCL2 translocations in those cell lines, as indicated at the bottom. Note that in SU-DHL-6 cells, somatic mutations in BCL2 impair its recognition by the OP60 antibody (n.a.) (65), but the protein is readily detected by antibody El 7. (C) Cell viability in the indicated cell lines in the presence or absence of ABT-199 (Venetoclax) was determined as in Fig. l (D) Cultures were treated with tigecycline and/or venetoclax at the indicated concentrations, and cell viability was determined as in Fig. 1.

Fig. 3. Therapeutic activity of tigecycline and venetoclax in DHL-xenografted mice. (A) Schematic representation of the general strategy, followed in panel B (B) The human cell lines SU- DHL-6, DOHH-2, OCI-LY8 (all with MYC and BCL2 translocations), or OCI-LY7 (MYC translocation only) were xenografted subcutaneously in CD 1 -nude mice. After about 2 weeks, tumor- bearing animals were randomized and treated with the indicated doses of tigecycline and/or venetoclax over a period of 12 days. Tumor volumes were measured at the indicated time points after treatment. For DOHH-2, OCI-LY8, and OCI-LY7, tumor growth is shown at every time point until termination. For SU-DHL6 tumors, only days 0-19 are included in the graph: of the eight animals treated with tigecycline + venetoclax, six reached the 120-day endpoint tumor-free, and two developed tumors and were sacrificed at day 50 (table 1). Error bars represent standard deviations; " p = 5xl0^"3; ⁺ p < 5xl0^"4; * p < 10^"5; ns: not significant.

Fig. 4. Therapeutic activity of tigecycline and venetoclax in a DHL patient-derived xenograft. The PDX line DFBL-69487-V3-mCLP, expressing luciferase, was expanded by intravenous injection in NSG mice. 15 days after seeding (day 0), tumor development was monitored by whole- body imaging: animals were randomized (5 animals/group), subjected to treatment with 75 mg/kg tigecycline and/or 50 mg/kg venetoclax over a period of 12 days (days 1 -12), and monitored by whole -body imaging at the indicated time points (before, during, and after treatment). (A) Tumor progression in the indicated groups of animals was followed by radiant efficiency, quantified over both femurs. (B) IVIS imaging (ventral and lateral) of all mice in the control (untreated) and tigecycline+venetoclax groups, shown at day 1 1. Other groups and time points are shown in fig. 12. Arrowheads point to the accumulation of tumor cells in the femurs (f.) and vertebrae (v.). The circles and rectangles appearing over the photographs define the areas that were used for quantification of luminescence over the femur and the corresponding values, respectively.

Fig. 5. Response of mouse Li-myclBCL2 lymphomas to tigecycline and venetoclax. (A) DNA- content histograms and (B) RT-PCR measurement of Cdknla mR A expression (normalized to the RplpO mRNA) after treatment of

lymphoma lines with 100 μΜ tigecycline for 72 hours. The RT-PCR results are shown as mean ± s.d. of technical triplicates. (C) Senescence- associated beta-galactosidase (SA-Bgal) staining after a 5 -day treatment with 100 μΜ tigecycline or 100 nM doxorubicin, the latter serving as a positive control for the induction of senescence in Εμ- myc/BCL2 lymphomas (20). Data are shown as % of positive cells (mean ± s.d.) from counts in 4 different fields. (D) Drug interaction landscapes and delta scores for synergy, based on the ZIP model in SynergyFinder (64), derived from the data of Fig. ID. The landscapes identify the specific dose regions where there is a synergistic (red) or antagonistic (green) drug interaction. Dashed black-and- white squares within the landscapes mark the regions of maximal synergy. A positive delta score signifies a synergistic interaction, while a score near 0 indicates that the drugs are not interactive. (E) Cell viability, determined as in Fig. 1 , after combined treatment with tigecycline and venetoclax as indicated, in Εμ-myc p53 null cells infected with EV, BCL2, or BCL-W retroviruses. The EV and BCL2 cultures in this experiment are derived from a series of infections distinct from those used in Figure ID.

Fig. 6. Synergistic effects of tigecycline and venetoclax against human DHL cell lines. Drug interaction landscapes and delta scores (as defined in fig. 5D) were derived from the data of Fig. 2D. Fig. 7. Similar effects of tigecycline, doxycycline, and tetracycline in DHL cells. (A) Immunoblot analysis of the mitochondrial proteins COX1 , COX2 (encoded by the mitochondrial genome), and SDHA (encoded in the nucleus) in SU-DHL-6 cells after 48 hours of treatment with 5 μΜ of tigecycline (tig), doxycycline (doxy), or tetracycline (tet). (B) Oxygen consumption rate (OCR), determined by polarographic analysis of SU-DHL-6 cells following the same treatments as in A. The data were normalized to total number of cells. (C-D) Cell viability (C, determined as in Fig. 1) and drug interaction landscapes (D, as defined in fig. 5D) after treatment of SU-DHL-6 cells with tigecycline, doxycycline, or tetracycline in combination with venetoclax, as indicated. The experiments were performed and analyzed as described in Figures 2D and 5D.

Fig. 8. Short-term effects of tigecycline and venetoclax in SU-DHL-6 tumors. Immunohistochemical analysis of subcutaneous SU-DHL-6 tumors collected 24 hours after a single treatment with the indicated drugs, stained with primary antibodies against cleaved caspase 3 (CC3) or Ki67. H&E: hematoxylin-eosin staining. Scale bars: 100 μηι.

Fig. 9. Kaplan-Meier representation of disease-free survival in PDX recipient mice. Animals bearing PDX-derived tumors and exposed to the indicated treatments (the same shown in Fig. 4) were sacrificed at the first observable signs of disease, including hind-limb paralysis and hunched posture.

Fig. 10. Reactive liver lesions caused by intraperitoneal delivery of tigecycline. CD 1 -nude mice bearing subcutaneous SU-DHL-6 tumors were treated with repeated doses of tigecycline delivered by either intraperitoneal (LP., bi-daily) or intravenous injection (I.V., every three days) for two weeks. (A) Liver morphology in mice sacrificed after the indicated treatments. Compacted liver lobes were observed in animals treated with tigecycline alone or in combination with venetoclax. (B) Hematoxylin-eosin stained sections show marked changes in the structure of the peritoneal membrane and liver tissue in mice after IP- but not IV-based delivery of tigecycline. In particular, the inventors observed a diffuse hyperplasia of mesothelial cells that were organized in irregular buds and papillae in the peritoneal membrane and a thickening of the liver capsule with areas of neutrophilic infiltration. In the liver, the lobular architecture was maintained, with diffuse centrilobular hepatocellular hypertrophy and hyperplastic foci. However, typical signs of liver toxicity (hepatocellular necrosis, periportal inflammatory infiltrates, or fibrosis) were absent, indicating that the observed lesions were reactive in nature. Scale bars: 100 μπι. (C) Tumor volumes after I.V. or LP. injection of tigecycline, as indicated. (D) Peripheral blood profiles and (E) alanine aminotransferase activity (in units/L) after treatment (day 12). Cell profiles are shown for white and red blood cells (as cells/ μί), as well as neutrophils, monocytes and lymphocytes (as % of total white blood cells), as indicated, n = 3 mice per group.

Fig. 11. Toxicology of tigecycline and venetoclax drug combination studies in vivo. Gross organ histology of nude mice treated with tigecycline (75 mg/kg twice a day by LP. injection) and/or venetoclax (50 mg/kg daily by oral gavage) or vehicle control. (A) Kidney, (B) heart, (C) spleen, (D) brain and (E) liver stained with hematoxylin and eosin are shown (scale bars: 100 μηι). Overall, the organs are normal with neither neoplastic nor inflammatory infiltrates, with the exception of animals treated with tigecycline (alone or in combination with venetoclax), in which the inventors observed changes in the liver and spleen. In the liver, the inventors noticed capsular fibrosis with subcapsular diffuse minimal inflammatory neutrophilic infiltrates, with a diffuse hydropic degeneration of hepatocyte cytoplasm and increased mitoses. In the spleen, the white pulp was expanded by increased numbers of periarteriolar lymph sheath (PALS) lymphocytes. The inventors also observed marginal zone atrophy, with a marked spleen hyperplasia in red pulp with diffuse neutrophilic inflammatory infiltrate.

Fig. 12. Whole-body imaging of PDX-derived tumors treated with tigecycline and/or venetoclax. A cohort of PDX-bearing mice was randomized and subjected to treatment with tigecycline and/or venetoclax, as indicated. IVIS images (ventral view) are shown here for all mice and time -points in the experiment. Note that the animals in the untreated and tigecycline + venetoclax groups are also shown in Fig. 4B at day 1 1. * Indicates that the animal died following anesthesia. Other missing animals succumbed to excessive tumor burden. Surviving animals were sacrificed upon disease manifestation. Note that the spleen was detectable only at advanced stages and in a fraction of the animals (day 19, sp.), suggesting that the PDX used here has poor tropism for this organ, and that no infiltration was detected in lymphnodes, which are underdeveloped in the NSG strain used as a recipient.

Fig. 13. Effect of rituximab with tigecycline and/or venetoclax on DHL xenografts. (A)

Schematic representation of the general strategy and treatment schedule. (B-C) Tumors derived from the indicated DHL cell lines (B) and the PDX (C) were treated as detailed in Figures 3 and 4, respectively. Where indicated, 10 mg/kg of rituximab was added to the treatment with a single intravenous injection on day 1. Control treatments with tigecycline or venetoclax alone are provided in Fig. 3 (for SU-DHL-6 and DOHH-2 tumors) and Fig. 4 (for PDX tumors). The IVIS images shown in (D) include all mice in the rituximab-only, as well as in the triple combination groups, shown at day 11. Other groups and time -points are shown in fig. 14. (E) Side -by-side quantifications of the effects of tigecycline + venetoclax with and without rituximab. * p = 2 x 10-4; ** p = 2 x 10-6; # p=0.05; $ p=5 x 10-5; + p < 10-6.

Fig. 14. Whole-body imaging of PDX-derived tumors treated with rituximab, tigecycline and/or venetoclax. The groups of mice shown here belong to the same cohort as in fig. 12 and were treated with rituximab in addition to tigecycline and/ or venetoclax, as indicated. Note that the animals in the rituximab and in the triple combination groups are also shown in fig. 13B at day 1 1.

Fig. 15 (A). Schematic summary of the nucleus-mitochondrial interaction. Oncogenic activation of Myc sensitizes to inhibition of the mitochondrial ribosome by tigecycline. (B). Kapan-Meier survival curve of Eu-Myc lymphoma-transplanted mice treated with tigecycline or vehicle (saline solution) (16).

Fig. 16. The human cell lines SU-DHL-6 (with MYC and BCL2 translocations), or OCI-LY7 (MYC translocation only) were xenografted subcutaneously in CD 1 -nude mice. After about 2 weeks, tumor- bearing animals were randomized and treated with the indicated doses of tigecycline and/or venetoclax over a period of 12 days. Tumor volumes were measured at the indicated time points after treatment. For OCI-LY7, tumor growth is shown at every time point until termination. For SU-DHL6 tumors, only days 0-19 are included in the graph: of the eight animals treated with tigecycline + venetoclax, six reached the 120-day endpoint tumor- free, and two developed tumors and were sacrificed at day 50 (table 1). Error bars represent standard deviations; " p = 5xl0^"3; ⁺ p < 5xl0^"4; * p < 10^"5; ns: not significant.

Detailed description of the invention

Materials and Methods

Study design

The objective of the inventors' study was to address the therapeutic interaction between at least one mitochondrially-targeted antibiotic and at least one inhibitor of BCL-2, for instance tigecycline and venetoclax, against MYC/BCL2 double-hit B-cell lymphoma (DHL) in a preclinical setting. For in vivo pharmacological studies, human DHL cell lines: SUDHL6 (ATCC CRL-2959), DOHH2 (D5M2 ACC-47), OCI-LY8 (CVCL-8803), OCI-LY7 (DSMZ ACC-688) and PDX (PDX are Patient Derived Xenografts: DFBL-69487 acquired from Proxe) were xeno-transplanted in female CD1- nude (086-CD1-Foxnl^nu Charles River Laboratories) and NSG mice (005557-NOD.Cg- Prkdc^scldI12rg^Tmlwjl/SzJ Charles River Lab), respectively. Tumors were allowed to develop to measurable sizes (about 2 weeks), followed by exclusion of outliers, blinded randomization of the experimental groups (day 0), and treatment (days 1 -12), as described in detail below. Although the number of biological replicates achieved depended upon the efficiency of engraftment and tumor growth, no fewer than 4-5 animals were included in any experimental group (with the exception of fig. IOC), to ensure statistical power. The endpoint of the experiment for subcutaneous tumors (with the DHL cell lines) was either a tumor size of 20 mm in any one dimension, or ulceration at any tumor size. For PDX-derived tumors, the endpoint was the first signs of disease manifestation (hind- limb paralysis, hunched posture).

Cell lines

The Εμ-myc p53-wt and p53-null lymphoma lines used here, LY27 and LY35, respectively, were the same as in the inventors' previous work and were cultured as described therein (16). The human lymphoma lines DOHH-2 and KARPAS-422 (CVCL-1325) were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1 % penicillin/streptomycin. The lines SU-DHL- 4, SU-DHL-6, OCI-LY7, and OCI-LY8 were cultured in IMDM (Iscove's modified Dulbecco's medium) with the same supplements. The information on BCL2 and MYC translocation reported in Fig. 2B was obtained from either ATCC (http://www.lgcstandards- atcc.org/Products/Cells_and_Microorganisms/Cell_Lines/Human.aspx) or DSMZ

(https://www.dsmz.de/catalogues/catalogue-human-and-animal-cell-lines.html) (62). Tigecycline (220620-09-7), doxorubicin (25316-40-9), venetoclax (1257044-40-8), Tetracycline (64-75-5) (all from Carbosynth) were dissolved in DMSO and added directly to the culture medium. Staining for senescence-associated beta-galactosidase activity (SA-Bgal) was performed as described previously (63). Viable cell numbers were assessed by Trypan blue exclusion with an automated cell counter (TC20, Bio-rad), following the manufacturer's instructions. Oxygen consumption of cultivated cells was determined by polarographic analysis using a Clark type Hansateck Oxygraph in a 1 ml chamber. Drug synergies were evaluated using the ZIP model in SynergyFinder (64).

Immunoblot and immunohistological analysis

Immunoblot and immunohistological analysis were performed as described (16). The following primary antibodies were used for immunoblotting: mouse monoclonal anti-vinculin (Abeam, cat. #abl 8058); mouse monoclonal anti-COXl (Abeam, cat. # abl4705); mouse monoclonal anti-COX2 (Invitrogen, cat. #12C4F12); mouse monoclonal anti-SDHA (Invitrogen, cat. #459200); rabbit monoclonal anti-MYC Y69 (Abeam, cat. #GR144732-28); rabbit monoclonal anti-BCL2 E17 (Abeam, cat. #ab32124); mouse monoclonal anti-BCL2 [OP60] (Calbiochem). The failure of the OP60 antibody to recognize BCL2 in SU-DHL-6 cells (Fig. 2B) has been reported previously (65). For immunohistological detection of Ki-67 and cleaved caspase-3, the inventors used antibodies #M7249 (Dako) and #9661 (Cell Signaling), respectively.

RNA extraction and analysis

Total RNA was purified and analyzed by RT-PCR as described (16), with the following primers: - human MYC:

CTAGATGGCATCATTCTTCC (Seq. ID No. 1) and GAAACTGGGCAAACAACAC (Seq. ID No. 2)

- human BCL2:

CACGCTGGGAGAACAGGGTA (Seq. ID No. 3) and GGATGTACTTCATCACTATCTCCCG (Seq. ID No. 4)

- mouse Cdknla:

CT GGG AGGGG AC AAG AG (Seq. ID No. 5) and GCTT GGAGTGAT AGAAAT CTG (Seq. ID No. 6)

- human and mouse RPLP0:

TTCATTGTGGGAGCAGAC (Seq. ID No. 7) and CAGCAGTTTCTCCAGAGC (Seq. ID No. 8)

Flow cytometry

For DNA content analysis, cells were fixed with 70% EtOH, resuspended in PBS with 50 μg/ml propidium iodide (PI) and 40 μg/ml RNAse A, and stained overnight at 4°C in the dark. Samples were acquired on a FACSCalibur flow cytometer (Becton Dickinson).

Peripheral blood profiles

For peripheral blood analysis, 50 μΐ of blood were collected by tail vein bleeding, and 10 μΐ 0.5 M EDTA was immediately added to prevent coagulation. Whole blood was analyzed using a Hematological Analyzer (Beckman Dickinson). Alanine aminotransferase activity was assayed with a specific kit (Sigma, MAK052).

Xenograft models of human DLBCL

10⁶ SU-DHL-6, DOHH-2, OCI-LY8, or OCI-LY7 cells were transplanted subcutaneously in irradiated (3 Gray) CD 1 -nude nu/nu female mice. Treatment started upon the appearance of measurable tumors. Tumor volumes were assessed from the start of the treatment every two days with a digital caliper and calculated as l/2(lengthxwidth²) (66). The following treatment schemes were used: twice a day intraperitoneal injection of tigecycline (spaced by ~8 hours) and/or daily oral gavage with venetoclax for 5 days, followed by two days off and a repeat of the same scheme, for a total of 12 days. A single intravenous injection of rituximab (Mabthera) 10 mg/kg on day 0 of the 12-days treatment scheme was used for the experiment in fig. 13. For the experiment in fig. 10B and C, tigecycline was delivered with a single intravenous injection every 3 days, for a total of 12 days. Tigecycline was dissolved in saline solution (0.9% NaCl); venetoclax was dissolved in 60% Phosal 50 PG (Lipoid), 30% polyethylene glycol (Aldrich), 10% ethanol. Solutions were freshly prepared from dry powder just before each injection.

The DHL PDX line DFBL-69487-V3-mCLP (33) was acquired from the Public Repository of Xenografts (www.proxe.org). 10⁶ cells were xenografted via tail vein injection into 6- to 8-week-old female NSG mice. The animals were monitored once a week by whole body imaging on an rVTS Lumina III platform after intraperitoneal injection of 150 mg/kg XenoLight D-Luciferin (PerkinElmer #122799) and anesthesia with isoflurane. The data were analyzed with the Living Image Software, version 4.2 (Caliper Life Sciences). Radiant efficiency was calculated based on the epifiuorescence signal, as indicated in the user manual.

Experiments involving animals were done in accordance with the Italian Law D.lgs. 26/2014, which enforces Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.

Statistics

All data are presented as the mean ± SD. Statistical analyses were performed by unpaired two-tailed Student's t-test. Differences were considered statistically significant atp < 0.05. Statistical evaluation of survival curves was performed using multiple t-tests for each time point, and the Holm- Sidak method.

Results

Tigecycline-induced apoptosis is suppressed by BCL2 and restored by venetoclax

Tigecyc line-induced cell death in mouse Εμ-myc lymphoma cell lines (16) was suppressed upon infection of the cells with a BCL2 -expressing retrovirus (Fig. 1 A). In these conditions, tigecycline suppressed proliferation, with concomitant Gl arrest and induction of the cell cycle-inhibitory gene Cdknla (Fig. IB and fig. 5, A and B). This antiproliferative effect was independent from the p53 status of the tumors and was not associated with appearance of the senescence-associated marker SA-Bgal, unlike observed with doxorubicin (20) (fig. 5C). Thus, BCL2 blocked the apoptotic effect of tigecycline without affecting its cytostatic action.

As seen with tigecycline, E i-myclBCL2 lymphomas were resistant to venetoclax alone (Fig. 1C), but were killed by the two drugs together, with strong dose-dependent and synergistic effects (Fig. ID, fig. 5D). This combined toxicity was p53 -independent, as previously shown for tigecycline- induced cell death (16). The action of venetoclax was on-target, as Εμ-myc lymphomas expressing BCL-W, a BCL2-related anti-apoptotic protein that is not inhibited by venetoclax (8), were resistant to the combined treatment (fig. 5E). Hence, venetoclax specifically restored tigecycline-induced cell death in ^ i-myclBCL2 lymphomas.

Tigecycline and venetoclax cooperate in killing human DHL cell lines

The inventors then addressed the effect of the drugs on five human MYC/BCL2 DHL cell lines, in which the inventors first confirmed the expression of either oncogene (Fig. 2, A and B). In three of these lines (Karpas-422, SU-DHL-6, andDOHH-2), neither tigecycline norvenetoclax alone showed significant pro-apoptotic activity, but the two drugs synergized when combined (Fig. 2C, 2D, fig. 6). In two others (SU-DHL-4 and OCI-LY8), venetoclax alone showed dose-dependent toxicity, but this was still enhanced by the addition of tigecycline. Finally, a DLBCL line with activation of MYC but not BCL2 (OCI-LY7) was fully resistant to the combination, implying that the effect of venetoclax was once again on-target (and incidentally that OCI-LY7 cells must carry a different anti-apoptotic lesion). As in Fig. 5D, the results shown in Fig. 6 demonstrate that Tigecycline and venetoclax cooperate in killing human DHL cell lines. Thus, as in mouse ^ i-myclY$ L2 lymphomas, tigecycline and venetoclax cooperated in killing human DHL lines.

The toxicity of tigecycline and related antibiotics to tumor cells (either rodent or human) has been associated with their inhibitory effect on mitochondrial translation and, hence, on respiratory activity (16, 18, 21-31). Consistent with this notion, treatment of SU-DHL-6 cells with tigecycline, doxy eye line, or tetracycline suppressed expression of the mitochondrion-encoded electron transport chain complex (ETC) IV subunits COX1 (or MTCOl) and COX2 (or MTC02), but not of the nucleus-encoded ETC II subunit SDHA (fig. 7A), and lowered oxygen consumption (fig. 7B). Finally, doxy eye line and tetracycline also synergized with venetoclax in killing SU-DHL-6 cells (fig. 7C, fig, 7D). Hence, concomitant inhibition of mitochondrial translation and of the pro-survival function of BCL2 allows effective killing of DHL cells.

Tigecycline and venetoclax allow eradication of DHL-derived tumors in mice

To address the therapeutic potential of tigecycline and venetoclax, the inventors xenografted the human DHL cell lines SU-DHL-6, DOHH-2, OCI-LY8 and OCI-LY7 in CDl-nude mice, let tumors develop for 2 weeks, and initiated treatment. Either drug alone partially slowed down tumor progression, but their combination showed strong anti-tumoral activity, causing either full regression (8/8 for SU-DHL-6; 3/9 for OCI-LY8) or stasis within the 12 days of treatment (Fig. 3B). The contribution of venetoclax was again on-target, as BCL2-negative OCI-LY7 tumors were resistant to the combination. Immunohistochemical analysis of SU-DHL-6 tumors after 1 day of treatment with the combined drugs showed decreased cell cohesiveness and intra-tumoral necrosis, associated with proliferative arrest and apoptosis (fig. 8). Follow-up of the animals upon cessation of treatment showed that OCI-LY8 and DOHH-2 tumors regrew over relatively short periods of time, requiring sacrifice of the animals within about 1 month (Fig. 3, Table 1).

Instead, most of the SU-DHL-6-xenografted animals remained tumor- free until the inventors' preset endpoint (120 days) and could thus be considered as cured (Table 1).

Table 1. Summary of all treatments performed on mice with SU-DHL-6-derived tumors

Column "n. mice (died < lw)" is the total numbers of mice treated and, in parentheses, those that died within the first week. The causes for these early deaths remain unclear and may be due to the overall experimental burden imposed on the animals; surviving animals showed no signs of distress. Regression: percentages of animals scored as showing tumor regression at the indicated time points, with the scored/total numbers in parentheses. At day 19, partial and complete regressions were defined as residual tumor volumes <50% and <10% of the initial volume, respectively. Animals showing complete regression at day 19 were followed until day 120 and sacrificed when tumors reached a diameter of 1.5 cm. Animals reaching the endpoint of 120 days had no detectable residual tumor. Compacted liver: "yes" indicates that post-mortem examination revealed a compacted liver lobule morphology. Mice sacrificed either after long-term survival with the highest doses of the combined drugs (day 120), or after short-term treatment with tigecycline alone (day 19) revealed a compacted liver morphology (Table 1 , fig. 10A, day 120). Histological analysis (performed at day 19) showed that this morphological change was associated with a thickening of the liver capsula and peritoneal membrane (fig. 10B). This was most likely a reactive lesion secondary to the twice a day intraperitoneal (LP.) injection of tigecycline, because it was absent after intravenous (I.V.) injection, the relevant route in the clinic (32), whereas both modalities were equally effective in slowing down tumor progression (fig. IOC). Peripheral blood profiles revealed no significant alterations in cell populations in treated vs. untreated animals (at day 12, fig. 10D). Combined treatment caused an increase in alanine aminotransferase activity (fig. 10E) consistent with liver injury, albeit without overt signs of toxicity such as necrosis or fibrosis (fig. 10B). Treated animals were overall in good health, histopathological analysis showing no alterations in major organs, with the exception of inflammatory infiltrates in the liver and spleen in the presence of tigecycline (alone or in combination) (fig. 1 1). Finally, the inventors note that a fraction of the mice treated with high doses of the combined drugs died within one week (Table 1). Although the causes underlying this effect remain to be addressed, lower dosage (75 mg/kg tigecycline and 50 mg/kg venetoclax) showed no toxicity yet retained substantial anti -tumoral effects.

Tigecycline and venetoclax synergize against a DHL patient-derived xenograft

A recent study described the establishment of DLBCL patient-derived xenografts (PDXs), some of which were classified as DHL (33). The inventors expanded one of these PDX lines (DFBL-69487- V3-mCLP, expressing luciferase) in NSG mice, and assessed its response to treatment. Cell suspensions (lxlO⁶) were transferred intravenously into recipient mice, and tumor development was monitored by whole -body imaging. Groups were randomized after two weeks (day 0) and treatment applied as above (days 1-12). Although they slowed down tumor progression, either tigecycline or venetoclax alone were insufficient to reverse the course of disease. In contrast, the combination caused rapid and marked tumor regression: in particular, luminescence in the femur (f.) and vertebrae (v.), clearly detectable in the controls, was suppressed by about two orders of magnitude, or became undetectable altogether between days 1 1 and 19 in double-treated animals (Fig. 4A, B, Fig. 12). Tumors were not eradicated, however, and re-grew at variable rates after cessation of treatment (Fig. 4A, Fig. 12, days 19-29). Taking disease manifestation (hind-limb paralysis, hunched posture) as the endpoint, untreated and single drug-treated animals had to be sacrificed at day 24, whereas combination-treated animals reached day 48 (Fig. 9). Thus, as observed above with the cell lines, tigecycline and venetoclax showed strong anti-tumoral effects with the DHL-derived PDX.

Tigecycline cooperates with rituximab

The anti-CD20 monoclonal antibody rituximab is a component of R-CHOP, the front-line immuno- chemotherapeutic regimen used to treat patients with DLBCL. However, those tumors that show MYC and BCL2 translocations, thus qualifying as DHL, show poor primary responses and high rates of relapse (2-7). In mice bearing SU-DHL-6 tumors, rituximab showed cooperation with either tigecycline or venetoclax in slowing tumor growth (fig. 13A, Table 2; compare with Fig. 3 for tigecycline or venetoclax alone).

Table 2. Effect of combined treatment on tumor regression (SU-DHL6)

In DOHH-2 tumors, rituximab cooperated neither with tigecycline nor with venetoclax: the latter appears in contrast with a previous report (8), but may be due to the lower dosage of venetoclax used in the inventors' experiment (50 vs. 100 mg/kg daily). Nevertheless, when added as a third component on top of tigecycline and venetoclax, rituximab clearly retarded the re-growth of DOHH- 2 tumors (fig. 13 A, 16 and 20 hours, Table 3).

Table 3. Effect of combined treatment on tumor regression (DOHH-2)

Treatment Regression: % tumor free (N)

Rituximab Tigecycline ABT-199 partial complete

n. mice dayl20

(l Omg kg) (75mg kg) (50mg kg) day 19 day 19

- - - 8 - - - + - - 8 - - -

+ - + 8 - - -

+ + - 8 - - -

+ + + 8 - - -

Finally, in PDX-xenografted mice, rituximab showed moderate cooperation with either tigecycline or venetoclax (fig. 13B, C and fig. 14). Hence, besides providing synergy against DHL as a two-drug combination, as shown in the previous sections, tigecycline and venetoclax have the potential to reinforce rituximab-containing therapies in the clinic.

Discussion

Among B-lymphoid malignancies, double -hit lymphomas (DHL) with concurrent MYC and BCL2 translocations (recently re-classified as high-grade B-cell lymphomas, or HGBL) (1) show the poorest prognosis. Hence, the development of additional therapeutic strategies represents a critical unmet need for patients who recur with this class of lymphomas (2-7). Previous work showed that MYC activation sensitized cells to tigecycline and that treatment with this antibiotic retarded the progression of MFC-driven lymphomas in mice (16, 17). Here, the inventors followed up on these findings to assess the activity of tigecycline against DHL, in combination with the BCL2 inhibitor venetoclax (8-10). The inventors first showed that over-expression of BCL2 in mouse Εμ-myc lymphomas blocked tigecycline-induced cell death, which was restored upon co-exposure to venetoclax. Consistent with this finding, the two drugs synergized in killing human DHL cells in vitro and showed strong anti-tumoral activity in vivo in mice xenografted with DHL cell lines or a PDX. With one line in particular (SU-DHL-6), the combined treatment achieved full disease eradication.

The anti-tumoral activity of tigecycline in either mouse or human cells is attributed to its inhibitory activity on mitochondrial translation and, as a consequence, on oxidative phosphorylation (16, 18, 25-31). Indeed, other antibiotics with similar properties were also toxic toward a variety of tumor cells ((21-25) and, as shown here for doxy- and tetracycline, cooperated with venetoclax in killing DHL cells. Tigecycline was also reported to inhibit WNT/B-Catenin (34) and PBK-Akt-mTOR signaling (30, 35, 36) and to induce AMPK signaling and autophagy (36, 37), but these effects may conceivably follow from mitochondrial dysfunction. Hence, although tigecycline and related antibiotics may inhibit other activities in eukaryotic cells, a comprehensive body of evidence points to the mitochondrial ribosome as their direct and critical target. The reciprocal is true as well, since compounds isolated as mitochondrial ribosome inhibitors also showed anti-bacterial activity (38). A recent study suggested that the sensitivity of several DLBCL cell lines to tigecycline was determined by their classification within the OxPhos as opposed to the BCR subtype (39), as defined by gene expression profiling (40). However, one of the OxPhos lines (Karpas-422) was resistant to tigecycline alone in the inventors' experiments (and was also the least sensitive of the OxPhos lines in the previous study). The inventors also note that OxPhos BCR are secondary to the alternative cell-of-origin based signatures in the clinic (ABC and GCB, for Activated and Germinal Center B cell-like, respectively) and that, regardless of these expression-based classifications, DLBCL cases show heterogeneous mutational landscapes (41, 42). Altogether, the determinants of tigecycline sensitivity, as well as its full molecular and metabolic effects in lymphomas remain to be fully characterized. This notwithstanding, the synergy with venetoclax reported here should be selective for MYC/BCL2 double-hit, and possibly double-expressor lymphomas (3).

In the clinic, tigecycline is among the antibiotics indicated for treatment of opportunistic, multi-drug resistant infections in cancer patients, with promising anti -bacterial responses and safety profiles (43- 47). In this setting, however, tigecycline's possible contribution to anti-tumoral responses was not considered. Based on the pre-clinical effect of tigecycline against acute myeloid leukemia (AML) (26, 48), a phase I study was undertaken in patients with relapsed AML (32), but no clinical response was reached so far, highlighting the need to reconsider the formulation and dosage of the antibiotic (32, 49), as well as its possible activity in combination regimens.

Preclinical studies reported that tigecycline cooperates with chemotherapeutic drugs in hepatocellular and renal cell carcinomas (29, 30), as well as with the BCR-ABL kinase inhibitor imatinib in chronic myelogenous leukemia (CML), where it contributed to the eradication of stem/progenitor cells (31), a feature that was independently reported for venetoclax (50, 51). Tigecycline also targeted cancer stem cells (CSCs) in AML (26), breast, and other tumor types (25, 28), pointing to a critical function of mitochondrial translation in CSCs. Other studies indicated that mitochondrial electron transport is critical for the survival of CSCs or cancer-repopulating cells (52, 53) and may itself be suppressed upon BCL2 inhibition (53, 54). Finally, MYC and the BCL2 -related protein MCL1 cooperated to promote mitochondrial respiratory activity and chemotherapy resistance in breast CSCs (55). Together with the inventors' data in DHL, these observations suggest that tigecycline and venetoclax (or other BH3 -mimetic drugs) (15) may complement current therapeutic regimens in a variety of tumor types. Multiple clinical studies have addressed the safety and efficacy profiles of venetoclax against hematological malignancies (10, 56-61), including lymphoma (57). Compared with other subtypes of lymphoma, venetoclax appears to be relatively ineffective against DLBCL (3, 10), although the impact of the MYC/BCL2 status on clinical responses was not reported. Venetoclax showed cooperativity with rituximab in patients with chronic lymphocytic leukemia (CLL) (58, 61), as well as in mice xenografted with DHL cell lines (8), as confirmed in the inventors' work. Finally, and most relevant for future clinical development, the effects of venetoclax and tigecycline in either DOHH-2 or PDX-derived tumors appeared to be reinforced by addition of rituximab in the inventors' study.

In conclusion, the inventors' preclinical data have uncovered a synergy between mitochondrially- targeted antibiotic such as tigecycline and an inhibitor of BCL-2 such as venetoclax against DHL, warranting the repurposing of these drugs in combination for secondary treatment of patients with refractory or relapsed MYC/BCL2 double-hit lymphomas. Moreover, the addition of both compounds to R-CHOP or other rituximab-based chemo-immunotherapeutic regimens (3) may improve the primary response of patients with this particularly aggressive lymphoma subtype.

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Claims

1- A combination of at least one mitochondrially-targeted antibiotic and at least one inhibitor of BCL-2 for use in the treatment of a MYC/BCL2 double positive B-cell lymphoma.

2- The combination for use according to claim 1 wherein the at least one mitochondrially-targeted antibiotic is selected from the group consisting of: erythromycin and derivatives thereof, tetracycline and derivatives thereof, glycylcycline and derivatives thereof, an anti-parasitic drug, and chloramphenicol and derivatives thereof.

3- The combination for use according to claim 2 wherein the erythromycin derivative is selected from the group consisting of: Azithromycin, Carbomycin, Cethromycin, Clarithromycin, Dirithromycin, Mitemcinal, Oleandomycin, flurithromycin, Roxithromycin, Spiramycin, Telithromycin and Tylosin, and/or wherein the tetracycline and/or glycylcycline derivative is selected from the group consisting of: Tigecycline, Tetracycline, Doxycycline, Chlortetracycline, Oxytetracycline, Demeclocycline, Lymecycline, Meclocycline, Methacycline, Minocycline and Rolitetracycline, and/or wherein the anti-parasitic drug is pyrvinium pamoate.

4- The combination for use according to any one of previous claim wherein the mitochondrially- targeted antibiotic is administered intravenously.

5- The combination for use according to any one of previous claim wherein the mitochondrially- targeted antibiotic is administered every two days.

6- The combination for use according to any one of previous claim wherein the inhibitor of BCL-2 is selected from the group consisting of: ABT-737, ABT-263, ABT-199 (Venetoclax).

7- The combination for use according to any one of previous claim wherein the inhibitor of BCL-2 is administered for at least one cycle of 5 days.

8- The combination for use according to any one of previous claim wherein the mitochondrially- targeted antibiotic is Tigecycline and the inhibitor of BCL-2 is ABT-199 or wherein the mitochondrially-targeted antibiotic is doxycycline and the inhibitor of BCL-2 is ABT-199 orwherein the mitochondrially-targeted antibiotic is tetracycline and the inhibitor of BCL-2 is ABT-199.

9- The combination for use according to any one of previous claim, wherein the MYC/BCL2 double positive B-cell lymphoma is a double -hit lymphoma or a double-expressor lymphoma. 10- The combination for use according to any one of previous claim further comprising at least one additional therapeutic agent.

11 - The combination for use according to claim 10 wherein the additional therapeutic agent is selected from the group consisting of: an anti-CD 20 antibody, an anti-CD22 antibody, an anti-VEGF antibody, an anti-CD52 antibody, Cyclophosphamide, Doxorubicin (Hydroxydaunomycin), Vincristine (Oncovin ®), Prednisolone and a combination thereof.

12- The combination for use according to claim 11 wherein the anti-CD 20 antibody is Rituximab, and/or wherein the anti-CD22 antibody is Epratuzumab, and/or wherein the anti-VEGF antibody is Bevacizumab, and or wherein the anti-CD52 antibody is Alemtuzumab.

13- The combination for use according to any one of claim 10 to 12 wherein the mitochondrially- targeted antibiotic is Tigecycline and the inhibitor of BCL-2 is ABT-199 and the additional therapeutic agent is rituximab.

14- The combination for use according to any one of previous claim wherein the mitochondrially- targeted antibiotic and the inhibitor of BCL-2 and optionally the least one additional therapeutic agent are administered simultaneously or sequentially.

15- A pharmaceutical composition comprising at least one mitochondrially-targeted antibiotic as defined in any one of claims 1 to 5 and 8, at least one inhibitor of BCL-2 as defined in any one of claims 1 , 6 to 8 , a pharmaceutically acceptable vehicle and optionally at least a further therapeutic agent as defined in any one of claims 10 to 12 for use in the treatment of a MYC/BCL2 double positive B-cell lymphoma.

16- The pharmaceutical composition for use according to claim 15, wherein the MYC/BCL2 double positive B-cell lymphoma is a double -hit lymphoma or a double-expressor lymphoma.

17- A kit comprising at least one mitochondrially-targeted antibiotic as defined in any one of claims 1 to 5 and 8, at least one inhibitor of BCL-2 as defined in any one of claims 1 , 6 to 8 and optionally at least a further therapeutic agent as defined in any one of claims 10 to 12 for use in the treatment of MYC/BCL2 double positive B-cell lymphomas.

18- The kit foruse according to claim 17, wherein the MYC/BCL2 double positive B-cell lymphoma is a double-hit lymphoma or a double-expressor lymphoma.