NZ722491B2 - Apilimod compositions and methods for using same - Google Patents

Abstract

The present invention relates to methods for treating cancer, specifically refractory or recurrent non-Hodgkin’s B cell lymphoma, with apilimod and related compositions and methods.

Description

APILIMOD COMPOSITIONS AND METHODS FOR USING SAME FIELD OF THE INVENTION The present ion relates to compositions comprising apilimod and methods of using same.

BACKGROUND OF THE INVENTION Apilimod, also referred to as STA-5326, hereinafter “apilimod”, is recognized as a potent transcriptional inhibitor of IL-12 and IL-23. See e.g., Wada et al. Blood 109 (2007): 1156-1164. IL-12 and IL-23 are inflammatory cytokines normally produced by immune cells, such as B-cells and macrophages, in response to antigenic stimulation. Autoimmune disorders and other disorders characterized by c inflammation are characterized in part by opriate tion of these cytokines. In immune cells, the selective tion of IL- 23 transcription by apilimod was recently shown to be mediated by apilimod’s direct g to phosphatidylinositolphosphate 5-kinase (PIKfyve). See, e.g., Cai et al. Chemistry and Biol. 20 (2013):912-921. PIKfyve plays a role in ike receptor signaling, which is important in innate immunity.

Based upon its activity as an immunomodulatory agent and a specific inhibitor of IL-12/IL-23, od has been ed as useful in treating autoimmune and inflammatory diseases and disorders. See e.g., US 6,858,606 and 733 (describing a family of pyrimidine compounds, including apilimod, purportedly useful for treating diseases and disorders characterized by IL-12 or IL-23 overproduction, such as rheumatoid arthritis, sepsis, Crohn’s e, multiple sclerosis, psoriasis, or insulin dependent diabetes mellitus). Similarly, apilimod was suggested to be useful for treating certain cancers based upon its activity to inhibit c-Rel or IL-12/23, particularly in cancers where these cytokines were believed to play a role in promoting aberrant cell proliferation. See e.g., and Baird et al., Frontiers in Oncology 3:1 (2013, respectively).

Each of three clinical trials of apilimod has focused on its potential efficacy in autoimmune and inflammatory diseases. The trials were conducted in patients having psoriasis, toid arthritis, and Crohn's disease. An open label clinical study in patients with psoriasis concluded that oral administration of apilimod showed immunomodulatory activity supporting the tion of IL-1 2/IL-23 sis for the treatment ofTHl - and TH17-mediated inflammatory es. Wada et al., PLOSOne 69 (April 2012). But the results of controlled trials in rheumatoid arthritis and Crohn's disease did not support the notion that IL- 12/IL-23 inhibition by apilimod translates into clinical ement in either of these indications. In a randomized, double-blind, placebo-controlled Phase II clinical trial of apilimod in patients with rheumatoid arthritis, od failed to alter synovial IL-12 and IL-23 expression. Krauz et al., Arthritis & Rheumatism 64:1750-1755 (2012). The authors concluded that the “results do not support the notion the lL-12/IL-23 inhibition by apilimod is able to induce robust al improvement in RA.” Similarly, a randomized, double-blind, placebo- controlled trial of apilimod for treatment of active Crohn's disease concluded that, although well tolerated, apilimod did not demonstrate efficacy over placebo. Sands et al Inflamm Bowel Dis. 2010 Jul;16(7):1209-18.

The mammalian target ofrapamycin (mTOR) pathway is an important cellular signaling pathway that is involved in multiple physiological functions, including cell growth, cell proliferation, metabolism, n synthesis, and autophagy (La Plante et al Cell 2012, (149 (2), pp .274-293). mTOR is a kinase that integrates intracellular and extracellular cues that signal the levels of amino acids, stress, oxygen, , and growth factors and regulates the cellular response to these environment cues. mTOR deregulation has been implicated in a wide range of disorders and diseases, including , obesity, diabetes, and egeneration. Certain components ofthe mTOR pathway have been explored as drug targets for treating some ofthese diseases. However, therapeutic y has been limited, for example, in the ent of some cancers, and some mTOR inhibitors have been shown to have an adverse effect on metabolism.

The tuberous sclerosis complex tumor suppressor genes, TSCl and TSC2, are negative tors ofmTOR.

SUMMARY OF THE INVENTION The present invention is based in part on the surprising ery that apilimod is a highly cytotoxic agent in TSC null cells. In these cells, the mTOR pathway is constitutively active. The mTOR pathway is activated in a number of cancers, and in fithher screening of over 100 cancer cell lines apilimod showed anti-proliferative activity in cell lines from diverse cancers. Among the apilimod sensitive cancer cell lines, B-cell lymphomas were the most sensitive. But, unexpectedly, the differential sensitivity ofB cell lymphomas to apilimod did not correlate with c-Rel expression, IL-12 expression, or lL-23 expression in these cells. This was surprising e earlier work had suggested apilimod would be usefill against cancers where c- Rel and/or IL-12/23 sion were critical in promoting aberrant cell proliferation. Instead, the present inventors demonstrated that apilimod’s cytotoxic ty in cancer cells was due to an inhibition of intracellular trafficking and a corresponding increase in apoptosis. This activity was not predicted based upon apilimod’s immunomodulatory activity via its inhibition of IL- 12/23 production. In addition, a screen of over 450 kinases identified PIKfyve as the only high affinity binding target (Kd=75 pM) for od in a human cancer cell line. The present invention provides new methods for the therapeutic use of apilimod, especially in treating cancer, and particularly in treating B cell lymphomas, and ally those that are resistant or refractory to standard chemotherapy regimens.

In one aspect, the present invention provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an apilimod composition ofthe invention, said composition sing apilimod, or a pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, prodrug, analog or derivative thereof. In one embodiment, the apilimod composition comprises apilimod free base or apilimod dimesylate. In one embodiment, the method fithher comprises administering at least one additional active agent to the t. The at least one additional active agent may be a eutic agent or a non-therapeutic agent. The at least one additional active agent may be administered in a single dosage form with the apilimod composition, or in a separate dosage form from the apilimod composition. In one embodiment, the at least one onal active agent is selected from the group consisting of an alkylating agent, an intercalating agent, a tublin binding agent, a corticosteroid, and combinations f In one embodiment, the at least one additional active agent is a eutic agent selected from the group consisting of nib, rituximab, doxorubicin, prednisolone, Vincristine, velcade, and imus, and ations thereof. In one embodiment, the at least one additional active agent is a therapeutic agent selected from cyclophosphamide, hydroxydaunorubicin (also referred to as doxorubicin or AdriamycinTM), Vincristine (also referred to as OncovinTM), prednisone, prednisolone, and ations thereof. In one embodiment, the at least one onal active agent is a non-therapeutic agent selected to ameliorate one or more side effects ofthe apilimod composition. In one embodiment, the non-therapeutic agent is selected from the group consisting of ondansetron, granisetron, dolasetron and palonosetron. In one embodiment, the non-therapeutic agent is selected from the group consisting ofpindolol and idone.

In one embodiment, the dosage form of the apilimod composition is an oral dosage form. In another ment, the dosage form ofthe apilimod composition is suitable for intravenous administration. In one embodiment, where the dosage form is le for intravenous administration, administration is by a single injection or by a drip bag.

In one embodiment, the subject is a human cancer patient. In one embodiment, the human cancer t in need of treatment with an od composition of the invention is one Whose cancer is refractory to a standard chemotherapy regimen. In one embodiment, the human cancer patient in need of treatment with an apilimod composition is one Whose cancer has recurred following treatment With a standard chemotherapy regimen. In one embodiment, the cancer is a lymphoma. In one embodiment, the cancer is a B cell lymphoma. In one embodiment, the B cell lymphoma is a non-Hodgkin’s B cell lymphoma. In one embodiment, the dgkin’s B cell lymphoma is selected from a diffuse large B cell lymphoma (DLBCL), a Burkitt’s ma, a mediastinal B cell ma, a mantle cell lymphoma, and a follicular lymphoma. In one embodiment, the non-Hodgkin’s B cell lymphoma is DLBCL. In one embodiment, the DLBCL is the GCB subtype.

In one embodiment, the standard chemotherapy regimen comprises one or more therapeutic agents selected from the group consisting of ibrutinib, rituximab, doxorubicin, prednisolone, vincristine, velcade, cyclophosphoamide, dexamethasone and everolimus. In one embodiment, the standard chemotherapy regimen is selected from CHOP, (cyclophosphamide, hydroxydaunorubicin, OncovinTM (vincristine), and prednisone or prednisolone), COOP (cyclophosamide, stine sulfate, procarbazine hydrochloride, prednisone), CVP (cyclophosamide, vincristine sulfate, sone), EPOCH (etoposide, prednisone, vincristine sulfate, cyclophosphamide, doxorubicin hydrochloride), Hyper-CVAD (cyclophosphamide, stine sulfate, doxorubicin hydrochloride, dexamethasone), ICE (ifosfamide, carboplatin, etoposide), R-CHOP imab, cyclophosamide, vincristine sulfate, procarbazine hydrochloride, prednisone, and R-CVP (rituximab, cyclophosamide, stine sulfate, prednisone).

In one embodiment, the method is a method ting a lymphoma using a combination therapy comprising an apilimod composition and a chemotherapy regimen for the ent ofthe lymphoma. In one embodiment, the chemotherapy regimen is the CHOP regimen. In another embodiment, the chemotherapy regimen is selected from COOP, CVP, EPOCH, Hyper-CVAD, ICE, R-CHOP, and R-CVP.

In some embodiments, apilimod compositions are provided herein that are usefill for treating s ated with TSC deficiency and/or mTOR activation. In some embodiments, methods are provided herein for treating TSC] - or TSC2-deficient s using an apilimod composition. In some embodiments, methods are provided for treating mTOR associated cancers using an apilimod composition. In some embodiments, an mTOR associated cancer is associated with a deletion, loss of on mutation, low sion or other TSCl or TSC2 deficiency. In some embodiments, an mTOR associated cancer is associated with a gain of function mutation that results in activation (e.g., tutive activation) of mTOR pathway activity in cells ofthe cancer. In some ments, methods are provided herein that filrther involve ining whether a subject having cancer is a ate for treatment with an apilimod composition based on the TSCl , TSC2 and/or mTOR status ofthe subject. For example, in some embodiments, a subject having a TSCl or TSC2 deficiency (e.g., an inactivating mutation in TSCl or TSC2) or constitutively active mTOR signaling is a candidate for treatment with an apilimod composition.

BRIEF DESCRIPTION OF THE DRAWINGS Figure l: TSC2 deficient cells are highly sensitive to apilimod (IC50 = 20 nM).

Figure 2A: Sensitivity ofcancer cell lines to apilimod (percentage of cell lines with IC50 less than 500 nM).

Figure 2B: NHL cell lines are particularly sensitive to apilimod (percentage of cell lines with IC50 less than 500 nM).

Figure 2C: Apilimod’s cytotoxic ty is selective for cancer cells over normal cells. Normal lung fibroblasts were insensitive to apilimod-induced cytotoxicity at concentrations as high as 10 micromolar.

Figure 3: a diffuse large B cell lymphoma, SUDHL-4, exhibited an IC50 of 50 Figure 4: Apilimod’s cytotoxic activity in NHL cells was a result of increased sis. Apoptotic (Caspase—3/7, middle bar) and necrotic (bis-AAF-Rl 10, right bar) markers in apilimod treated diffuse large B cell lymphoma cells 48 hours afier addition of od to the culture media; left bar shows viability marker (GF-AFC). figm: od induces autophagy in a dose-dependent . figm: Volanco plot ofsignificant captured hits applying CT-689 at 0.1 uM concentration under optimized capture conditions.

Ligw: Apilimod binds with high affinity to PIKfyve (Kd = 75 pM). figm: REL CCLE expression in apilimod ive (lines #1-13) and insensitive (lines #14-22) B cell lymphoma lines. figm: ILl2A CCLE expression in apilimod sensitive (#1-23 lines) and insensitive (#24-75 lines) cancer cell lines.

Figure 10: ILl2B CCLE expression in apilimod sensitive (#1-23 lines) and insensitive (#24-75 lines) cancer cell lines.

Figure 11: ILl2RBl CCLE expression in apilimod sensitive (#1 -23 lines) and insensitive (#24-75 lines) cancer cell lines Figure 12: ILl2RB2 CCLE expression in apilimod sensitive (#1 -23 lines) and itive (#24-75 lines) cancer cell lines.

Figure 13: IL23R CCLE expression in od sensitive (#1-23 lines) and insensitive (#24-75 lines) cancer cell lines.

Figure 14: IL23A CCLE expression in apilimod sensitive (#1-23 lines) and insensitive (#24-75 lines) cancer cell lines.

Figure 15: IL-23A expression is not a statistically significant predictor of sensitivity in Non-Hodgkin's B cell lymphoma. Shown are apilimod sensitive NHB cell lines (bottom, dark) and insensitive (top, light).

Figure 16: Apilimod inhibits the growth of SU—DHL-6 DLBCL afi tumors; Top line shows vehicle saline (diamond, light grey solid lines) QD x5, 2 days off, QD x5 i.v.; 0.5% cellulose (triangle, solid dark grey lines) QD x5, 2 days off, QD x5 p.o.; apilimod late (square, dashed lines) 67.5 mg/kg (47 mg/kg free base) QD x5 iv, 2 days off, QD x5; od free base (square, light grey solid lines) 150 mg/kg QD x5, 2 days off, QD x5 p.o; apilimod free base (cross, solid lines) 75 mg/kg BID x5, 2 days off, BID x5 po.

Figure 171Antitumor activity ofapilimod in combination with ibrutinib on DLBCL tumors in viva; Top line shows vehicle (diamond, light grey solid lines) QD x5, 2 days off, QD x5 p.o. + i.v.; ibrutinib (triangle, solid dark grey lines) 10 mg/kg QD x12 i.v.; apilimod free base (square, dashed lines) 75 mg/kg QD x5, 2 days off, QD x5 p.o.; ibrutinib (cross, solid dark line) 20 mg/kg QD x12 i.V.; apilimod free base 75 mg/kg QD x5, 2 days off, QD x5 p.o. + ibrutinib 10 mg/kg QD X12 i.V. (square, solid light grey lines); apilimod free base 75 mg/kg QD x5, 2 days off, QD x5 p.o. + ibrutinib 10 mg/kg QD x12 i.V. (circle, solid medium grey lines).

Figure 18: Screening SU-DHL-4 cells with a manually curated library of 93 drugs with and without apilimod (lOnM) identified nib as a drug that when combined with apilimod exerts synergistic activity.

Figure 19: Apilimod induces vacuolization ofa representative cancer cell line.

Left: Untreated cell. Right: Live cells treated with 500nM apilimod 24h.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides itions and methods related to the use of apilimod for treating cancer in a subject, ably a human subject, in need of such treatment.

The present invention generally relates to new uses of apilimod based upon the sing discovery of apilimod’s cytotoxic activity against a range of cancer cells ofboth id and non-lymphoid origin, an activity that is not clearly related to, or table from, apilimod’s known immunomodulatory and IL-1 2/23 inhibitory activity. In addition, the present invention es novel therapeutic approaches to cancer treatment based upon combination therapy utilizing apilimod and at least one additional therapeutic agent. The ation therapies described herein exploit the unique cytotoxic activity of apilimod which is shown to provide a synergistic effect when combined with other therapeutic , including for e, anti- cancer agents.

As used herein, the term “an apilimod composition” may refer to a composition comprising apilimod itself (free base), or may encompass pharmaceutically acceptable salts, solvates, clathrates, hydrates, rphs, prodrugs, analogs or derivatives of apilimod, as described below. The structure of apilimod is shown in Formula]: 0 (1) The chemical name of apilimod is 2-[2-Pyridinyl)-ethoxy]N'-(3-methyl- benzilidene)—hydrazino](morpholin-4—yl)-pyrimidine (IUPAC name: (E)(6-(2-(3- methylbenzylidene)hydrazinyl)—2-(2-(pyridiny1)ethoxy)pyrimidinyl)morpholine), and the CAS number is 5415500.

Apilimod can be ed, for example, ing to the methods described in US. Patent Nos. 7,923,557, and 7,863,270, and .

As used herein, the term aceutically acceptable salt," is a salt formed from, for example, an acid and a basic group of an apilimod composition. Illustrative salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, , nitrate, bisulfate, phosphate, acid ate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, besylate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (e.g., l,l '-methylene-bis-(2-hydroxy naphthoate)) salts. In a preferred embodiment, the salt of apilimod comprises methanesulfonate.

The term aceutically acceptable sal " also refers to a salt prepared from an od composition having an acidic functional group, such as a carboxylic acid fianctional group, and a pharmaceutically able inorganic or organic base.

The term "pharmaceutically acceptable sal " also refers to a salt prepared from an apilimod composition having a basic functional group, such as an amino filnctional group, and a pharmaceutically able inorganic or organic acid.

The salts ofthe compounds described herein can be synthesized from the parent compound by conventional chemical methods such as s described in Pharmaceutical Salts: Properties, Selection, and Use, P. Hemrich Stalil (Editor), Camille G. Wermuth r), ISBN: 3026-8, August 2002. Generally, such salts can be prepared by reacting the parent compound with the appropriate acid in water or in an organic solvent, or in a mixture ofthe two.

One salt form of a nd described herein can be converted to the free base and optionally to another salt form by methods well known to the skilled person. For example, the free base can be formed by passing the salt solution through a column containing an amine stationary phase (e.g. a Strata-NHZ column). Alternatively, a solution ofthe salt in water can be treated with sodium bicarbonate to decompose the salt and precipitate out the free base. The free base may then be combined with another acid using routine methods.

As used herein, the term "polymorph" means solid crystalline forms of a compound ofthe present invention (e.g., 2-[2-Pyridinyl)-ethoxy]N'-(3 l- benzilidene)—hydrazino](morpholin—4-yl)-pyrimidine) or complex thereof. Different polymorphs ofthe same compound can exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to stability (e.g., to heat or light), compressibility and density (important in ation and product manufacturing), and dissolution rates (which can affect bioavailability). Differences in stability can result from changes in chemical reactivity (e.g., differential oxidation, such that a dosage form ors more rapidly when comprised of one polymorph than when sed of another polymorph) or mechanical characteristics (e.g., tablets crumble on storage as a kinetically d polymorph converts to thermodynamically more stable polymorph) or both (e.g., tablets of one polymorph are more susceptible to breakdown at high humidity). Different physical properties of rphs can affect their sing. For e, one polymorph might be more likely to form solvates or might be more difficult to filter or wash free of impurities than another due to, for example, the shape or size distribution icles of it.

As used herein, the term "hydrate" means a compound of the present invention (6.g. , 2- [2 -Pyridinyl)—ethoxy] N'-(3-methyl-benzilidene)-hydrazino](morpholinyl)- pyrimidine) or a salt thereof, which fiarther includes a stoichiometric or non-stoichiometric amount ofwater bound by non-covalent intermolecular forces.

As used herein, the term "clathrate" means a compound of the present invention (6.g. , 2- [2 -Pyridinyl)—ethoxy] N'-(3—methyl-benzilidene)-hydrazino](morpholinyl)- pyrimidine) or a salt thereof in the form of a crystal lattice that contains spaces (e.g., channels) that have a guest molecule (e.g., a t or water) trapped within.

As used herein, the term "prodrug" means a derivative of a compound described herein (e.g. , 2-[2-Pyridin-2 -yl)-ethoxy]-4—N'-(3-methyl—benzilidene)-hydrazino](morpholin yl)-pyrimidine) that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to e a compound ofthe invention. Prodrugs may only become active upon such reaction under biological conditions, or they may have activity in their unreacted forms.

Examples ofprodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of a compound described herein (e.g., 2—[2-Pyridinyl)-ethoxy]N’-(3-methyl- benzilidene)—hydrazino](morpholinyl)-pyrimidine) that comprise biohydrolyzable moieties such as rolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues.

Other examples ofprodrugs include derivatives of compounds of any one ofthe formulae disclosed herein that comprise -NO, -N02, —ONO, or —ON02 moieties. Prodrugs can typically be prepared using well-known methods, such as those described by Burger’s Medicinal Chemistry and Drug Discovery (1995) 8, 949—982 (Manfred E. Wolff ed., 5th ed).

As used herein, the term te” or "pharmaceutically acceptable solvate," is a solvate formed from the association ofone or more solvent molecules to one ofthe compounds disclosed herein (e.g., 2-[2-Pyridinyl)-ethoxy]N'-(3-methyl-benzilidene)-hydrazino] (morpholinyl)-pyrimidine). The term e includes hydrates (e.g., hemi-hydrate, mono- hydrate, dihydrate, trihydrate, tetrahydrate, and the like).

As used herein, the term “analog” refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one fianctional group by another fianctional group). Thus, an analog is a compound that is similar or comparable in on and appearance, but not in structure or origin to the nce compound. As used herein, the term ative” refers to compounds that have a common core structure, and are tuted with various groups as described herein.

Methods of Treatment The t invention provides methods for the treatment of cancer in a subject in need thereofby administering to the subject a therapeutically effective amount of an apilimod composition ofthe invention, said ition comprising apilimod, or a pharmaceutically acceptable salt, solvate, ate, hydrate, polymorph, prodrug, analog or tive thereof In one embodiment, the apilimod composition comprises apilimod free base or apilimod dimesylate. The present invention further provides the use ofan apilimod composition for the preparation of a medicament usefiil for the ent of cancer.

In one ment, the cancer is brain cancer, glioma, sarcoma, breast cancer, lung cancer, non-small-cell lung , mesothelioma, appendiceal cancer, genitourinary cancers, renal cell carcinoma, prostate cancer, bladder cancer, testicular cancer, penile cancer, cervical cancer, ovarian cancer, von Hippel Lindau disease, head and neck cancer, gastrointestinal cancer, hepatocellular oma, adder cancer, esophageal cancer, gastric _10_ cancer, colorectal cancer, pancreatic cancer, neuroendocrine tumors, thyroid tumor, pituitary tumor, adrenal tumor, hematological malignancy, or leukemia.

In one embodiment the cancer is a lymphoma. In one embodiment, the lymphoma is a B cell lymphoma. In one embodiment, the B cell ma is selected from the group consisting of a Hodgkin’s B cell lymphoma and a non-Hodgkin’s B cell lymphoma. In one embodiment, the B cell lymphoma is a non-Hodgkin’s B cell lymphoma selected from the group consisting L, follicular lymphoma, marginal zone lymphoma (MZL) or mucosa associated lymphatic tissue lymphoma (MALT), small cell lymphocytic lymphoma (overlaps with chronic lymphocytic ia) and mantle cell lymphoma. In one embodiment, the B cell lymphoma is a non-Hodgkin’s B cell lymphoma selected from the group consisting of Burkitt lymphoma, Burkitt lymphoma, Primary mediastinal (thymic) large B-cell ma, Lymphoplasmacytic lymphoma, which may manifest as Waldenstrom macroglobulinemia, Nodal marginal zone B cell ma (NMZL), Splenic marginal zone lymphoma (SMZL), ascular large B-cell lymphoma, Primary effiJsion lymphoma, Lymphomatoid granulomatosis, T cell/histiocyte-rich large B-cell lymphoma, Primary central nervous system lymphoma, Primary cutaneous diffuse large B-cell lymphoma, leg type (Primary cutaneous DLBCL, leg type), EBV positive diffuse large B-cell lymphoma ofthe elderly, Diffilse large B- cell lymphoma associated With inflammation, Intravascular large B-cell ma, ALK- positive large B-cell ma, and Plasmablastic lymphoma.

Combination Therapy The present ion also provides methods comprising combination therapy.

As used herein, “combination therapy” or “co-therapy” es the administration of a therapeutically effective amount of an od composition with at least one additional active agent, as part of a specific treatment regimen ed to provide a beneficial effect from the co- action ofthe od ition and the additional active agent. “Combination therapy” is not intended to ass the administration oftwo or more therapeutic compounds as part of separate monotherapy regimens that incidentally and arbitrarily result in a beneficial effect that was not intended or predicted.

In one embodiment, the method is a method oftreating cancer using a combination therapy comprising an apilimod composition and a chemotherapy regimen for the treatment of cancer. In one embodiment, the chemotherapy regimen is the CHOP regimen. _11_ CHOP refers to a regimen lly used in the treatment of non-Hodgkin’s lymphoma consisting ofthe following active agents: (C)yclophosphamide, an alkylating agent which damages DNA by binding to it and causing the formation of cross-links; (H)ydroxydaunorubicin (also called doxorubicin or Adriamycin), an intercalating agent which damages DNA by inserting itselfbetween DNA bases; (O)ncovin (Vincristine), which ts cells from duplicating by binding to the protein tubulin; and (P)rednisone or nisolone, which are corticosteroids. In another embodiment, the chemotherapy regimen is ed from COOP (cyclophosamide, Vincristine sulfate, procarbazine hydrochloride, prednisone), CVP phosamide, Vincristine sulfate, prednisone), EPOCH (etoposide, prednisone, Vincristine sulfate, cyclophosphamide, doxorubicin hydrochloride), Hyper-CVAD (cyclophosphamide, Vincristine sulfate, doxorubicin hloride, dexamethasone), ICE (ifosfamide, carboplatin, etoposide), R-CHOP imab, cyclophosamide, Vincristine e, procarbazine hydrochloride, prednisone, and R-CVP (rituximab, hosamide, Vincristine sulfate, prednisone).

The at least one additional active agent may be a therapeutic agent, for example an anti-cancer agent or a cancer herapeutic agent, or a non-therapeutic agent, and combinations thereof With respect to eutic agents, the beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of eutically active compounds. With t to non- therapeutic agents, the beneficial effect ofthe combination may relate to the mitigation of a toxicity, side effect, or adverse event associated with a therapeutically active agent in the combination.

In one embodiment, the at least one onal agent is a non-therapeutic agent which mitigates one or more side effects of an apilimod composition, the one or more side effects selected from any of nausea, vomiting, headache, dizziness, lightheadedness, drowsiness and stress. In one aspect ofthis embodiment, the non-therapeutic agent is an antagonist of a serotonin receptor, also known as 5-hydroxytryptamine receptors or 5-HT receptors. In one aspect, the non-therapeutic agent is an antagonist of a 5-HT3 or 5-HT]a receptor. In one aspect, the non-therapeutic agent is ed from the group consisting of ondansetron, granisetron, dolasetron and palonosetron. In another aspect, the non-therapeutic agent is selected from the group consisting of pindolol and risperidone. _12_ In one embodiment, the at least one additional agent is a therapeutic agent. In one embodiment, the therapeutic agent is an anti-cancer agent. In one embodiment, the anti-cancer agent is ibrutinib. In one embodiment, an apilimod composition is administered along with ibrutinib in a single dosage form or in separate dosage forms. In one embodiment, the dosage form is an oral dosage form. In another embodiment, the dosage form is suitable for intravenous administration.

In one embodiment, the anti-cancer agent is a drug that is approved for use in treating ma. Non-limiting examples of such drugs include abitrexate trexate), adcetris uximab vedotin), ambochlorin ambucil), orin (chloramucil), arranon (nelarabine), becenum (carmustine), beleodaq (belinostat), belinostat, bendamustine hydrochloride, bexxar (tositumomab and Iodine I 131 tositumomab), BiCNU (carmustine), blenoxane ycin), bleomycin, bortezomib, brentuximab vedotin, carmubris (carmustine), carmustine, chlorambucil, clafen phosphamide), cyclophosphamide, cytoxan (cyclophosphamide), denileukin diftitox, DepoCyt (liposomal cytarabine), doxorubicin hydrochloride, foleX (methotrexate), folotyn (pralatrexate), ibritumomab tiuxetan, ibrutinib, idelalisib, imbruVica (ibtrutinib), intron A (recombinant interferon Alfa-2b), istodax (romidepsin), lenalidomide, leukeran ambucil), linfolizin (chlorambucil), liposomal cytarabine, mechlorethamine hydrochloride, methotrexate, methotrexate LPF (methotrexate), mexate (methotrexate), mexate —AQ (methotrexate), mozobil (perixafor), mustargen (mechlorethamine hydrochloride), nelarabine, neosar (cyclophosphamide), ontak (denifleukin diftitox), perixafor, pralatrexate, prednisone, recombinant eron Alfa—Zb, revlimid (lenalidomide), rituxan (rituximab), rituximab, romidepsin, tositumomab and iodine I 131 tositumomab, treanda mustine hydrochloride), velban (Vinblastine sulfate), velcade (bortezomib), velsar (Vinblasinte sulfate), vinblastine sulfate, Vincasar PFS (Vincristine sulfate), Vincristine sulfate, vorinostat, zevalin (ibritumomab triuxetan), zolinza ostat), and zydelig (idelalisib).

In one embodiment, the ancer agent is selected from an inhibitor ofEZHZ, e.g., EPZ-6438. In one embodiment, the anti-cancer agent is selected from taxol, stine, bicin, temsirolimus, carboplatin, ofatumumab, mab, and combinations thereof.

In one embodiment, the at least one additional agent is a B cell receptor pathway inhibitor. In some embodiments, the B cell receptor pathway inhibitor is a CD79A inhibitor, a CD79B tor, a CD 19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blnk _13_ inhibitor, a PLCy inhibitor, a PKCP tor, or a combination thereof In some embodiments, the at least one additional agent is an antibody, B cell receptor signaling inhibitor, a PI3K tor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA damaging agent, a proteosome tor, a e deacetylase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jakl/2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP tor, or a combination thereof.

In one embodiment, the at least one additional agent is selected from mbucil, ifosphamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, umab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a ation thereof.

In one embodiment, the at least one additional agent is a monoclonal antibody such as, for example, alemtuzumab, bevacizumab, catumaxomab, cetuximab, edrecolomab, gemtuzumab, ofatumumab, mumab, rituximab, trastuzumab, eculizumab, efalizumab, muromab-CD3, zumab, adalimumab, afelimomab, certolizumab pegol, golimumab, infliximab, basiliximab, canakinumab, daclizumab, mepolizumab, tocilizumab, ustekinumab, ibritumomab tiuxetan, tositumomab, abagovomab, adecatumumab, alemtuzumab, anti-CD30 onal antibody 13, anti-MET monoclonal antibody , apolizumab, apomab, arcitumomab, basiliximab, bispecific antibody 2B1, blinatumomab, brentuximab vedotin, capromab pendetide, cixutumumab, claudiximab, conatumumab, dacetuzumab, denosumab, eculizumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, fresolimumab, mab, ganitumab, gemtuzumab icin, glembatumumab, ibritumomab, inotuzumab ozogamicin, ipilimumab, lexatumumab, lintuzumab, lintuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, monoclonal antibody CC49, necitumumab, nimotuzumab, ofatumumab, oregovomab, pertuzumab, rimab, ranibizumab, siplizumab, sonepcizumab, tanezumab, tositumomab, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, Visilizumab, volociximab, and zalutumumab.

In the context of combination therapy, administration of the od composition may be aneous with or tial to the administration ofthe one or more additional active agents. In another embodiment, administration of the different components of a combination therapy may be at different frequencies. The one or more additional agents may be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, _14_ 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a compound ofthe present invention.

The one or more additional active agents can be formulated for co-administration with an apilimod composition in a single dosage form, as described in greater detail herein. The one or more additional active agents can be administered separately from the dosage form that comprises the compound ofthe present invention. When the additional active agent is administered separately from the apilimod composition, it can be by the same or a ent route of administration as the apilimod composition.

Preferably, the administration ofan apilimod ition in combination with one or more additional agents provides a synergistic response in the subject being treated. In this context, the term “synergistic” refers to the efficacy of the combination being more effective than the ve effects of either single therapy alone. The istic effect of a combination therapy according to the invention can permit the use oflower s and/or less frequent administration of at least one agent in the combination compared to its dose and/or frequency outside ofthe combination. Additional beneficial effects ofthe combination can be manifested in the nce or reduction of adverse or unwanted side effects ated with the use of either therapy in the combination alone (also ed to as monotherapy).

“Combination therapy” also embraces the stration ofthe compounds ofthe present invention in further ation with non-drug therapies (e.g., surgery or radiation treatment). Where the combination therapy fithher comprises a non-drug treatment, the non- drug treatment may be conducted at any suitable time so long as a beneficial effect from the co- action ofthe combination of the therapeutic compounds and non—drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non—drug treatment is temporally removed from the administration ofthe eutic compounds, perhaps by days or even weeks.

The non—drug treatment can be selected from chemotherapy, ion therapy, hormonal therapy, anti-estrogen therapy, gene therapy, and surgery. For example, a non-drug therapy is the removal of an ovary (e.g., to reduce the level of estrogen in the body), thoracentesis (e.g., to remove fluid from the , paracentesis (e.g., to remove fluid from the _15_ n), surgery to remove or shrink angiomyolipomas, lung transplantation (and optionally with an antibiotic to prevent infection due to transplantation), or oxygen therapy (e.g., through a nasal cannula containing two small c tubes or prongs that are placed in both nostrils, through a face mask that fits over the nose and mouth, or through a small tube ed into the windpipe through the front ofthe neck, also called transtracheal oxygen therapy).

The present invention also provides methods oftreating mTOR-related diseases, disorders, and conditions, in a subject in need thereofby administering to the subject a therapeutically effective amount of an apilimod composition of the invention. Such diseases and disorders include, for example, cancers in which mTOR is dysregulated. mTOR dysregulation has been implicated in 70 % of all cancers. See e.g., Menon et a] Oncogene 27 (2009):S43-SSl.

Specific cancers having a component ofmTOR ulation include brain tumors such as gliomas (e.g., glioblastoma multiforme), a, breast cancer, lung cancer (e.g., non-small-cell lung cancer), mesothelioma, appendiceal cancer, genitourinary cancers (e.g., renal cell carcinoma, prostate, r, testicular, penile, cervical , ovarian cancer, von Hippel Lindau disease), head and neck cancer, gastrointestinal tumors (e.g., hepatocellular carcinoma, gallbladder cancer, geal cancer, gastric cancer, colorectal cancer, or pancreatic cancer), neuroendocrine tumors (NETs), thyroid tumor, pituitary tumor, l tumor, hematological malignancy (e.g., Non-Hodgkin’s lymphoma, mantle cell lymphoma, myeloma, B-cell lymphoma, leukemia, Hodgkin’s lymphoma), or metastatic forms of one or more ofthe cancers described herein. See e.g., Laplante et al. Cell 149 (2012):274-293.

In the context ofthe methods described herein, the amount of an od composition administered to the subject is a therapeutically effective amount. The term “therapeutically ive amount” refers to an amount sufficient to treat, ameliorate a m of, reduce the severity of, or reduce the duration of the e or disorder being d, or enhance or improve the therapeutic effect of another therapy, or sufficient to exhibit a detectable therapeutic effect in the subject. In one embodiment, the therapeutically effective amount of an apilimod composition is the amount ive to inhibit PIKfyve kinase activity.

An effective amount of an apilimod composition can range from about 0.001 mg/kg to about 1000 mg/kg, about 0.01 mg/kg to about 100 mg/kg, about 10 mg/kg to about 250 mg/kg, about 0.1 mg/kg to about 15 mg/kg; or any range in which the low end of the range is any amount between 0.001 mg/kg and 900 mg/kg and the upper end ofthe range is any amount between 0.1 mg/kg and 1000 mg/kg (e.g., 0.005 mg/kg and 200 mg/kg, 0.5 mg/kg and 20 mg/kg). Effective doses will also vary, as recognized by those skilled in the art, depending on the diseases treated, route of administration, ent usage, and the possibility of co-usage with other therapeutic treatments such as use of other . See, e.g., US. Patent No. 7,863,270, incorporated herein by nce.

In more specific aspects, an apilimod composition is administered at a dosage regimen of30-1000 mg/day (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day) for at least 1 week (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, 36, 48, or more weeks). Preferably, an apilimod ition is administered at a dosage regimen of 100-1000 mg/day for 4 or 16 weeks. Alternatively or subsequently, an apilimod composition is administered at a dosage regimen of 100 mg-300 mg twice a day for 8 weeks, or optionally, for 52 weeks. atively or subsequently, an apilimod composition is administered at a dosage regimen of 50 mg-1000 mg twice a day for 8 weeks, or optionally, for 52 weeks.

An ive amount ofthe od composition can be administered once daily, from two to five times daily, up to two times or up to three times daily, or up to eight times daily.

In one embodiment, the apilimod composition is administered thrice daily, twice daily, once daily, fourteen days on (four times daily, thrice daily or twice daily, or once daily) and 7 days off in a 3-week cycle, up to five or seven days on (four times daily, thrice daily or twice daily, or once daily) and 14-16 days off in 3 week cycle, or once every two days, or once a week, or once every 2 weeks, or once every 3 weeks.

In accordance with the methods described herein, a “subject in need of’ is a subject having a disease, disorder or condition, or a subject having an increased risk of developing a disease, disorder or condition relative to the population at large. The t in need f can be one that is “non-responsive” or “refractory” to a currently available therapy for the disease or disorder, for example cancer. In this context, the terms “non-responsive” and “refractory” refer to the subject’s response to therapy as not clinically adequate to relieve one or more symptoms associated with the disease or er. In one aspect ofthe s described here, the subject in need thereof is a subject having cancer whose cancer is refractory to standard therapy or whose cancer has recurred following standard treatment.

A “subject” includes a mammal. The mammal can be e.g., any mammal, e.g., a human, primate, vertebrate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or a pig. Preferably, the mammal is a human. The term “patient” refers to a human subject. _17_ The present invention also provides a monotherapy for the treatment of a disease, er or ion as described herein. As used herein, “monotherapy” refers to the administration of a single active or therapeutic compound to a subject in need thereof.

As used herein, “treatment”, “treating” or “treat” describes the ment and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of an apilimod composition to alleviate the symptoms or complications of a disease, condition or disorder, or to ate the disease, condition or disorder.

As used herein, ntion”, “preventing” or “prevent” describes reducing or ating the onset of the symptoms or cations of the disease, condition or er and includes the administration of an apilimod composition to reduce the onset, development or recurrence of symptoms ofthe disease, condition or disorder.

In one embodiment, the administration of an apilimod composition leads to the elimination of a symptom or complication of the disease or disorder being treated, however, ation is not required. In one embodiment, the severity of the symptom is decreased. In the context of cancer, such symptoms may include clinical markers of severity or progression including the degree to which a tumor s growth factors, degrades the extracellular matrix, becomes vascularized, loses adhesion to juxtaposed tissues, or metastasizes, as well as the number ofmetastases.

Treating cancer according to the methods described herein can result in a reduction in size of a tumor. A reduction in size of a tumor may also be referred to as “tumor regression”. ably, after treatment, tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more ably, d by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.

Treating cancer ing to the methods described herein can result in a ion in tumor volume. Preferably, after treatment, tumor volume is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor volume is reduced by 10% or greater; more preferably, d by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by r than 75% or greater. Tumor volume may be measured by any ucible means ofmeasurement.

Treating cancer according to the methods described herein can result in a decrease in number oftumors. Preferably, after treatment, tumor number is d by 5% or greater relative to number prior to treatment; more preferably, tumor number is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. Number oftumors may be measured by any reproducible means of measurement. The number oftumors may be measured by counting tumors Visible to the naked eye or at a ed magnification. Preferably, the specified magnification is 2X, 3X, 4x, 5x, 10x, or 50x.

Treating cancer ing to the methods bed herein can result in a decrease in number of metastatic lesions in other tissues or organs distant from the primary tumor site.

Preferably, after treatment, the number of atic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by % or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. The number of metastatic lesions may be measured by any reproducible means ofmeasurement. The number of metastatic lesions may be measured by counting metastatic s visible to the naked eye or at a ed magnification. Preferably, the specified magnification is 2X, 3X, 4X, 5X, 10X, or 50X.

Treating a disorder, e or condition according to the methods bed herein can result in an increase in e survival time of a population oftreated subjects in comparison to a population receiving carrier alone. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any ucible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active nd. An increase in average survival time of a population may also be measured, for example, by ating for a population the average length of survival following completion of a first round oftreatment with an active compound. _19_ Treating a disorder, disease or condition according to the methods described herein can result in an increase in average survival time of a population oftreated subjects in comparison to a population of untreated subjects. Preferably, the average survival time is increased by more than 30 days; more ably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a tion may be measured by any ucible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival ing initiation of treatment with an active compound. An increase in e survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round oftreatment with an active compound.

Treating a disorder, disease or condition according to the methods described herein can result in increase in e survival time of a population oftreated subjects in comparison to a population receiving monotherapy with a drug that is not an apilimod composition as described . Preferably, the average survival time is increased by more than days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a tion may be measured, for example, by calculating for a population the average length of survival ing initiation of treatment with an active nd. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival ing completion of a first round of treatment with an active compound.

Treating a disorder, disease or condition according to the methods bed herein can result in a decrease in the mortality rate of a population oftreated subjects in comparison to a population receiving carrier alone. Treating a disorder, disease or condition according to the methods bed herein can result in a decrease in the mortality rate of a population of treated subjects in comparison to an ted population. Treating a disorder, disease or condition according to the methods described herein can result in a se in the mortality rate of a population of treated subjects in comparison to a tion receiving monotherapy with a drug that is not an apilimod composition. ably, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than _20_ %; and most ably, by more than 25%. A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation oftreatment with an active nd.

A decrease in the ity rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of ent with an active compound.

Treating a disorder, disease or ion according to the methods described herein can result in a decrease in tumor growth rate. Preferably, after treatment, tumor growth rate is reduced by at least 5% relative to number prior to treatment; more preferably, tumor growth rate is d by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. Tumor growth rate may be measured by any ucible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time. In one embodiment, after treatment the tumor growth rate may be about zero and is determined to maintain the same size, e.g., has stopped g.

Treating a er, disease or condition according to the s described herein can result in a se in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%. Tumor regrowth may be measured by any reproducible means ofmeasurement. Tumor regrowth is measured, for e, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by failure oftumors to reoccur after treatment has stopped.

Treating or preventing a cell proliferative disorder according to the methods described herein can result in a reduction in the rate of cellular proliferation. Preferably, after treatment, the rate of cellular proliferation is d by at least 5%; more preferably, by at least %; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The rate of cellular proliferation may be measured by any reproducible means ofmeasurement. The rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder ing to the methods described herein can result in a reduction in the proportion ofproliferating cells. Preferably, after treatment, the proportion iferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The proportion of erating cells may be measured by any reproducible means ofmeasurement. Preferably, the proportion ofproliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample. The proportion of proliferating cells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder according to the methods described herein can result in a decrease in the size of an area or zone of cellular proliferation. ably, after treatment, size of an area or zone of cellular proliferation is reduced by at least % relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, d by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, d by at least 50%; and most preferably, reduced by at least 75 %. The size of an area or zone of cellular proliferation may be measured by any reproducible means of measurement. The size of an area or zone of cellular proliferation may be measured as a diameter or Width of an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder according to the methods described herein can result in a decrease in the number or proportion of cells having an al appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5% ve to its size prior to treatment; more ably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more ably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, d by at least 75%. An abnormal cellular appearance or morphology may be measured by any reproducible means urement. An abnormal cellular morphology can be measured by microscopy, e.g., _22_ using an inverted tissue e microscope. An abnormal cellular morphology can take the form of r pleiomorphism.

As used herein, the term “selectively” means g to occur at a higher frequency in one population than in another population. The compared populations can be cell populations. Preferably, an apilimod composition as described herein acts selectively on hyperproliferating cells or abnormally proliferating cells, compared to normal cells. As used herein, a “normal cell” is a cell that cannot be classified as part of a “cell proliferative disorder”. A normal cell lacks unregulated or abnormal growth, or both, that can lead to the development of an unwanted ion or disease. Preferably, a normal cell possesses normally functioning cell cycle oint control mechanisms. Preferably, an apilimod composition acts selectively to modulate one molecular target (e.g., a target kinase) but does not significantly modulate another molecular target (e.g., a non-target ). The invention also provides a method for ively inhibiting the activity of an enzyme, such as a kinase. ably, an event occurs selectively in population A relative to population B if it occurs greater than two times more frequently in population A as compared to tion B. An event occurs selectively if it occurs greater than five times more frequently in population A. An event occurs selectively if it occurs greater than ten times more frequently in population A; more preferably, greater than fifty times; even more preferably, greater than 100 times; and most preferably, greater than 1000 times more frequently in population A as compared to population B. For example, cell death would be said to occur selectively in diseased or hyper-proliferating cells if it occurred greater than twice as frequently in diseased or hyper-proliferating cells as compared to normal cells.

Pharmaceutical itions and Formulations The present invention provides apilimod compositions that are preferably pharmaceutically acceptable compositions suitable for use in a mammal, preferably a human. In this context, the compositions may r comprise at least one pharmaceutically acceptable excipient or carrier, wherein the amount is effective for the treatment of a disease or disorder. In one embodiment, the disease or disorder is cancer, preferably a lymphoma, and most preferably a B cell lymphoma. In one embodiment, the disease or disorder is an mTOR disease or er.

In one embodiment, the od composition comprises apilimod free base or od dimesylate. _23_ In one embodiment, the apilimod composition is combined with at least one onal active agent in a single dosage form. In one embodiment, the composition filrther comprises an antioxidant.

In one embodiment, the at least one additional active agent is selected from the group consisting of an alkylating agent, an alating agent, a tublin binding agent, a corticosteroid, and combinations thereof. In one embodiment, the at least one additional active agent is a eutic agent selected from the group consisting of ibrutinib, rituximab, doxorubicin, prednisolone, vincristine, velcade, and everolimus, and combinations f. In one embodiment, the at least one additional active agent is a therapeutic agent selected from wdqfimflmmfiqhwhmwhmmmhdn@bomknafiowdmmmfldnmAmmmwmnfi vincristine (also referred to as OncovinTM), prednisone, prednisolone, and combinations thereof.

In one ment, the at least one additional active agent is a non-therapeutic agent selected to ameliorate one or more side effects ofthe apilimod composition. In one embodiment, the non- therapeutic agent is selected from the group consisting of ondansetron, granisetron, dolasetron and palonosetron. In one embodiment, the non-therapeutic agent is selected from the group consisting ofpindolol and risperidone.

In one embodiment, the at least one additional active agent is selected from an tor of the mTOR pathway, a PI3K inhibitor, a dual PI3K/mTOR inhibitor, a SRC inhibitor, a VEGF inhibitor, a Janus kinase (JAK) inhibitor, a Raf inhibitor, an Erk tor, a farnesyltransferase inhibitor, a histone deacetylase inhibitor, an itotic agent, a multi-drug resistance efflux inhibitor, an antibiotic, and a therapeutic antibody. In one embodiment, the at least one additional active agent is selected from a farnesyltransferase inhibitor (e.g., tipifamib), an anti-mitotic agent (e.g., docetaxel), a histone deacetylase tor (e.g., vorinostat), and a multi-drug resistance effluX inhibitor.

In one embodiment, the mTOR inhibitor is selected from the group consisting of rapamycin (also referred to as sirolimus), everolimus, temsirolimus, ridaforolimus, umirolimus, zotarolimus, AZD8 055, INK128, WYE-l32, Torin-1, pyrazolopyrimidine analogs PP242, PP30, PP487, PPl2l , 794, KU-BMCL1, Wyeth-BMCL9b, INK-128, XL388, AZD8055, P2281, and P529. See, e.g., Liu et a3. Drug Disc. Today Ther. Strateg., 6(2): 47-55 (2009).

In one embodiment, the mTOR inhibitor is trans[4-amino(7-methoxy-lH- indolyl)imidazo[5,l -f][l ,2,4]triazinyl]cyclohexane carboxylic acid (also known as 081- _24_ 027), and any salts, solvates, hydrates, and other al forms, crystalline or amorphous, thereof. See US 2007/0112005. OSI—027 can be prepared according to US 2007/0112005, incorporated herein by nce. In one embodiment, the mTOR inhibitor is OXA-01. See e.g., WO 2013152342 A1.

In one embodiment, the PI3K inhibitor is selected from the group consisting of GS-1101 lisib), GDC0941 (Pictilisib), LY294002, BKM120 (Buparlisib), PI—103, TGX- 221, IC-87114, XL 147, ZSTK474, , AS-605240, PIK-75, 3-methyladenine, A66, PIK- 93, PIK-90, 2, IPI-145 (Duvelisib), TG100-115, AS-252424, PIK294, AS-604850, GSK2636771, BAY 80-6946 lisib), CH5132799, 05, PIK-293, TG100713, CZC24832 and HS—173.

In one embodiment, the dual PI3K/mTOR inhibitor is selected from the group consisting of, 4, WAY-001, WYE-354, WAY-600, WYE-687, Wyeth-BMCL- 200910075-16b, Wyeth-BMCL27, 794 and KUBMCL5 , NVP- BEZ235, XL-765, PF-04691502, GDC-0980 (Apitolisib), GSK1059615, PF-05212384, BGT226, PKI—402, VS-558 and GSK2126458. See, e.g., Liu et a1. Drug Disc. Today Ther.

Strateg, 6(2): 47-55 (2009), incorporated herein by reference.

In one embodiment, the mTOR pathway inhibitor is a polypeptide (e.g., an antibody or fragment thereof) or a nucleic acid (e.g., a double-stranded small interfering RNA, a short hairpin RNA, a micro-RNA, an antisense oligonucleotide, a locked nucleic acid, or an aptamer) that binds to and ts the expression level or activity or a protein (or nucleic acid encoding the protein) in the mTOR pathway. For example, the polypeptide or c acid inhibits mTOR Complex 1 (mTORCl ), tory-associated protein ofmTOR (Raptor), mammalian lethal with SEC13 n 8 (MLST8), proline-rich Akt substrate of40 kDa (PRAS40), DEP domain-containing mTOR-interacting n (DEPTOR), mTOR Complex 2 (mTORC2), rapamycin-insensitive companion ofmTOR (RICTOR), G protein beta subunit-like (GBL), mammalian stress-activated protein kinase interacting protein 1 (mS]N1), paxillin, RhoA, Ras-related C3 num toxin substrate 1 (Racl), Cell division control protein 42 homolog (Cdc42), protein kinase C 0t (PKCOL), the serine/threonine protein kinase Akt, oinositide 3- kinase (PI3K), p7OS6K, Ras, and/or eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4EBPs), or the nucleic acid encoding one of these proteins.

In one embodiment, the SRC inhibitor is selected from the group consisting of bosutinib, saracatinib, dasatinib, ponatinib, KX2-391, XL-228, TG100435/TG100855, and _25_ DCC2036. See, e.g., Puls et al. gist. 2011 May; 16(5): 566—578. In one embodiment, the SRC inhibitor is a polypeptide (e.g., an antibody or fragment thereof) or nucleic acid (e.g., a double-stranded small ering RNA, a short n RNA, a micro-RNA, an antisense oligonucleotide, a locked nucleic acid, or an aptamer) that binds to and inhibits the sion level or activity ofthe SRC protein or a c acid encoding the SRC protein.

In one embodiment, the VEGF inhibitor is selected from zumab, sunitinib, pazopanib, axitinib, sorafenib, fenib, inib, and motesanib. In one embodiment, the VEGF inhibitor is a polypeptide (e.g., an antibody or fragment thereof) or nucleic acid (e.g., a -stranded small interfering RNA, a short hairpin RNA, a micro-RNA, an antisense oligonucleotide, a morpholino, a locked nucleic acid, or an r) that binds to and inhibits the expression level or activity of a VEGF protein, a VEGF receptor protein, or a nucleic acid encoding one ofthese proteins. For example, the VEGF inhibitor is a soluble VEGF receptor (e.g., a soluble VEGF-C/D receptor (sVEGFR—3)).

In one embodiment, the JAK inhibitor is selected from facitinib, ruxolitinib, tinib, CYT3 87 (CAS number 10566344), lestaurtinib, pacritinib, and TG101348 (CAS number 936091 8). In one embodiment, the JAK inhibitor is a polypeptide (e.g., an antibody or nt thereof) or nucleic acid (e.g., a double-stranded small interfering RNA, a short hairpin RNA, a micro-RNA, an antisense oligonucleotide, a morpholino, a locked nucleic acid, or an aptamer) that binds to and inhibits the expression level or activity of a JAK (e.g., JAKl JAK2, JAK3, or TYK2) or a nucleic acid ng the JAK protein.

In one embodiment, the Raf inhibitor is selected from PLX4032 (vemurafenib), sorafenib, PLX-4720, GSK2118436 (dabrafenib), GDC-0879, RAF265, AZ 628, NVP-BHG712, SB90885, ZM 336372, GW5074, TAK-632, CEP-32496 and LGX818 (Encorafenib). In one embodiment, the Raf inhibitor is a polypeptide (e.g., an antibody or fragment thereof) or nucleic acid (e.g., a double-stranded small interfering RNA, a short hairpin RNA, a micro-RNA, an antisense oligonucleotide, a morpholino, a locked nucleic acid, or an aptamer) that binds to and ts the expression level or activity of a Raf (e.g., A-Raf, B-Raf, C-Raf) or a nucleic acid encoding the Raf protein. In one embodiment, the MEK inhibitor is selected from AZD6244 (Selumetinib), PD0325901, GSKl 120212 (Trametinib), U0126-EtOH, PD184352, RDEA119 (Rafametinib), PD98059, BIX 02189, MEK162 (Binimetinib), AS-703026 (Pimasertib), SL-327, BIX02188, 0, TAK-733 and PD318088. In one embodiment, the MEK inhibitor is a polypeptide (e.g., an antibody or fragment thereof) or nucleic acid (e.g., a double-stranded small interfering RNA, a short hairpin RNA, a micro-RNA, an antisense oligonucleotide, a morpholino, a locked nucleic acid, or an aptamer) that binds to and inhibits the expression level or activity of a MEK (e.g., MEK-l or a nucleic acid encoding the MEK protein.

, MEK-2) In one embodiment, the Akt inhibitor is selected from MK-2206, KRX-040l osine), GSK690693, GDC-0068 (Ipatasertib), AZD5363, CCT128930, A-674563, PHT- 427. In one embodiment, the Akt inhibitor is a polypeptide (e.g., an antibody or fragment thereof) or nucleic acid (e.g., a double-stranded small interfering RNA, a short hairpin RNA, a micro-RNA, an antisense oligonucleotide, a morpholino, a locked nucleic acid, or an aptamer) that binds to and inhibits the sion level or activity of a Akt (e.g., Akt-l or a , Akt-2, Akt-3) nucleic acid ng the Akt protein.

In one embodiment, the farnesyltransferase inhibitor is selected from LB42708 or tipifarnib. In one embodiment, the farnesyltransferase inhibitor is a polypeptide (e.g., an antibody or fragment thereof) or nucleic acid (e.g., a double-stranded small interfering RNA, a short hairpin RNA, a micro-RNA, an antisense oligonucleotide, a morpholino, a locked nucleic acid, or an aptamer) that binds to and inhibits the expression level or activity of farnesyltransferase or a nucleic acid encoding the farnesyltransferase protein. In one embodiment, the histone modulating inhibitor is selected from anacardic acid, C646, MGl49 (histone acetyltransferase), GSK J4 Hcl ne demethylase), GSK343 (active against EZH2), BIX 01294 (histone methyltransferase), MK0683 (Vorinostat), M8275 (Entinostat), LBHS 89 (Panobinostat), Trichostatin A, MGCD0103 (Mocetinostat), Tasquinimod, TMP269, Nexturastat A, , PDXlOl (Belinostat).

In one embodiment, the anti-mitotic agent is selected from Griseofillvin, vinorelbine tartrate, paclitaxel, docetaxel, vincristine, vinblastine, Epothilone A, Epothilone B, ABT-751, CYT997 (Lexibulin), ine tartrate, tabulin, 364, ON-019 10 (Rigosertib), Ro3280, B12536, NMS-P937, B16727 (Volasertib), HMN-2 14 and MLN0905.

In one embodiment, the polyether antibiotic is selected from sodium monensin, nigericin, mycin, salinomycin.

A aceutical composition” is a ation containing the compounds described herein in a ceutically acceptable form suitable for administration to a subject.

As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, als, compositions, carriers, and/or dosage forms which are, Within the scope of sound l judgment, suitable for use in contact With the tissues n beings and animals Without _27_ excessive toxicity, irritation, allergic se, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that is usefill in preparing a pharmaceutical composition that is lly safe, xic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. Examples ofpharmaceutically acceptable excipients include, without limitation, sterile s, water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), oils, detergents, suspending , carbohydrates (e.g., glucose, lactose, sucrose or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or suitable mixtures thereof.

A pharmaceutical composition can be provided in bulk or in dosage unit form. It is especially ageous to formulate pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. The term “dosage unit form” as used herein refers to physically te units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the d therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active nd and the particular therapeutic effect to be achieved. A dosage unit form can be an ampoule, a via], a suppository, a dragee, a tablet, a capsule, an IV bag, or a single pump on an l inhaler.

In therapeutic applications, the dosages vary depending on the agent, the age, weight, and clinical condition ofthe recipient patient, and the experience and judgment ofthe clinician or practitioner administering the therapy, among other s affecting the selected dosage. Generally, the dose should be a therapeutically effective amount. Dosages can be provided in mg/kg/day units ofmeasurement (which dose may be adjusted for the patient’s weight in kg, body surface area in m2, and age in years). An effective amount of a ceutical composition is that which provides an objectively identifiable improvement as noted by the clinician or other ed observer. For example, alleviating a m of a disorder, disease or condition. As used herein, the term “dosage ive manner” refers to amount of a pharmaceutical composition to e the desired biological effect in a subject or cell.

For example, the dosage unit form can comprise l nanogram to 2 milligrams, or 0.1 milligrams to 2 grams; or from 10 milligrams to 1 gram, or from 50 rams to 500 milligrams or from 1 microgram to 20 rams; or from 1 microgram to 10 milligrams; or from 0.1 rams to 2 rams.

The pharmaceutical compositions can take any suitable form (e.g, liquids, aerosols, solutions, inhalants, mists, sprays; or solids, powders, ointments, pastes, creams, lotions, gels, patches and the like) for administration by any desired route (e.g, pulmonary, inhalation, intranasal, oral, buccal, sublingual, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrapleural, intrathecal, transdermal, transmucosal, rectal, and the like). For example, a pharmaceutical composition ofthe invention may be in the form of an aqueous solution or powder for l administration by inhalation or insufflation (either through the mouth or the nose), in the form of a tablet or capsule for oral administration;; in the form of a sterile aqueous solution or dispersion suitable for administration by either direct injection or by addition to sterile infusion fluids for intravenous infusion; or in the form of a lotion, cream, foam, patch, suspension, solution, or suppository for transdermal or ucosal administration.

A pharmaceutical ition can be in the form of an orally acceptable dosage form including, but not limited to, capsules, tablets, buccal forms, troches, lozenges, and oral liquids in the form of emulsions, aqueous suspensions, dispersions or solutions. Capsules may contain mixtures of a compound ofthe present invention with inert fillers and/or ts such as the pharmaceutically acceptable starches (eg, corn, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline oses, flours, gelatins, gums, etc. In the case oftablets for oral use, carriers which are commonly used include e and corn starch. Lubricating agents, such as magnesium stearate, can also be added. For oral administration in a capsule form, useful diluents include lactose and dried corn . When aqueous suspensions and/or emulsions are administered orally, the compound of the t invention may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending . If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

A pharmaceutical ition can be in the form of a tablet. The tablet can comprise a unit dosage of a compound of the present invention together with an inert diluent or r such as a sugar or sugar alcohol, for example lactose, sucrose, sorbitol or mannitol. The _29_ tablet can fithher comprise a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium ate, or a cellulose or derivative thereof such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch. The tablet can fithher comprise g and granulating agents such as nylpyrrolidone, disintegrants (e.g. swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (6.g. stearates), preservatives (e.g. parabens), antioxidants (e.g. BHT), buffering agents (for e phosphate or citrate buffers), and effervescent agents such as citrate/bicarbonate res.

The tablet can be a coated tablet. The coating can be a protective film coating (e.g. a wax or varnish) or a coating designed to control the release ofthe active agent, for example a delayed release (release ofthe active after a predetermined lag time following ingestion) or release at a ular location in the gastrointestinal tract. The latter can be ed, for example, using enteric film coatings such as those sold under the brand name Eudragit®.

Tablet formulations may be made by conventional compression, wet granulation or dry granulation s and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, surface ing agents (including surfactants), suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, rystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starches and powdered sugar. Preferred surface modifying agents include nonionic and c surface modifying agents. Representative es of surface modifying agents include, but are not d to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, crogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine.

A pharmaceutical composition can be in the form of a hard or soft gelatin capsule.

In accordance with this formulation, the compound of the present invention may be in a solid, semi-solid, or liquid form.

A pharmaceutical composition can be in the form of a sterile s solution or dispersion le for parenteral administration. The term parenteral as used herein includes _30_ subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infilsion ques.

A pharmaceutical composition can be in the form of a sterile aqueous solution or dispersion suitable for administration by either direct injection or by addition to sterile infusion fluids for intravenous infusion, and comprises a solvent or sion medium containing, water, l, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, or one or more vegetable oils. Solutions or suspensions ofthe compound of the present invention as a free base or acologically acceptable salt can be prepared in water suitably mixed with a surfactant. Examples of suitable surfactants are given below. sions can also be prepared, for example, in glycerol, liquid polyethylene glycols and mixtures ofthe same in oils.

The pharmaceutical itions for use in the methods ofthe present invention can fithher comprise one or more additives in addition to any carrier or diluent (such as lactose or mannitol) that is present in the formulation. The one or more additives can comprise or consist of one or more surfactants. tants typically have one or more long aliphatic chains such as fatty acids which s them to insert directly into the lipid structures of cells to enhance drug penetration and absorption. An empirical parameter commonly used to characterize the relative hydrophilicity and hydrophobicity of surfactants is the hydrophilic- lipophilic balance (“HLB” value). tants with lower HLB values are more hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have greater solubility in aqueous solutions. Thus, hydrophilic surfactants are lly considered to be those compounds having an HLB value greater than about 10, and hydrophobic surfactants are generally those having an HLB value less than about 10. However, these HLB values are merely a guide since for many tants, the HLB values can differ by as much as about 8 HLB units, depending upon the empirical method chosen to determine the HLB value.

Among the surfactants for use in the compositions ofthe ion are polyethylene glycol fatty acids and PEG-fatty acid mono and rs, PEG glycerol esters, l-oil transesterification products, polyglyceryl fatty acids, propylene glycol fatty acid esters, sterol and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar and its derivatives, polyethylene glycol alkyl phenols, polyoxyethylene-polyoxypropylene (POE-POP) block copolymers, sorbitan fatty acid esters, _31_ ionic surfactants, fat-soluble vitamins and their salts, water-soluble Vitamins and their amphiphilic derivatives, amino acids and their salts, and organic acids and their esters and anhydrides.

The present invention also provides packaging and kits comprising pharmaceutical compositions for use in the methods ofthe present invention. The kit can comprise one or more containers selected from the group consisting of a , a vial, an ampoule, a blister pack, and a syringe. The kit can fithher include one or more of instructions for use in treating and/or ting a disease, condition or disorder of the t invention, one or more syringes, one or more applicators, or a e solution suitable for reconstituting a pharmaceutical composition of the present invention.

All percentages and ratios used herein, unless otherwise indicated, are by weight.

Other features and advantages ofthe present invention are apparent from the different examples.

The ed examples illustrate different components and methodology useful in practicing the present ion. The examples do not limit the claimed invention. Based on the present disclosure the d artisan can identify and employ other components and methodology usefiJl for practicing the present invention.

EXAMPLES Example 1: Apilimod is a highly ive inhibitor of TSC2 null cell proliferation Apilimod was identified in a high throughput cell Viability screen using TSC2-/— mouse embryonic fibroblasts (MEF-EV) cells. TSC2 null cells have constitutively active mTOR. Briefly, MEF cells derived from TSC2 —/— knockout mouse embryos (Onda et al., J.

Clin. . 104(6):687-95, 1999) were infected with a retrovirus vector encoding the hygromycin antibiotic resistance gene (MEF-EV) or the same retrovirus vector also encoding TSC2 (MEF-TSC2). The MEF-EV and MEF—TSC2 line were then established by hygromycin selection.

Cells were ed in DMEM containing 10% FBS (Omega Scientific) and 2mM L-Glutamine. Frozen stocks of cells were ed for direct use in the HTS assay. Cells were ted, pelleted and then resuspended in 95% FBS & 5% DMSO at a concentration 1X107 cells/ml. One ml aliquots were rate frozen to -80 at a rate of 1 degree per minute. These stocks were then transferred to vapor phase liquid nitrogen for long term storage. _32_ For ing, Vials were thawed at 37°C with continuous agitation until just thawed then re-suspended in room temperature assay media and centrifuged at 1,000 rpm for 5 minutes. The resulting pellet was re-suspended in appropriate volume and counted using an automated cell counter and diluted accordingly to a final count of 40,000 cells/ml.

Test compounds (5 ul stock solution, 6 x desired final well concentration) were dispensed to 384-well assay plates (Coming 3712) using a Biomek FX liquid handler. MEF-EV cells (1000 cells per well in 25 uL of media) were added to these pre-formatted plates using a Therrno Wellmate, non-contact dispensing system with a standard bore te head. Plates were incubated for 72h at 37°C under an here of5% C02 in a fied incubator.

Cell Viability was ined with CellTiter-Glo® luminescence assay (Promega) as per the manufacturer’s instructions. Viability was sed as a percentage ofuntreated control cells. As an example, for apilimod, MEF-EV cell Viability (Mean +/- StDev, n=3) was 2.16 +/- 0.36% @ 0.5 uM and 1.94 +/- 0.07% @ 5 uM.

The activity of apilimod on TSCZ deficient cells was further demonstrated by performing 10 point dose response on the MEF—EV and MEF-TSC2 lines described above as well as three additional pairs of isogenic lines: (1) (TSC2 -/-, ) and (TSC2 +/—, 1953 -/-) MEF lines were established from (TSC2 -/-, p53-/—) or (TSC2 +/—, 1953 -/-) embryos according to standard methods. See e.g., Zhang et a]. J. Clin. Invest. 112, 1223-33, 2003. (2) ELT3-EV and ELT3-TSC2 lines were established from the ELT3 rat tumor cell line. The ELT3 line is an established rat tumor model for LAM/TSC. See e.g., Howe et al., Am. J. Path. 146, 1568-79, 1995. These cells harbor an inactivating mutation in TSC2, which leads to constitutive activation ofthe mTOR pathway. To p an isogenic pair of cells ELT3 cells were infected with a retrovirus vector encoding the hygromycin antibiotic resistance gene (ELT3-EV) or the same retrovirus vector also encoding TSC2 TSC2). The ELT3-EV and ELT3-TSC2 line were then established by hygromycin selection. (3) TRI—AML102 and AML103 lines were established from a TSC2 null primary human AML sample provided by Dr. Elizabeth Henske (Fox Chase Cancer , Philadelphia, PA). The cells were infected with amphotropic retrovirus LXSN16E6E7 that encodes the HPV16 E6 and E7 open reading frames and neomycin resistance cassette. Cells were ed and neomycin-selected. Individual clones were isolated and frozen down. The coding sequence for the human Telomerase gene (hTERT) with hygromycin resistance cassette (pLXSN hyg plasmid) was stably expressed into a TSC2'/' confirmed E6E7 AML clone using Fugene6 transfection reagent (Roche Applied Science, Indianapolis, IN). TRI-AML102 was generated by stable incorporation of a control zeomycin selection plasmid 3 .1 -zeo), while TRI—AML103 expresses the human TSC2 cDNA pcDNA3.1-zeo plasmid. As a result ofthese engineering ses, both TR1102 and TR1103 are neomycin, hygromycin, and zeomycin resistant lines.

For 10-point dose response, 750 MEF, 2000 ELT3, or 2000 AML cells in 100 [L of growth media (DMEM (CellGro 10CV) PBS 10% (Sigma Aldrich F2442-500ML, Lot 12D370) Penicillin/Streptomycin (100X) (CellGro Ref 30-002) were plated per well of a 96 well plate. 24 hours after plating cells, the media was removed and apilimod dilutions (1-500 nM, 2- fold dilutions) in 100 [L of growth media were added (0.1% final DMSO concentration). 72 hours after compound addition, relative cell viability was ined by CellTiter—Glo® scence assay (Promega) and expressed as a percentage relative to vehicle (DMSO) treated control cells. IC50 values were then calculated from the dose response curves using XLFIT (lDBS).

The TSC2 deficient cells were highly sensitive to apilimod (IC50 = 20 nM, Ligm 1). TSC2 -/- p53-/— MEFs demonstrated sed sensitivity to apilimod compared to the TSC2 +/- p53 -/- MEFs as indicated by a selectivity ratio above 1 (2.45).

Table 1: IC50 (viability) of apilimod in various cell types MEF MEF AML ELT3 Ce11 tWe".

TSC2 -/- TSC2 -/- TSC2 +/- 053-/- 053 —/— IC50 TSC2 rescue 20.10 70.70 132.00 16.05 IC50s (nM) calculated from 10-p0int dose response on TSC2-/- deficient and rescue lines. IC50s are calculated from the average oftwo experiments. The selectivity ratio is ated by dividing the IC50 of the TSC2 rescue line by the TSC2-/- line. rmore, higher concentrations ofapilimod had higher potency on the TSC2-/- MEF-EV cells compared to the TSCZ rescue MEF-TSC2 cells. This data, coupled to the fact that apilimod is not xic on peripheral blood mononuclear cells (Wada er al., Blood 109, 1156-64, 2007), nor on a variety of other cancer lines including U937, HELA, Jurkat, and THP-l (PCT Publication No. WC 2006/128 129) suggests that there will be a high therapeutic index for treating — cancer cells with od (Figure 2A-2C). _34_ Example 2: Apilimod is a highly selective cytotoxic agent in cancer cells The cytotoxic activity of apilimod was evaluated using a standard cell viability assay such as CellTiterGloTM ing to the manufacturer’s instructions. 122 human cancer cell lines were evaluated for sensitivity to apilimod. A cell line was called as apilimod sensitive ifthe IC50 was less than 500 nM. 35 cell lines were identified as sensitive to apilimod-induced cytotoxicity. Apilimod was also highly selective for cancer cells compared to normal cells, which had ICso’s ranging from 20-200 fold higher than the cancer cells e 2A-2C).

Figure 2A shows that the apilimod-sensitive cells included cells derived from several different cancers including non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, colorectal cancer, and lung cancer. The most sensitive of those tested were dgkin’s Lymphoma (NHL) cell lines. Just over 50% of the NHL cell lines tested were sensitive to od. NHL represents a diverse group ofhematopoietic malignancies that vary in severity, with subtypes ranging from slow growing to aggressive. Subtypes ofNHL include diffuse large B cell lymphoma (DLBCL), Burkitt’s lymphoma, mantle lymphoma, and follicular B cell lymphoma.

DLBCL is divided into two subtypes, GCB and ABC, based on gene expression and cell of origin. The GCB are germinal center B cell type, arising from normal germinal center B cells, and the ABC are activated B cell type, arising from post-germinal center B cells in the process of differentiating into plasma cells. In the present study, we found that certain subtypes ofNHL were extremely sensitive to od, with IC50 values of less than 100 nM (compared to the cutoff for sensitive/insensitive in the screen, which was 500 nM). These ed a human t's lymphoma (ST486), a human mantle cell lymphoma l) and a human DLBCL (SUDHL-4, IC50 = 50 nM). See Figure 2B. These results te that apilimod may be effective against many NHL cancers, including the more aggressive subtypes that are ofien refractory to standard ents.

As ed in Examples 6 and 7, infra, we investigated the biological mechanisms underlying apilimod’s selective cytotoxicity against cancers cells and found that it is due to an inhibition of intracellular trafficking and a corresponding increase in apoptosis in those cells.

Example 3: Apilimod synergizes with components of CHOP As discussed above, NHL cells demonstrated particular ivity to apilimod in our cancer cell line screen. DLBCL is the most common type ofNHL, accounting for 30-40% of _35_ lymphomas in n countries. DLBCL is an aggressive neoplasm of mature B cells.

Approximately 40% of all DLBCL patients relapse alter first line treatment. Many refractory DLBCL-GCB cancers exhibit single and double translocations ofMYC and BCL2. Patients with these genetic variants tend to have a poorer prognosis due at least in part to overexpression of MYC and BCL2. Notably, apilimod was ive even in DLBCL-GCB cell lines exhibiting these translocations (M), supporting a role for od in the treatment of even aggressive subtypes ofNHL, either alone, as monotherapy, or in combination with standard ents.

Table 2. Bcl-2 and c-myc ocation status for B Cell Lymphoma Lines and their ivity to apilimod. ND= No Data Number B Cell Lymphoma Model Cell Line IC50 Bcl-2 C-myc (n M) 7 Human DLBCL-GCB SUDHL—4 25 Yes Yes 8 Human DLBCL-GCB 6 80 Yes No 9 Human DLBCL-GCB DB 150 No No Human DLBCL-GCB Toledo 270 ND ND 11 Human DLBCL-GCB SUDHL—10 20 Yes Yes 12 Human DLBCL-GCB WSU-DLCL2 160 Yes No 13 Human DLBCL-GCB OCI-Ly19 380 Yes No Human DLBCL-GCB HT 642 ND ND 21 Human DLBCL-GCB er 2,620 ND ND To filrther evaluate the effectiveness ofapilimod against aggressive NHL tumors, the ability of apilimod to act synergistically with any of a number of chemotherapeutic agents that comprise the standard first line treatment for many such cancers was tested. These included, for example, hosphamide, doxorubicin, vincristine and prednisone (referred to as the “CHOP” herapy regimen), and rituximab, which is sometimes combined with CHOP (R- CHOP), as well as the chemotherapeutic agents velcade, which is indicated for relapsed mantle cell lymphoma, and everolimus, an inhibitor ofmTOR.

For synergy studies the following DLBCL-GCB cell lines were used: WSU- DLCL2, SUDHL-4 and SUDHL-6. Cells were seeded in 96 well plates at their optimum density.

Cells were treated with apilimod alone (7.8 - 1000 nM), bicin (3.13 — 400 nM), vincristine (0.08 — 10 nM), prednisone (19.5 — 2500 nM), velcade (0.16 — 20nM), or everolimus (0.23 — 500 nM), either alone or in a combination with apilimod. In each case, the dilutions were 2-fold With a total of 8 dilutions over the drug concentration range.

Cells were treated for 72 h before proliferation was assessed using CellTiterGlo® (Promega). For calculation of synergy, CalcuSyn (version 2.11, Biosoft) was used to determine the combination index (CI) as defined by Chou et (1]., Adv. Enzyme. Regul. (1984) 22 227—55.

Thus, drug ations producing CI values > 1 were defined as antagonistic, CI = l as additive, and CI < l as synergistic.

As shown inm, apilimod demonstrated synergistic activity with 5 of 6 agents tested (doxorubicin, prednisolone, vincristine, velcade, and everolimus) in the SUDHL-6 cell line and was synergistic with vincristine in all three cell lines. In on, apilimod was synergistic with prednisolone, velcade, and everolimus in at least two of the three cell lines tested. These results demonstrate that combination therapy with apilimod represents a promising new approach for addressing the unmet medical need for treatments that benefit patients who relapse after or who are refractory to standard chemotherapy regimens.

Table 3 Combination Treatment WSU-DLCL2 -4 SU-DHL—6 Cyclophosphamide Apilimod ND ND ND Standard Doxorubicin Apilimod Antagonistic ND Synergistic of care 50-400 nM 125-500 nM Prednisolone Apilimod Synergistic ND Synergistic 62-1667 nM 62.5-500 nM Vincristine Apilimod Synergistic istic Synergistic 0.6-10 nM 62.5-1000 nM Other therapies Velcade Apilimod Additive Synergistic Synergistic 1.3-5 nM 625—250 nM Everolimus Apilimod Synergistic ND Synergistic 18.5-55.6 nM 625—250 nM y ofdrug combination effects of apilimod and individual components ofCHOP (excluding cyclophosphamide), Velcade or Everolimus in DLBCL—GCB cell lines. Combination index (CI) was used to determine combination effects, where C1 > 1 is antagonistic, CI = 1 is additive and CI < l is synergistic. The range of concentrations of apilimod in combination with either CHOP components, Velcade or Everolimus to produce the described effect is shown (italics). \ Example 4: Synergistic activity n od and ibrutinib Studies in SUDHL-4 cells were also undertaken to screen for other drugs that could act synergistically with apilimod. A ly curated y of 93 drugs including both FDA approved and oved drugs was used in the . Cells were grown in the presence of drug, with or without apilimod (at IC20 = 10 nM), with each drug ofthe y being tested in a 10 point-concentration response curve (1.5 — 30,000 nM; 3 —fold dilutions). 4 cells were grown in RPMI Medium 1640 containing (Sigma h F2442-500ML, Lot l2D370) _37_ Penicillin/Streptomycin (100X) (CellGro Ref ). Cells were seeded into 96 well plates at a y of 19,000 cells per well, in a final volume of 50 uL. 50 uL ofthe 10 point drug dilution series (at 2x) was added to the cells to give the final concentrations stated above. Plates were incubated at 37°C under an atmosphere of5% C02 in a humidified incubator. 72 hours after compound addition relative cell viability was determined by CellTiter—Glo® luminescence assay (Promega) as per the manufacturer’s instructions, and values were expressed as a percentage ve to vehicle (DMSO) treated control cells (set to 100%).

The viability of cells treated with individual compound in the drug library was ed to the viability of cells treated with each library drug + apilimod (ICQO) and significant combinations were identified. Ibrutinib was identified as cantly reducing SUDHL-4 cell viability in the presence of apilimod compared with either ibrutinib or apilimod alone. See Figure 18. Ibrutinib is an FDA-approved drug targeting B-cell malignancies and indicated for monotherapy in treating mantle cell lymphoma and chronic lymphocytic leukemia. It is also known as PC1-32765 and marketed under the trade name ImbruvicaTM. Ibrutinib is a selective and covalent tor of the enzyme Bruton's tyrosine kinase (BTK). BTK is a key or of at least three critical B-cell pro-survival mechanisms occurring in parallel - regulation of apoptosis, cell adhesion and cell migration and homing. The synergistic ty of apilimod with ibrutinib filrther indicate that apilimod is a promising agent for use in combination therapy with other chemotherapy agents, especially those targeted against B-cell lymphomas. e 5: Anti-tumor activity of apilimod in combination with ibrutinib on DLBCL tumors in vivo The ability of apilimod to inhibit tumor growth in viva, either alone or in ation with ibrutinib was tested next. As described below, apilimod alone significantly reduced tumor growth and the combination of od and ibrutinib provided greater growth inhibition than either agent alone.

The study objective was to evaluate pre—clinically the in viva therapeutic efficacy of apilimod in the treatment of a subcutaneous SUDHL-6 human DLBCL cancer xenograft model alone, and in combination with ibrutinib.

In the first arm of the study, apilimod was tested alone. The SUDHL-6 cell line was maintained in RPMI—l 640 medium supplemented with 10% fetal bovine serum and L- glutamine (2 mM) at 37°C in an here of5% C02. The tumor cells were sub-cultured twice weekly and harvested during ntial growth for tumor ation. NOD-SCID mice were )f-irradiated 24 hrs before inoculation. Each mouse was inoculated subcutaneously in the right flank with SU-DHL-6 tumor cells (5 X 106) in 0.1 m1 ofPBS with Matrigel (1:1). The tumors were then grown to a mean size of approximately 80-120 mm3 and the mice were then split into groups and treated as detailed in the Table 4.

Table 4: Xenografi Model ofDLBCL tumors route of mice 1 Vehicle(Sa|ine ) - QDXS -2days off-QDXS i.v. 6 2 Apilimod 67.5mg/kg QDX5 -2days off-QDXS i.v. 6 Dimesylate (47mg/kg Free Base} 3 0.5% - BIDXS -2days DXS p.0. 6 Methylcellulose 4 Apilimod Free g BIDXS -2days off-Ble5 p.0. 6 Base Apilimod Free 150mg/kg QDX5 -2days e5 p.0. 6 Base Tumor size was measured twice a week in two dimensions using a r, and the volume is expressed in mm3 using the formula: V = 0.5 a X b2 where a and b are the long and short diameters of the tumor, respectively. The mice were monitored for 29 days and significant growth inhibition was observed in all apilimod treatment arms. Intravenous administration reduced tumor size by 58% (47 mg/kg) and oral dosing reduced growth by 68 % (150 mg/kg split dose) or by 64% (150 mg/kg single dose) with negligible effect on body weight (see F_igm m). Thus, intravenous and oral administrations of od displayed similar efficacy in impairing the growth of -6 tumors in vivo.

The second arm ofthe study evaluated efficacy of apilimod when combined with ibrutinib in the same SUDHL-6 human DLBCL cancer xenografi model using the same protocol as described above. Each mouse was inoculated subcutaneously in the right flank with SU-DHL-6 tumor cells (5 x 106) in 0.1 m1 ofPBS with Matrigel (1:1). The tumors were then grown to a mean size of approximately 80-120 mm3 and the mice were then split into 6 groups and treated as detailed in the Table 5.

Table 5: SUDHL-6 cell line xenograft experiment Group Treatment Dose Dosing schedule Administration Number route of mice 1 Vehicle NA days off— p.o. + i.V. 6 QDx5 2 Apilimod Free Base 75 mg/kg QDx5-2days off— p.0. 6 QDx5 3 Ibrutinib 10 mg/kg QD>< 12 i.V. 6 4 Ibrutinib 20 mg/kg QD>< 12 i.V. 6 Apilimod Free Base + 75 mg/kg + QDx5-2 days off— p.o. + i.V. 6 Ibrutinib 10 mg/kg QDx5 + QD>< 12 6 Apilimod Free Base + 75 mg/kg + days off— p.o. + iv 6 Ibrutinib 20 mg/kg QDx5 + QD>< 12 Tumor size was measured twice a week in two dimensions using a caliper, and the volume is expressed in mm3 using the formula: V = 0.5 a X b2 where a and b are the long and short diameters of the tumor, respectively. The mice were red for 31 days and significant growth inhibition was observed in the 75 mg/kg apilimod (57%), 10 mg/kg nib (54%), and mg/kg ibrutinib (64%) treatment arms. The ation of75 mg/kg apilimod with ibrutinib fithher reduced tumor growth in a dose dependent manner; 10 mg/kg ibrutinib (65%) and 20 mg/kg ibrutinib (70%)(see Figure 17).

Example 6: Apilimod is a highly selective binder of PIKfyve Kinase In order to identify the cellular target of apilimod in cancer cells, whole cell lysate prepared from human neuroglioma cells was used to identify its binding partners using chemical capture mass spectrometry (CCMS). This work was performed at Caprotec Bioanalytics GmbH, Berlin Germany. See Michaelis el al., J. Med. Chem, 55 3934-44 (2012) and references cited therein. Briefly, two capture compound variants employing apilimod as ivity function ed in a single orientation were synthesized and analyzed by LC-MS and lH-NMR to ensure identity and purity. Capture conditions were optimized in whole cell lysate, e.g. zation ofnon-specific interactions ofthe proteins with capture compounds, concentration of ts and proteins to obtain maximum binding of proteins and capture compounds, etc. _40_ One capture compound was selected to identify specific n s in the CCMS experiments using apilimod as a competitor ligand. Proteins that are detected by LC-S in the capture assay and that are significantly diminished in competition control experiments are considered to be specific binders. These specific binders were further subjected to stringent data analysis criteria to determine specificity after unbiased data evaluation. Specific protein binders were ranked according to their fold change (FC) values in the capture experiments. Only two proteins were identified as high probability candidate target proteins of apilimod: PIKfyve and Vac14. FC and p-values for these proteins in the four different capture compound concentration experiments are shown in Table 6.

Table 6. wwfimxaw‘u \\ ‘ 0 6.3 6.2 4 1 4.

PIKfyve 2 10g10(pValue)37285139 10g (FC) M53“ 5 6 Inf 3 9 Vacl4 W3\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ -log10(p-value) 3 9 3 8 1 9 3 6 In a separate study, protein kinase ng ofapilimod was ted to identify kinase targets (DiscoveRx, Fremont, CA). A dissociation constant (Kd) study was performed using apilimod at increasing concentrations (0.05 — 3000 nM) against PIKfyve, a known target of apilimod. The experiment was performed in duplicate and the Kd was determined to be 0.075 nM (range 0.069 — 0.081 nM) (F_igLe7).

Next, od was screened against a comprehensive panel ses ve not ed). In total, 456 kinases, including disease-relevant kinases, were assayed for their y to bind with apilimod. The screening tration ofapilimod was 1 HM, a concentration that is >l0,000 times greater than the K1 for apilimod against PIKfyve. The results from the screen showed that apilimod did not bind to any ofthe 456 s tested.

Together, these results demonstrate that apilimod binds with high selectivity in cancer cells to a single cellular kinase, PIKfyve. PIKfyve is an enzyme that binds to PI(3)P and _41_ catalyzes the formation ofthe lipid second messengers PI(3,5)P2 and PI(5)P and others have shown that apilimod is also a potent and specific inhibitor ofthis kinase PIKnye in normal cells.

Cai X et al., Chem Biol. 2013 Jul 25 ;20(7):912-21. As sed in more detail below, in order to understand the mechanism of apilimod’s selective cytotoxicity against cancer cells, we conducted a series of experiments aimed at elucidating its biological activity in cancer cells.

Example 7: Mechanism of Anti-cancer Activity of Apilimod Apilimod is known to be a potent inhibitor of the inflammatory cytokines IL-12 and IL-23. To the extent apilimod was indicated for ng a e or disorder, it was predicated on this activity. Although the clinical testing ofapilimod focused on its ial efficacy in autoimmune and inflammatory diseases such as psoriasis, rheumatoid arthritis, and Crohn's disease, there were a few published tions that apilimod might be useful against cancers, and specifically against cancers in which c-rel or IL—12/23 were acting as pro- proliferative factors. See e.g., and Baird el al., Frontiers in Oncology 3:1 (2013), respectively. singly, and contrary to these expectations predicated on apilimod’s IL-12/23 inhibitory actiVity, we found no correlation between any of c-Rel expression (c-Rel is a transcription factor for the IL-12/23 genes), IL-12, or IL-23 sion and ivity to apilimod in the tested cell lines (see Figures 8-14).

Briefly, gene expression data from the Cancer Cell Line Encyclopedia (CCLE) was analyzed for the 22 B cell lymphoma lines for which we obtained dose response curves against apilimod (see Table 7).

Table 7. 22 B Cell ma Lines analyzed for gene sion and response to apilimod.

Epstein Barr status and nuclear cREL status is noted. ND= No Data Number B Cell Lymphoma Model Cell Line |C50 EBV Nuclear (n M) REL 1 Human Burkitt's lymphoma ST486 25 No ND 2 Human Burkitt's lymphoma Daudi 200 Yes Yes 3 Human Burkitt's lymphoma E31 174 Yes ND 4 Human Burkitt's lymphoma GA-lO 382 No ND Human Mantle Cell Lymphoma Rec-1 300 No ND 6 Human Mantle Cell Lymphoma JeKo—l 70 No ND 7 Human e Large B Cell Lymphoma —GCB SU DHL—4 25 No Yes 8 Human Diffuse Large B Cell Lymphoma —GCB SU DHL—6 80 No ND 9 Human Diffuse Large B Cell Lymphoma —GCB DB 150 No ND _42_ Human Diffuse Large B Cell ma —GCB Toledo 270 No ND 11 Human Diffuse Large B Cell Lymphoma —GCB SU DHL-10 20 No ND 12 Human Diffuse Large B Cell Lymphoma —GCB WSU-DLCL2 160 No ND 13 Human Diffuse Large B Cell Lymphoma —GCB OCl—Ly19 380 Yes ND 14 Human Burkitt's ma Namalwa 600 Yes ND Human Burkitt's lymphoma CA46 >10,000 No ND 16 Human Burkitt's lymphoma Raji >10,000 Yes Yes 17 Human Mantle Cell Lymphoma GRANTA—519 >10,000 Yes ND 18 Human Follicular B Cell Lymphoma RL >10,000 ND ND 19 Human Follicular Lymphoma - DLBCL-GCB DOHH-2 700 No ND Human Diffuse Large B Cell Lymphoma -GCB HT 642 No ND 21 Human Diffuse Large B Cell Lymphoma -GCB Pfeiffer 2,620 ND ND 22 Human Diffuse Large B Cell Lymphoma —GCB KARPAS-422 >10,000 No ND Expression of c—REL was compared in sensitive (IC 50 less than 500 nM) and insensitive (ICso greater than 500 nM) lines by ed t-test. No statistically significant relationship between c-REL expression and sensitivity was found (p=0.97). Furthermore, no detection of a cant relationship between sensitivity to od and either the presence of tutive nuclear c-REL or infection with Epstein Barr virus in cell lines for which data has been published was found. The cell lines tested included the following apilimod ive (#1 -l3) and insensitive (#14-22) B cell lymphoma lines: Human Burkitt’s lymphoma cell lines l-4 (ST486, Daudi, EBl Human Mantle Cell Lymphoma 5-6 (Rec-l Human , GA-lO), , JeKo-l), Diffilse Large B Cell Lymphoma —GCB 7—13(SUDHL-4, SUDHL-6, DB, Toledo, SUDHL-lO, WSU-DLCL2, OCl-Lyl9), Human Burkitt’s ma 14-16 (Namalwa, CA46, Raj i), Human Mantle Cell Lymphoma l7 (GRANTA-Sl9), Human Follicular B Cell Lymphoma 18 (RL), Human ular Lymphoma-DLBCL-GCB l9 (DOHH-2), Human Diffuse Large B Cell Lymphoma —GCB (HT, Pfeiffer, KARPAS-422).

The expression ofIL-12A, IL-12RB1, IL-12RB2, IL-l2B, IL-23A and IL-23R was further analyzed in a diverse group of75 cancer cell lines, ing the aforementioned 22 lymphoma lines (see Table 8).

Table 8. Various Cancer cell lines Cancer Model Cell Line |C50 (nM) Human Burkitt's lymphoma ST486 HumanManue Ce" Lymphoma Human Diffuse Large B Cell Lymphoma —GCB SU DHL-4 _43_ 4 Human e Large B Cell ma —GCB SUDHL-6 80 Human Burkitt's lymphoma Daudi 200 6 Human cytic lymphoma U937 106 7 Human lung carcinoma A549 110 8 Human colorectal cancer HCT116 125 9 Human B-cell lymphoma DB 150 Human Diffuse Large B Cell Lymphoma —GCB WSU—DLCL2 160 11 Human Colorectal HCT—15 200 12 Human Colorectal SW480 90 13 Human Colorectal COLO-205 380 14 Human Colorectal SW620 90 Human T-cell leukemia Jurkat 200 16 Human lioma H4 250 17 Human Diffuse Large B Cell ma —GCB Toledo 270 18 Human B cell Non-Hodgkin's Lymphoma Rec-1 300 19 Human Hodgkin's lymphoma KMH—2 181 Human Burkitt's lymphoma E31 174 21 Human Diffuse Large B Cell Lymphoma —GCB SUDHL-10 20 22 Human Burkitt's lymphoma GA—10 382 23 Human Diffuse Large B Cell Lymphoma —GCB OCl—Ly19 380 24 Human Diffuse Large B Cell Lymphoma -GCB HT 642 Human Diffuse Large B Cell Lymphoma -GCB Pfeiffer 2,620 26 Human Burkitt's lymphoma Namalwa 600 27 Human Follicular B Cell Lymphoma-GCB DOHH-2 700 28 Human Bladder carcinoma (GATOR -/—) SW780 1000 29 Human colorectal cancer MDST8 1000 Human Burkitt's lymphoma Raji 10,000 31 Human Hodgkin's lymphoma HD-MyZ >1000 32 Human Hodgkin's lymphoma L540 >1000 33 Human Hodgkin's lymphoma HDLM—2 >1000 34 Human Burkitt's lymphoma CA46 >10,000 Human Anaplastic Large Cell Lymphoma l 590 36 Human lung carcinoma H1734 1500 37 Human colorectal cancer SW1116 1500 38 Human ctal COLO—320DM 2,060 39 Human neuroblastoma A172 2000 40 Human lung carcinoma H1693 2000 41 Human lung carcinoma H460 > 2000 42 Human lung carcinoma H358 >2000 43 Human pancreatic cancer CAPANZ >2000 44 Human pancreatic cancer PANCl >2000 45 Human pancreatic cancer MiaPaCa-Z >2000 _44_ 46 Human pancreatic cancer AsPCl >2000 47 Human prostate cancer DU145 >2000 48 Human acute myelogenous leukemia KG—l >2500 49 Human prostate cancer LnCap 3000 50 Human T-cell ma HH 3,300 51 Human T-cell leukemia MOLT-4 3,300 52 Human prostate cancer 22RV1 >5000 53 Human colorectal cancer DLD—l >5000 54 Human myelogenous leukemia K562 >5000 55 Human colorectal cancer RKO >5000 56 Human ovarian TOV-21G 7000 57 Human prostate cancer PC-3 10,000 58 Human Hodgkin's lymphoma L428 10,000 59 Human plasmacytoma RPMl—8226 >10,000 60 Human lung carcinoma NCI—1975 >10,000 61 Human breast cancer CAMAl >10,000 62 Human neuroblastoma SW1088 >10,000 63 Human neuroblastoma M0591K >10,000 64 Human neuroblastoma U-118 MG >10,000 65 Human neuroblastoma U—87 MG >10,000 66 Human acute monocytic leukemia THP1 >10,000 67 Human Diffuse Large B Cell Lymphoma -GCB KARPAS-422 >10,000 68 Human Follicular B Cell Lymphoma RL 0 69 Human Mantle Cell Lymphoma GRANTA-519 >10,000 70 Human bronchioalveolar NCl-H1650 >20,000 71 Human ioalveolar SW1573 >20,000 72 Human bronchioalveolar NCl-H1781 >20,000 73 Human bronchioalveolar 666 20,000 74 Human Colorectal LOVO >10,000 75 Human Colorectal HT—29 >10,000 Briefly, gene expression data from the CCLE was analyzed for the 75 cancer cell lines for which dose response curves t apilimod were obtained. The expression of each interleukin gene was compared in sensitive (IC50 less than 500 nM) and insensitive (IC50 greater than 500 nM) lines by unpaired t-test. No tically significant relationship was found with the sole ion of lL-23A (p = 0.022). lL-23A has been previously noted to be elevated in apilimod sensitive non small cell lung cancer lines, and recombinant IL-23A was noted to increase proliferation ofnon small cell lung cancer lines (see Baird el al. 2013, supra).

Importantly, the statistical significance of lL-23A sion in sensitive cancer lines appears to _45_ be driven entirely by just two colon cancer lines. Furthermore IL-23A expression is not a statistically significant predictor of ivity in Non-Hodgkin's B cell lymphoma (Figure 15).

Global gene expression data from the CCLE database was ed for a reliable two gene biomarker for apilimod sensitivity in the 22 B cell lymphoma lines.

Additional experiments demonstrated that od’s cytotoxic activity was based at least in part on its inducing cellular apoptosis. Apoptosis was quantified and distinguished from is using the Apotox-Glo x assay (Promega, Inc.) according to the manufacturer’s instructions. In this assay, viabillity, apoptosis, and necrosis are assessed simultaneously using three different markers (GF-AFC, Caspase-3/7, and bis-AAF-Rl 10, respectively). Figure 4 shows apoptotic (middle bar) and necrotic (right bar) s in apilimod treated in diffuse large B cell lymphoma cells 48 hours after addition of apilimod to the culture media. The left bar shows the viability marker.

The mechanism of apilimod’s cytotoxic activity was further investigated by ng for autophagic vacuoles after 72 hours of ent in an H4 neuroglioma cell line (IC50 250-300 nM). Autophagy was quantified using the Cyto—ID Autophagy detection kit (Enzo) ing to manufacturer’s directions. Figure 5 shows that apilimod induced autophagy in a dose-dependent manner.

PIKfyve is associated with the cytosolic leaflet of early endosomes and its activity is required for mbrane homeostasis, sosomal fianction and proper retrograde transport from the endosome to the trans-Golgi network. Introduction of a kinase dead mutant into cells s a swollen vacuole phenotype that can be rescued by the injection of PI(3 ,5)P2.

Inhibition ofPIKfyve by pharmacological methods as well as RNAi also produces n vacuoles and disruption of endomembrane dynamics. As shown in Figure 21 , pharmacological disruption ofPIKfyve with apilimod induces selective lethality of specific cancer cell lines through disruption of intracellular trafficking.

Claims (19)

What is claimed is:

1. Use of a pharmaceutical composition comprising apilimod, or a pharmaceutically acceptable salt thereof, in the cture of medicament for treating refractory or ent non-Hodgkin’s B cell lymphoma in a subject, wherein the ment is formulated to be administered either alone or in combination with one or more additional active agents.

2. The use of claim 1, wherein the pharmaceutically acceptable salt is selected from a sulfate, e, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, ate, maleate, besylate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (e.g., 1,1'- ene-bis-(2-hydroxynaphthoate)).

3. The use of claim 2, wherein the pharmaceutically acceptable salt is selected from a chloride, mesylate, fumarate, lactate, maleate, pamoate, phosphate, and tartrate.

4. The use of claim 1, wherein the apilimod is apilimod free base or apilimod dimesylate.

5. The use of any one of the preceding claims, wherein the pharmaceutical composition is an oral dosage form or a dosage form le for intravenous administration.

6. The use of any one of the preceding claims, wherein the non-Hodgkin’s B cell lymphoma is ed from diffuse large B cell ma (DLBCL), Burkitt’s lymphoma, ular lymphoma, mediastinal B cell lymphoma, and mantle cell lymphoma.

7. The use of claim 6, wherein the non-Hodgkin’s B cell lymphoma is DLBCL.

8. The use of claim 7, wherein the DLBCL is DLBCL-GCB.

9. The use of claim 6, wherein the non-Hodgkin's B cell lymphoma is follicular lymphoma. [Link] http://en.wikipedia.org/wiki/Alkylating_antineoplastic_agent [Link] http://en.wikipedia.org/wiki/Alkylating_antineoplastic_agent [Link] http://en.wikipedia.org/wiki/Intercalating_agent [Link] /en.wikipedia.org/wiki/Intercalating_agent [Link] http://en.wikipedia.org/wiki/Cyclophosphamide [Link] http://en.wikipedia.org/wiki/Cyclophosphamide [Link] http://en.wikipedia.org/wiki/Hydroxydaunorubicin [Link] http://en.wikipedia.org/wiki/Hydroxydaunorubicin [Link] http://en.wikipedia.org/wiki/Vincristine [Link] http://en.wikipedia.org/wiki/Vincristine [Link] http://en.wikipedia.org/wiki/Oncovin [Link] /en.wikipedia.org/wiki/Oncovin [Link] http://en.wikipedia.org/wiki/Prednisone [Link] http://en.wikipedia.org/wiki/Prednisone [Link] http://en.wikipedia.org/wiki/Prednisolone [Link] http://en.wikipedia.org/wiki/Prednisolone

10. The use of any one of the preceding claims, wherein it is suitable to administer the apilimod in combination with at least one additional active agent.

11. The use of claim 10, wherein the at least one additional active agent is formulated to be administered in a single dosage form with the od, or in a te dosage form from the od.

12. The use of claim 11, wherein the at least one additional active agent is selected from the group consisting of an alkylating agent, an intercalating agent, a tubulin binding agent, a corticosteroid, and combinations thereof.

13. The use of claim 11, wherein the at least one additional active agent is a therapeutic agent selected from the group consisting of ibrutinib, rituximab, doxorubicin, prednisolone, stine, velcade, and everolimus, and combinations thereof.

14. The use of claim 11, wherein the at least one additional active agent is a therapeutic agent selected from cyclophosphamide, hydroxydaunorubicin (also referred to as doxorubicin or Adriamycin™), stine (also referred to as Oncovin™), prednisone, prednisolone, and combinations thereof.

15. The use of claim 10 or 11, wherein the at least one additional active agent is rituximab.

16. The use of claim 10 or 11, wherein the at least one additional active agent is nib.

17. The use of claim 11 or 12, wherein the at least one additional active agent is selected to ameliorate one or more side effects of the apilimod composition.

18. The use of claim 17, n the agent is selected from the group consisting of ondansetron, granisetron, dolasetron and palonosetron.

19. The use of claim 17, wherein the agent is selected from the group consisting of pindolol and risperidone.