BACKGROUND OF THE INVENTION

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor, characterized by a short doubling time, high growth fraction, and early development of widespread metastases (2). Around two thirds of patients present with extensive stage (ES) disease, defined by spread beyond a tolerable radiation field (3) limiting treatment options to platinumbased chemotherapy. Platinum-based doublet chemotherapy is effective in 60-80% of ES- SCLC, but these responses are short lived (4). Almost all ES-SCLC patients relapse with drug resistant disease within months for which there is no effective second line therapy. Together this culminates in an appalling overall survival rate of less than 5% (1). The only substantive change in front line treatment over the last four decades has been the addition of the immune checkpoint inhibitors Atezolizumab or Durvalumab, however this only achieves a modest 2 month increase in overall survival (5,6). There is a need for new and effective drugs to treat SCLC including agents that re-sensitize or prolong the response to platinum-based chemotherapy.

SUMMARY OF THE INVENTION

The present invention relates to methods of treating small cell lung cancer with a compound of Formula I, or a pharmaceutically acceptable salt thereof, where A is optionally substituted phenyl, optionally substituted pyridyl or optionally substituted pyrimidyl. A preferred compound of Formula I has the structure

and is referred to herein as fimepinostat. The compounds of Formula I are dual inhibitors of PI3 kinase and histone deacetylases and have been shown to have significant inhibitory activity in cancer lines. Inhibition of each of these targets has been shown to reduce MYC levels in cancer cells, and fimepinostat has shown particular clinical activity in MYC-altered diffuse large B-cell lymphoma.

In a first embodiment, the invention provides a method for treating small cell lung cancer in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a compound of Formula I, and a therapeutically effective amount of a platinum anticancer agent. In one embodiment, the SCLC is platinum responsive. In another embodiment, the SCLC is platinum resistant.

In another embodiment, the invention provides a method of treating platinum resistant SCLC in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula I.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings.

Figure 1: (A) Cell lines derived from the Rbl ^_/ :Trp53 ^/_ mouse model of small cell lung cancer were continuously cultured in an LD50 dose of carboplatin and platinum sensitivity of both platinum naive and resistant clones determined following treatment with the indicated doses of carboplatin for 7 days. Data is the mean+/-SD in three independent experiments. (B) MYC expression in naive and platinum resistant mouse SCLC cell lines was determined by western blot. Data is representative of 3 independent experiments. (C) Rbl ^/_ :Trp53 ^/_ mouse SCLC cell lines were stably transduced with empty vector (EV) or MYC encoding retrovirus (MYC) and MYC expression confirmed by western blot. Data is representative of 3 independent experiments. (D) The impact of stable MYC overexpression on platinum sensitivity was determined following 7 days exposure to the indicated doses of carboplatin. Data is the mean+/-SD in three independent experiments.

Figures 2(A)-(D) present data showing that MYC-expression is increased following platinum treatment in vivo. (A) Schematic overview of experimental approach. (B) Kaplan- Meier plot showing that carboplatin and etoposide treatment significantly increases the survival in the RP mouse model of SCLC. ****p<0.0001 Log-rank Mantel-Cox test. (C) representative immunohistochemical analysis of MYC expression in vehicle and carboplatin / etoposide (Pt / etop). (D) The % of MYC positive cells per tumor area in each tissue section was determined using the automated positive cell detection feature in QuPath for each animal in each cohort (n>7 mice per cohort). ****P<0.0001 students t-test.

Figures 3(A)-(C) present data showing that MYC-expression drives platinum resistance in vivo. (A) Schematic overview of experimental approach. (B) Representative CT images of RPM mice at time-points following Cre inhalation but before tumor development (Baseline), when tumor was detected, and treatment initiated (Treatment) and when mice reached ethical endpoint (Endpoint). The area surrounded by the red-dashed line is the heart and the area surrounded by the yellow-dashed line is tumor. CT images were used to generate a 3D reconstruction of the lung. Normal lung is shown in red and tumor in green. Images are representative of at least 8 mice per group. (C) Kaplan-Meier plot showing that carboplatin and etoposide treatment has no significant impact on the survival in the RPM mouse model of SCLC (Log-rank Mantel-Cox test).

Figures 4(A)-(D) present the drug screening identifies fimepinostat as an effective treatment for small cell lung cancer in vitro. (A) Platinum resistant mouse SCLC cell line EN84R was screened for sensitivity to 355 kinase inhibitors at lOOnM for 7 days and the average viability z-score of three independent experiments plotted. Drugs achieving a Z-score of -3>z>-2 are shown in blue and drugs where z<-3 are shown in red. Fimepinostat is highlighted. (B) Drug screening was performed in triplicate on the indicated cell lines and a heat map of average Z scores for the top 10 hits shown in all cell lines. (C) Dose response curves for fimepinostat sensitivity in a panel of human SCLC cell lines and PDX lines. Data represents the % viability at indicated dose (Log2 nM) and LD50 (nM) calculated using Prism.

(D) Fimepinostat LD50 (nM) doses determined for matched platinum naive and resistant mouse SCLC cell lines and primary (1°) mouse lung epithelium. Statistically significant differences in viability between 1° epithelium and SCLC cell lines were determined by 2-way ANOVA (*p<0.05, **p<0.005, ***p<0.001, ****p<0.0001).

Figures 5(A) and (B) show Western blot analyses showing reduction in MYC expression, phosphorylated (S473) AKT and increased acetylation of histone H3 following a 24-hour treatment with the indicated dose of fimepinostat in platinum resistance mouse SCLC cell lines (A) B37R and (B) EN84R.

Figures 6(A)-(E) show that fimepinostat is an effective SCLC treatment in vivo. Platinum resistant mouse SCLC cell lines (A) B37R and (B) EN84R, human cell line (C) NCLH209 or PDX cell lines (D) PDX102 and (E) PDX109 were transplanted subcutaneously into the flanks of recipient mice. When tumors reached 150-200mm ³ mice were treated with fimepinostat (70mg/Kg, daily, per os) and tumor volume measured with calipers. Plotted data is the mean +/- SEM for cohorts of at least 8 mice per group. Statistically significant tumor reduction was calculated by paired student’s t-test (**p<0.005, ***p<0.001, ****p<0.0001).

Figures 7(A)-(E) show that the combination of fimepinostat with standard of care chemotherapy significantly reduces SCLC tumor burden and increases survival in vivo. (A) Schematic outline of experimental design (B) Representative CT images of RPM mice at time-points following Cre inhalation but before tumor development (Baseline), when tumor was detected, and treatment initiated (Treatment) and when mice reached ethical endpoint (Endpoint). CT images are shown for the fimepinostat alone and fimepinostat/carboplatin/etoposide groups. The area surrounded by the red-dashed line is the heart and the area surrounded by the yellow-dashed line is tumor. Images are representative of at least 8 mice per group. (D) Representative H&E images and immunohistochemical staining for PCNA are shown. Scale bar = 5mm in whole lung image and 50pm in 40X magnification.

(E) The % of PNCA positive cells per tumor area in each tissue section was determined using the automated positive cell detection feature in QuPath for each animal in each cohort (n>8 mice per cohort). Data is the mean +/-S.D for each treatment group. Statistically significant differences were calculated by students t-test (*p<0.05, ***p<0.001, ****p<0.0001). (F) Kaplan-Meier plot showing that fimepinostat is superior to carboplatin and etoposide treatment and that the combination of fimepinostat with carboplatin and etoposide provides a very significant survival advantage in the RPM mouse model of platinum-resistant SCLC (Log-rank Mantel-Cox test, (*p<0.05, ****p<0.0001).

Figures 8 (A) and (B) show that JQ1 has no effect on MYC expression in platinum resistant mouse SCLC cell lines. (A) B37R and (B) EN84R cells were treated with the indicated doses of JQ1 and MYC expression determined by western blot. Blots were reprobed with an actin antibody as a loading control. Data is representative of three independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating small cell lung cancer with a compound of Formula I. The invention relates to the discovery that fimepinostat, a particular compound of Formula I restores platinum responsiveness in platinum refractory SCLC cells and inhibits or delays the development of platinum resistance in platinum naive cells.

In a preferred embodiment of the compounds of Formula I, A is phenyl, pyridyl or pyrimidyl substituted with methoxy, amino or N-methylamino. More preferably, A is one of the groups set forth below.

In a preferred embodiment, the compound of Formula I is selected from fimepinostat and Compounds 1 and 2 below.

The present invention provides methods of treating SCLC in a subject in need thereof comprising administering the subject a therapeutically effective amount of a compound of Formula I. In certain embodiments, the SCLC is platinum refractory. In certain embodiments, the SCLC was initially responsive to a platinum anticancer agent in the absence of fimepinostat but subsequently relapsed despite continued administration of the platinum anticancer agent. In other embodiments, the SCLC is platinum responsive. In this embodiment, the SCLC can be platinum naive or SCLC that has been treated with a platinum anticancer agent without developing platinum resistance.

The SCLC to be treated can be limited stage SCLC or extensive stage SCLC.

In certain embodiments, the compound of Formula I is administered in combination with a therapeutically effective amount of a platinum anticancer agent.

In preferred embodiments, the methods of the invention further comprise administering to the subject a therapeutically effective amount of a platinum anticancer agent and a therapeutically effective amount of a chemotherapeutic agent.

In certain embodiments, the methods of the invention further comprise administering to the subject a therapeutically effective amount of an immuno-oncology agent. In particularly preferred embodiments, the methods of the invention further comprise administering to the subject an effective amount of a platinum anticancer agent, a chemotherapeutic agent and an immuno-oncology agent.

The platinum anticancer agent can be any platinum anticancer agent which is effective in treating platinum responsive SCLC, including platinum naive SCLC. Suitable platinum anticancer agents include, but are not limited to, carboplatin, cisplatin, oxaliplatin, nedaplatin, lobaplatin, hextaplatin, satraplatin, picoplatin, lipoplatin and Prollndac. A preferred platinum anticancer agent is carboplatin.

The chemotherapeutic agent is an agent that inhibits cancer cell replication and is preferably an agent having clinical activity against SCLC. In certain embodiments, the chemotherapeutic agent is a topoisomerase II inhibitor, preferably etoposide.

The immuno-oncology agent is an immune checkpoint inhibitor, such as an inhibitor of PD-1 signaling, for example, an inhibitor of PD-1 or PD-L1. Suitable immuno-oncology agents include, but are not limited to, YW243.55.S70, MPDL3280A, MEDL4736, MSB- 0010718C, MDX-1105, AMP-224, pembrohzumab (KEYTRUDA™), mvolumab (OPDIVO™), atezolizumab (TECENTRIQ™), avelumab (BAVENCIO™), durvalumab (IMFINZI™), and pidilizumab. Preferred immuno-oncology agents are atezolizumab and durvalumab.

In certain embodiments, the invention provides a method of treating platinum sensitive SCLC in a subject in need thereof, comprising administering to the subject the compound of Formula I, preferably fimepinostat, carboplatin, etoposide and optionally a PD-L1 inhibitor selected from durvalumab and atezolizumab, wherein the compound of Formula I, carboplatin, etoposide and optional PD-L1 inhibitor are administered in amounts which in combination are therapeutically effective. Preferable, the compound of Formula I is administered in an amount which is effective to inhibit the development of platinum resistance by the SCLC.

In certain embodiments, the invention provides a method of treating platinum resistant SCLC in a subject in need thereof, comprising administering to the subject a compound of Formula I, preferably fimepinostat, carboplatin, etoposide and optionally a PD- L1 inhibitor selected from durvalumab and atezolizumab, wherein the compound of Formula I, carboplatin, etoposide and optional PD-L1 inhibitor are administered in amounts which in combination are therapeutically effective. Preferably, the compound of Formula I is administered in an amount which is effective to sensitize the SCLC to platinum.

In one embodiment, the compound of Formula I is used as a maintenance therapy following induction therapy with a platinum anticancer agent, a chemotherapeutic agent, and an optional immuno-oncology agent. In this embodiment, the subject has been previously treated with the platinum anticancer agent, the chemotherapeutic agent and, optionally, the immuno-oncology agent. The method comprises administering to the subject effective amounts of the compound of Formula I, the platinum anticancer agent, the chemotherapeutic agent and the optional immuno-oncology agent.

In another embodiment, the compound of Formula I is used in re-induction therapy. In this embodiment the subject has been previously treated with a platinum anticancer agent, a chemotherapeutic agent and, optionally, an immuno-oncology agent and relapsed at least 4, 5 or 6 months following initiation of therapy. The method comprises administering to the subject effective amounts of fimepinostat, the platinum anticancer agent, the chemotherapeutic agent and the optional immuno-oncology agent.

In another embodiment, the present invention provides a method of treating SCLC in a subject in need thereof, where the subject was previously treated with a platinum anticancer agent, a chemotherapeutic agent and, optionally, an immuno-oncology agent but relapsed early, for example, less than 4 month, or less than 3 months following initiation of therapy. The method comprises administering to the subject an effective amount of a compound of Formula I. Preferably, the compound of Formula I is administered as a single agent or in combination with an effective amount of an immuno-oncology agent.

An amount of the compound of Formula I, such as fimepinostat, which inhibits the development of platinum resistance in SCLC is an amount which when administered in combination with a platinum anticancer agent to treat platinum sensitive SCLC is effective to prevent or delay the development of platinum resistance compared to administration of the platinum anticancer agent in the absence of the compound of Formula I. When the compound of Formula I delays the development of platinum resistance, such delay is clinically meaningful, that is, the subject receives a clinical or therapeutic benefit from the treatment with the compound of Formula I compared to treatment without the compound of Formula I. The dose, dosing frequency and total daily dose of the compound of Formula I, and, where applicable, the platinum anticancer agent, the chemotherapeutic agent and/or the immune-oncology agent can be determined for an individual subject by one of skill in the art, for example, by the attending physician within the scope of sound medical judgment. The specific dose or doses for any particular subject will depend upon a variety of factors including the disease being treated and the severity of the disease; the activity of the specific agent, the health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

The compound of Formula I, such as fimepinostat, the platinum anticancer agent, the chemotherapeutic agent and the immuno-oncology agent can each be administered by any suitable means, including, without limitation, parenteral, intravenous, intramuscular, subcutaneous, implantation, oral, sublingual, buccal, nasal, pulmonary, transdermal, topical, vaginal, rectal, and transmucosal administrations or the like. Topical administration can also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. Pharmaceutical preparations include a solid, semisolid or liquid preparation (tablet, pellet, troche, capsule, suppository, cream, ointment, aerosol, powder, liquid, emulsion, suspension, syrup, injection, etc.) containing a compound of Formula I or a pharmaceutically acceptable salt thereof, a PD- 1 signaling inhibitor, or both which is suitable for selected mode of administration. In one embodiment, the pharmaceutical compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets, sachets and effervescent, powders, and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment of the present invention, the composition is formulated in a capsule. In accordance with this embodiment, the compositions of the present invention comprise in addition to the active compound and the inert carrier or diluent, a hard gelatin capsule.

The compound of Formula I can be administered as the neutral compound or in the form of a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid or inorganic acid. Examples of pharmaceutically acceptable nontoxic acid addition salts include, but are not limited to, salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid lactobionic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. Certain salts such as the sodium, potassium and choline base salts as well as acidic salts such as sulfate and methanesulfonate salts have been found to improve the solubility of compounds of Formula I in pharmaceutically acceptable aqueous media. In one embodiment, the pharmaceutically acceptable salt of fimepinostat is the choline salt. Preferred salts of fimepinostat include the sodium salt and the potassium salt. Other preferred salts include the sulfate and methanesulfonate salts. Particularly preferred salts of fimepinostat are the methanesulfonate and benzenesulfonate salts.

The compound of Formula I and other therapeutic agents of use in the present methods are each preferably administered in the form of a pharmaceutical composition comprising the therapeutic agent and a pharmaceutically acceptable carrier or excipient. When the fimepinostat or other therapeutic agent is administered orally, it is formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets, sachets and effervescent, powders, and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment of the present invention, the composition is formulated in a capsule. In accordance with this embodiment, the compositions of the present invention comprise in addition to the active compound and the inert carrier or diluent, a hard gelatin capsule.

Any inert excipient that is commonly used as a carrier or diluent may be used in the formulations of the present invention, such as for example, a gum, a starch, a sugar, a cellulosic material, an acrylate, or mixtures thereof. A preferred diluent is microcrystalline cellulose. The compositions may further comprise a disintegrating agent (e.g., croscarmellose sodium) and a lubricant (e.g., magnesium stearate), and may additionally comprise one or more additives selected from a binder, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetener, a film forming agent, or any combination thereof. Furthermore, the compositions of the present invention may be in the form of controlled release or immediate release formulations.

For liquid formulations, pharmaceutically acceptable carriers may be aqueous or nonaqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solutions or suspensions can also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

In addition, the compositions may further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), buffers (e.g., tris-HCI., acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol, polyethylene glycol), a glidant (e.g., colloidal silicon dioxide), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hydroxypropylmethyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents (e.g., peppermint, methyl salicylate, or orange flavoring), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral compositions in unit dosage form for ease of administration and uniformity of dosage. “Unit dosage form”, as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

Formulations of the invention intended for oral administration can include one or more permeation enhancers, including long chain fatty acids or salts thereof, such as decanoic acid and sodium decanoate.

In one preferred embodiment, the therapeutic agent is formulated in an aqueous solution for intravenous injection. In one embodiment, solubilizing agents can be suitably employed. A particularly preferred solubilizing agent includes cyclodextrins and modified cyclodextrins, such as sulfonic acid substituted P-cyclodextrin derivative or salt thereof, including sulfobutyl derivatized-P-cyclodextrin, such as sulfobutylether-7-P-cyclodextrin which is sold by CyDex, Inc. under the tradename CAPTISOL®.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Daily administration may be repeated continuously for a period of several days to several years. Oral treatment may continue for between one week and the life of the subject. Preferably the administration may take place for five consecutive days after which time the subject can be evaluated to determine if further administration is required. The administration can be continuous or intermittent, e.g., treatment for a number of consecutive days followed by a rest period. The compounds of the present invention may be administered intravenously on the first day of treatment, with oral administration on the second day and all consecutive days thereafter.

The preparation of pharmaceutical compositions that contain an active component is well understood in the art, for example, by mixing, granulating, or tablet-forming processes. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, the active agents are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic, or oily solutions and the like as detailed above.

In certain embodiments, the amount of the compound of Formula I that is administered to the subject is less than the amount that causes a concentration of the compound in the subject's plasma to equal or exceed the toxic level of the compound. Preferably, the concentration of the compound in the subject's plasma is maintained at about 10 nM. In one embodiment, the concentration of the compound in the subject's plasma is maintained at about 25 nM. In one embodiment, the concentration of the compound in the subject's plasma is maintained at about 50 nM. In one embodiment, the concentration of the compound in the subject's plasma is maintained at about 100 nM. In one embodiment, the concentration of the compound in the subject's plasma is maintained at about 500 nM. In one embodiment, the concentration of the compound in the subject's plasma is maintained at about 1000 nM. In one embodiment, the concentration of the compound in the subject's plasma is maintained at about 2500 nM. In one embodiment, the concentration of the compound in the subject's plasma is maintained at about 5000 nM. The optimal amount of the compound that should be administered to the subject in the practice of the present invention will depend on the particular compound used and the type of cancer being treated.

In preferred embodiments, the platinum anticancer agent is carboplatin and is administered by IV infusion. In certain embodiments, carboplatin is administered such that the carboplatin exposure, represented by the area under the curve (AUC) of plasma carboplatin concentration versus time, is 5 mg/mL X time (minutes). AUC is converted to a subject- specific carboplatin dose (in mg) according to renal function by using the Calvert formula, total dose (mg) = (target AUC) x (GFR + 25), where GFR is the subject’s measured glomerular filtration rate. If using measured serum creatinine, the maximal GFR for the calculation is limited to 125 mL/min.

In preferred embodiments, the chemotherapeutic agent is etoposide and is preferably administered by IV infusion, for example at a dose of 100 mg/m ² .

In preferred embodiments, carboplatin is administered on day 1 of a 21 -day cycle and etoposide is administered on days 1, 2 and 3 of a 21 -day cycle. The number of 21 -day cycles can be determined by a physician, based, for example, on the subject’s response to the therapy. In certain embodiments the number of cycles is from about 2 to about 10, for example 6.

In certain embodiments, the compound of Formula I is fimepinostat, which is administered at a dose of 60 mg orally at a schedule of 5 days on/2 days off. In another embodiment, fimepinostat is administered at a dose of 60 mg orally at a schedule of 3 days on/4 days off. Fimepinostat can be administered for a number of weeks as determined by the subject’s physician. In certain embodiments, fimepinostat dosing is continued for the duration of the 21 -day cycles of carboplatin and etoposide administration. Fimepinostat doses described herein are free base equivalent doses, which are independent on the form of fimepinostat administered, i.e., as the free base or as a pharmaceutically acceptable salt. In certain embodiments, fimepinostat is administered in the form of a methanesulfonate salt.

DEFINITIONS

Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

The term “platinum anticancer agent” refers to a coordination complex of Pt(II) which is useful as an antineoplastic agent. Platinum anticancer agents include, but are not limited to carboplatin, cisplatin, oxaliplatin, nedaplatin, lobaplatin, heptaplatin, satraplatin, picoplatin, ProLindac (AP 5346) and lipoplatin. Preferred platinum anticancer agents include carboplatin, cisplatin, oxaliplatin, nedaplatin, lobaplatin, and heptaplatin. A particularly preferred platinum anticancer agent is carboplatin. A method of treating platinum-resistant SCLC in a subject in need thereof, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein A is optionally substituted phenyl, optionally substituted pyridyl or optionally substituted pyrimidyl.

The term “platinum sensitive SCLC’ as used herein, refers to limited stage or extensive stage SCLC which responds clinically to a platinum anticancer agent when the agent is administered in doses which are judged by a health care provider to be effective with an acceptable risk profile. A clinical response is stable disease, a partial response, or a complete response, as these terms are understood in the art.

The terms “platinum resistant SCLC’ and “platinum refractory SCLC” as used herein, are interchangeable and refer to limited stage or extensive stage SCLC which had been platinum sensitive but has stopped responding clinically to a platinum anticancer agent.

As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration, such as sterile pyrogen-free water. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. The terms “subject” and “patient” as used herein are interchangeable and refer to an individual animal. Preferably the animal is a mammal. More preferably the mammal is a human. A subject can also be a domestic, livestock or companion animal, for example, a dog, a cat, a horse, a cow, or a pig.

The terms “therapeutically effective amount" and “effective amount” of a therapeutic agent, including fimepinostat, a platinum anticancer agent, a chemotherapeutic agent or an immuno-oncology agent, or combination of two or more thereof, is meant an amount of such agent or the amount of each such agent in the combination which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to treatment of small cell lung cancer. A therapeutically effective amount of an agent may be different when used as a single agent that when used in combination with one or more other agents. In addition, a therapeutically effective amount of an agent may depend on the specific combination of agents to be administered. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). Therapeutically effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or contemporaneously with the specific compound employed; and like factors well known in the medical arts. In preferred embodiments, the therapeutically effective amount of the combination of the compound of Formula I or pharmaceutically acceptable salt thereof and the PD- 1 signaling inhibitor, exhibits synergism in the cancer type to be treated.

The total daily dose of each compound in the combination therapy of this invention administered to a human or other animal in single or in divided doses can be in amounts, for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1 to 25 mg/kg body weight. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. In general, treatment regimens according to the present invention comprise administration to a subject in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses.

Each compound in the combination therapy of the invention can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or by inhalation, with a dosage ranging from about 0.1 to about 500 mg/kg of body weight, alternatively dosages between 1 mg and 1000 mg/dose, every 4 to 120 hours, or according to the requirements of the particular drug. The methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with pharmaceutically excipients or carriers to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations may contain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular subject will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the subject’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.

Upon improvement of a subject’s condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level. Subjects may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms. EXEMPLIFICATION

Materials and Methods

Cell culture

Mouse tumor cell lines were generated following a protocol previously reported (14). NCLH146, NCLH209 and NCLH69 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and fidelity confirmed by short tandem repeat (STR) profiling. SCLC cell lines were cultured in Advanced RPMI supplemented with 1% FCS and 2mM glutamax in humidified incubators at 37°C and 5% CO2.

Drug screening

Cells were plated in clear bottom 384 well plates (Greiner bio-one) in culture media using liquid handling robotics (Beckman Coulter, Biomek NXP). Cells were treated with 355 kinase inhibitors (Selleckchem Kinase library, L2000) at a final concentration of lOOnM. Plates were sealed with gas permeable tape (Roll-Seal, Sigma- Aldrich) to stop evaporation and incubated in a humidified culture incubator at 37°C and 5% CO2 for 7 days. Alamar blue viability dye (ThermoFisher) was added using liquid handling robotics and fluorescence measured using a ClarioStar plate reader (BMG Labtech). Background fluorescence was calculated as the mean of wells containing media alone and subtracted from well fluorescence. The mean fluorescence in vehicle control wells was considered 100% viable and fluorescence values for all drug wells expressed as a percentage viability. Z-scores were calculated for each well and a drug considered to be a hit if the Z score was lower than -3. Experiments were performed in duplicate, and a quality control cut off correlation between replicate plates of 0.75 was used. Cell number was optimized for each cell line based on Alamar blue fluorescence after a 7-day incubation.

Mice

Rbl^J- rpS^ andRbl^- rpSS^- yt ¹ ^ mice have been described previously (15,16). NSG mice were purchased from Australian BioResources (Garvan Institute of Medical Research, Sydney, Australia). All animals were housed in a specific-pathogen-free (SPF) vivarium at the Monash Medical Centre (Clayton, Victoria, Australia). All mouse studies were performed in accordance with the ethics approval from the Hudson Institute Animal Ethics Committee. For spontaneous SCLC models, 6-8-week-old mice were anaesthetized with an intraperitoneal injection of 1.25% (v/v) Avertin (Sigma- Aldrich, Missouri, USA) and subsequently inoculated intranasally with 10 ⁶ pfu Ad5-CGRP-Cre (Viral Vector Core Facility, University of Iowa, IA, USA). For xenograft studies, mice were subcutaneously injected with 10 ⁶ cells resuspended 50% (v/v) Matrigel (Corning, NY, USA). Tumor measurements were taken with digital calipers and tumor volume in mm ³ calculated as (width ² x length)/2. Animals were sacrificed upon reaching ethical endpoints that include, but are not limited to, breathing difficulties, >20% body weight loss or 800mm ³ tumor volume. Drug treatments in flank models began when tumors reached 150-200mm ³ . Drug treatment in GEMMs was commenced when tumors were visible by computed tomography. Fimepinostat, 70mg/kg was administered daily per. os. in a 30% captisol solution (CyDex pharmaceuticals). Mice in the platinum/etoposide cohort were injected with carboplatin (60mg/Kg in PBS) and etoposide (lOmg/kg) intraperitoneally once a week for three weeks with one day between agents. On the day of carboplatin injection mice received an intraperitoneal injection of ImE of PBS to minimize kidney toxicity.

Isolation of primary lung epithelium

Lungs were removed and rinsed in PBS containing MgCh (lOOmg/L) and CaCh (lOOmg/L) and diced. Tissue was digested in collagenase IV (2.5mg/mL) and DNAse I (lOOpg/mL) in PBS at 37°C for 90 minutes with rotation. Cells were resuspended in fetal calf serum, passed sequentially through 70pm and 40pm filters and plated in DMEM/F12 (Gibco). Cells were allowed to attach for 90 minutes in a humidified incubator with 5%CC>2 and media containing epithelial cells was removed and viable lung epithelial cell cells plated for drug treatments.

Computed Tomography

Mice were anaesthetized with isoflurane and Computed Tomography (CT) images were captured using a Siemens Inveon Small Animal PET/SPECT/CT scanner at 39.95pm resolution, 80KV, with 500pA current. Mice were fitted with a heart rate and respiration monitor (BioVet) and scans gated to maximum expiration. Images were processed with Fiji and Analyze 12.0 (AnalyzeDirect).

Western blotting

Cell lysates were resolved through acrylamide gels using SDS-PAGE and transferred to PVDF-FL membranes (Millipore). Membranes were blocked in Odyssey blocking buffer (Li-Cor) and incubated in specific primary antibodies including MYC (Abeam, 32072), pAkt (Abeam, 38449), Acetyl-Histone H3 (Abeam, 4729) and actin (Abeam, 3280). Membranes were incubated with IRDye fluorescent-conjugated secondary antibodies (Li-Cor), and protein expression was detected using Odyssey Infrared imaging system (Li-Cor).

Histology and immunohistochemistry

Lungs were inflation-fixed in 10% neutral buffered formalin for 24 h prior to paraffin embedding. Histological assessment was performed on H&E-stained sections. For immunohistochemical analysis, sections were dewaxed, re-hydrated and subjected to microwave-based antigen retrieval with 20 min boiling in citrate buffer (10 mM citrate, 0.05% Tween-20, pH 6.0) under pressure. Sections were probed with primary antibodies against

MYC (Abeam, Cambridge, UK) or PCNA (Dako). Blocking and secondary antibody staining were performed using Vectastain ABC Elite kits (Vector Laboratories, Burlingame, CA, USA).

Results

MYC expression drives platinum resistance in vitro and in vivo.

The frequency of MYC amplification is increased in relapsed SCLC (11). To determine whether MYC expression is similarly increased in SCLC cell lines after platinum resistance is acquired two independent cell lines derived from a genetically engineered mouse model driven by the loss of Rbl and Trp53 from lung neuroendocrine cells (RP mouse model) were repeatedly treated with an LD50 dose of carboplatin and cells considered resistant if an increase in LD50 of at least 5 -fold was achieved. The LD50 of naive and resistant clones (denoted “R”) was shown to exceed this threshold: B374pg/mL, B37R 24 pg/mL, EN842 pg/mL, EN84R 23 pg/mL. Western blot analysis for MYC showed that increased platinum resistance was co-incident with an increase in MYC expression (Eig. IB). To determine whether MYC expression is associated with platinum treatment in a more clinically relevant model, SCLC was initiated in the RP mouse model by intranasal inoculation of adenoviral Cre -recombinase under control of a neuroendocrine promoter (Ad5-CGRP-Cre) (15,16). These mice develop SCLC with a median survival of -200 days. 80 days after disease initiation, mice were randomized into two groups, one received vehicle (PBS) and the other received three cycles of carboplatin (60mg/kg) and etoposide (lOmg/kg). After three cycles treatment ceased and mice were monitored until they reached ethical endpoint (Pig. 2A). Platinum-etoposide chemotherapy significantly increased the overall survival of RP mice (vehicle 180.5 days, Pt/Etop 319 days, p<0.0001 Log-Rank Mantel-Cox test), but mice did relapse and ultimately succumb to SCLC (Pig. 2B). Immunohistochemical analysis of MYC expression showed very few MYC positive cells in the primary lung tumors from vehicle control mice (mean of 2.28%) but following platinum and etoposide treatment a significant increase in the proportion of MYC positive cells in tumors was observed (mean of 16.14%, p<0.0001, students t-test). Together these data confirm that increased MYC expression is observed following platinum-based chemotherapy and that MY C expression is coincident with acquired platinum resistance in vitro and in vivo. However, these data do not directly assess whether enforced MYC expression drives platinum resistance. To address this, mouse SCLC cell lines derived from RP mice were engineered to stably overexpress MYC and expression confirmed by western blot (Fig. 1C). Three independent matched pairs of cells were treated with titrating concentrations of platinum for 7 days and in each pair of cell lines the overexpression of MY C alone resulted in a significant increase in platinum resistance (Fig ID). These data confirm that MYC expression is not only coincident with but is also a driver of platinum resistant SCLC in vitro. To determine whether sustained MYC expression drives platinum resistance in vivo we took advantage of a mouse model of SCLC driven by the loss of Rbl and Trp53 combined with sustained expression of M YC exclusively in neuroendocrince cells of the lung (RPM mice) (15). These mice rapidly develop SCLC and have a median survival of -100 days. Disease was initiated in RPM mice by intranasal inoculation of Ad5-CGRP-Cre and disease onset and progression monitored by computed tomography (CT). A baseline scan was performed 25 days after Cre delivery and mice were subsequently monitored every 10 days by CT (Fig. 3A). When tumor was observed mice were randomized into vehicle and platinum-etoposide treatment groups and treated as described for the RP mouse model. Strikingly, RPM mice were completely refractory to platinum-etoposide treatment. No reduction in tumor volume was observed in RPM mice treated with platinum and etoposide (Fig. 3B). A subtle, but insignificant increase in median survival was observed (vehicle: 23 days on treatment. Platinum and etoposide: 31 days on treatment). Together these data show that elevated MYC expression is not only coincident with platinum resistance but is a potent driver of resistance both in vitro and in vivo.

Drug screening identifies fimepinostat as a potent inhibitor of platinum resistant SCLC Identification of drugs that are effective against platinum resistant SCLC, or that will re-sensitize patients to platinum-based therapy are amongst the most urgent unmet clinical needs in this disease. To identify novel drugs, a library of 355 kinase inhibitors was screened for their ability to kill platinum resistant mouse-derived SCLC cell lines at a dose of lOOnM in 7 days. Viability z-scores were calculated in response to each agent and drugs that achieved a z-score less than -3 were prioritized. We identified drugs that have previously been shown to be effective against SCLC including PLK1, Aurora kinase A and CDK inhibitors validating the screening approach. The most effective drug in this screen was the novel dual PI3K and HD AC inhibitor fimepinostat (Fig. 4A). Importantly, fimepinostat was the most effective drug against all mouse derived cell lines irrespective of their platinum resistance or MYC expression status (Fig. IB) suggesting that fimepinostat may be broadly effective in the treatment of SCLC. To determine whether fimepinostat efficiently kills human SCLC cell lines we determined the LD50 dose of fimepinostat in four established cell lines representative of the recently described neuroendocrine subgroups of SCLC defined by ASCL1 or NEURODI expression (17) (SCLC-N: NCI-H82. SCLC-A: NCI-H209, NCLH146 and NCL H69) and two patient derived xenograft (PDX) lines (18). Like the observation in mouse derived SCLC cell lines, all human SCLC cell lines were sensitive to fimepinostat with LD50 doses between 5.93 and 23.45nM. This was irrespective of prior chemotherapy treatment, MYC status or SCLC subgroup. The two PDX lines tested were exquisitely sensitive to fimepinostat with LD50 values of 0.00025 and 0.039nM (Fig. 4C). Drugs like PLK1 inhibitors have previously been shown to be effective against SCLC in cell lines but have ultimately failed in clinical testing due to toxicity. Fimepinostat has undergone clinical testing and has been granted orphan drug approval for the treatment of diffuse large B-cell lymphoma (19) and therefore has a known safety profile. However, to indicate whether fimepinostat is likely to have a useful therapeutic window in SCLC the LD50 dose was determined for a matched pair of platinum naive and resistant mouse derived SCLC cell lines and primary mouse lung epithelial cells. We observe significantly less toxicity in normal lung epithelium than observed in either SCLC line (Fig. 4D) suggesting that fimepinostat could be an effective treatment for SCLC whilst preserving normal lung epithelium.

Fimepinostat reduces MYC expression

MYC is often amplified via gene doubling, tandem duplication, or chromosomal translocation. Indeed, the MYC gene is amplified in 40% of human cancers, including breast (20), ovarian (21), prostate (22), hepatocellular (23), colon (24), and lung cancer (25). MYC has largely been considered undruggable through direct approaches which has led to alternative and indirect approaches to treat MYC expressing tumors. One such approach is the inhibition of bromodomain and extra-terminal domain (BET) family of proteins. BET proteins bind acetylated histone lysine residues, leading to recruitment of P-TEFb via its BRD4 domain to sites of active transcription of genes such as MYC. The prototypical BRD2/4 inhibitor, JQ-1 has been shown to decrease MYC expression in other tumor indications (26,27). However, we found that treatment of two independent, MYC expressing, platinum resistant mouse derived SCLC cell lines had no impact on MYC expression even at super- physiological doses (Fig. 8).

Fimepinostat is a dual histone deacetylase (HDAC1/HDAC2/HDAC3/HDAC10) and phosphoinositide 3-kinase (PI3Ka/PI3Kp/PI3K5) inhibitor. Both pathways are the focus of intense clinical investigation and have seen agents approved by the FDA for cancer treatment (28,29). The PI3K pathway and HD AC activity are implicated in MYC expression. PI3K signaling leads to activation of AKT and inhibition of GSK3P, which phosphorylates MYC at Thr-58 leading to degradation of MYC protein. Hence, inhibition of PI3K prevents inhibition of GSK3P thereby promoting MYC turnover (30-32). HD AC inhibitors lead to acetylation of MYC at lysine 323 and decreased MYC mRNA and protein expression (33). Therefore, to determine whether fimepinostat reduced MYC expression in SCLC, two MYC expressing, platinum resistant mouse-derived SCLC cell lines were treated with titrating concentrations of fimepinostat for 24 hours. This timepoint precedes the initiation of cell death observed following fimepinostat treatment. Reduction in MYC expression was observed in response to doses as low as lOnm in B37R2 cells and 500nm in EN84R2 cells (Fig. 5). This is consistent with studies showing fimepinostat decreases MYC expression in diffuse large B-cell lymphoma and NUT midline carcinoma (34,35). Fimepinostat reduced Akt phosphorylation and increased histone H3 acetylation confirming inhibition of PI3K and HD AC respectively (Fig. 5). Importantly, these doses are lower than the drug concentration achieved in patient serum following treatment (36). Together these data show that fimepinostat is an efficient PI3K and HDAC inhibitor and that it reduces MYC expression. Moreover, we show that fimepinostat efficiently kills SCLC cell lines in vitro and that this is not restricted by MYC expression or platinum resistance status.

Fimepinostat is an effective SCLC therapy in vivo.

Our data show single agent efficacy of fimepinostat at concentrations that are consistent with those that are achievable in patients. Therefore, to determine whether fimepinostat can reduce tumor volume in vivo, SCLC cell lines were injected into the flanks of host mice. Mouse derived SCLC cell lines B37R and EN84R were transplanted subcutaneously into the flanks of immune competent C57BL/6 mice. The human SCLC cell line NCI-H209 and PDX lines 102 and 109 were transplanted subcutaneously into the flanks of NOD. Cg-Prkdc ^scid Il2rg ^tmlWjl /SzJ

(NSG) mice. Once tumor volume reached 150-200mm ³ mice received 70mg/Kg fimepinostat or vehicle by daily oral gavage for 28 days or until tumors reached 800mm ³ defined as an ethical endpoint. Fimepinostat treatment significantly reduced tumor growth in vivo in all models tested which is remarkable given the rapid growth of the vehicle control cohorts (Fig. 6A-E). The previously untreated NCI-H209 and two PDX lines had the most dramatic response to fimepinostat (Fig. 6C-E). Together these data show the single agent efficacy of fimepinostat against tumor cell lines that are representative of the genetic diversity observed in patients. However, these models are very aggressive, are grown in the flank rather than the lung and in the case of the human derived lines are transplanted into immunocompromised mice. Our data show that MYC is a potent driver of platinum resistance (Fig 1-3) and that fimepinostat efficiently reduces MYC expression. Therefore, we hypothesized that whilst fimepinostat efficacy is not dependent on MYC expression it will have the dual capacity to reduce MYC expression extending the duration of response to platinum in addition to its single agent efficacy. To address this, disease was initiated in the autochthonous RPM mouse model driven by deletion of Trp53, Rbl and gain of MYC expression in pulmonary neuroendocrine cells and in the context of an intact immune system. Disease progression was monitored by CT imaging and once tumor was detected mice were randomized into four groups who received (i) vehicle, (ii) carboplatin and etoposide, (iii) fimepinostat or (iv) carboplatin, etoposide and fimepinostat (Fig. 7A). As observed previously no appreciable decrease in tumor size was detected in the platinum / etoposide cohort. In contrast, fimepinostat alone reduced tumor volume (Fig. 7B) and fimepinostat in combination with carboplatin and etoposide led to a very significant reduction in tumor mass (Fig. 7B). Histological and immunohistochemical analysis of lungs showed tumor cell death following each of the treatment arms which was most pronounced in the fimepinostat, carboplatin and etoposide cohort (Fig. 7C). This was accompanied by a significant decrease in cell proliferation based on the percentage of PCNA positive cells within the tumor mass (Fig. 7C, D). Interestingly, in mice treated with fimepinostat, carboplatin and etoposide we observed large regions of disrupted lung tissue architecture likely due to the destruction of tumor that previously occupied the lung (Fig. 7C). No overall survival benefit was observed following carboplatin and etoposide treatment alone. In contrast, fimepinostat monotherapy provides a significant increase in overall survival (p <0.0001, log -rank Mantel-Cox test) and in this model is superior to standard of care chemotherapy. The combination of fimepinostat, carboplatin and etoposide produce a very significant increase in overall survival, taking the median survival from the 8 days on treatment observed in the vehicle group to 54.5 days in the combination therapy group (p<0.0001, log-rank Mantel-Cox test) (Fig. 7E).

Together these data directly show that elevated MYC expression drives platinum resistant SCLC in vitro and in vivo. We identify fimepinostat as a drug that efficiently reduces MYC expression and has single agent efficacy against SCLC in the low nM range. Finally, our data show that the combination of fimepinostat with platinum and etoposide provides a significant survival benefit in an autochthonous mouse model of platinum resistant SCLC.

Discussion

Small cell lung cancer is an aggressive neuroendocrine tumor with a devastating overall survival rate. Most patients present clinically with extensive-stage SCLC limiting their treatment options to platinum-based doublet chemotherapy. 60-80% of patients initially respond to this chemotherapy regime, however there is nearly universal relapse with platinum resistant disease for which there is currently no effective second line treatment (4,37). Amplification of the MYC-family of oncogenes occurs in 20% of SCLC and has been associated with worse prognosis and platinum resistance (7,8,10). An elegant recent study has directly shown that MYCN and MYCL are drivers of platinum resistance in mouse models of SCLC (13). However, the evidence of a role for MYC in platinum resistant SCLC remains circumstantial, primarily derived from the observation that MYC amplification is more frequent in cell lines derived from platinum resistant patients than treatment naive lines (10). In this study we confirm that MYC-overexpression is more frequent following platinum - etoposide chemotherapy in vivo in a mouse model of SCLC and that platinum resistant SCLC cell lines have higher MYC expression than matched platinum naive cell lines. We provide direct evidence that MY C is a driver of platinum resistance in vitro and in vivo. Cell lines engineered to stably over-express MYC were significantly more resistant to carboplatin than matched control lines. Importantly, we show that an autochthonous mouse model of SCLC driven by the loss of Trp53, Rbl and gain of MYC expression are refractory to platinum- etoposide chemotherapy. In contrast platinum-etoposide treatment of a mouse model driven by the loss of Trp53, Rbl alone provides a significant survival advantage. Together these data provide the first direct evidence of the ability of MYC to drive platinum resistance, highlighting the clinical potential to target MYC or MYC-dependent processes to overcome platinum resistant SCLC.

Developing direct MYC inhibitors has been challenging. Instead, we took an unbiased screening approach to identify drugs with single agent efficacy against platinum resistant, MYC expressing SCLC cell lines and identified the dual PI3K-HDAC inhibitor fimepinostat. Fimepinostat has been shown to efficiently reduce MYC protein expression (19), and indeed it was given orphan drug designation for the treatment of relapsed/refractory diffuse B-Cell lymphoma, and has entered phase I clinical testing for pediatric brain tumors with a high incidence of MYC amplification (NCT03893487). We show that fimepinostat efficiently reduces MYC expression in platinum resistant SCLC. Moreover it efficiently kills mouse and human SCLC and PDX lines with a low nanomolar LD50 which is in close agreement with an recent independent study in SCLC (38). It is important to note that in our study we observe that fimepinostat is effective irrespective of the MYC-expression status of the cell line. Together, our demonstration of the role of MYC in platinum resistant SCLC, the ability of fimepinostat to reduce MYC expression and to have single agent efficacy independent of MYC expression suggests that fimepinostat will be an effective drug for the treatment of SCLC through multiple mechanisms. It is likely to reduce MYC expression in MYC amplified tumors presumably restoring platinum sensitivity in addition to its efficacy in killing platinum naive and resistant SCLC. Indeed, in an autochthonous mouse model of platinum resistant SCLC we show that fimepinostat significantly increases survival. However, the combination of fimepinostat with standard of care platinum - etoposide chemotherapy provides a greater survival advantage than either agent achieving a durable response that is an increase of -50% of the lifespan of this model.

Histological analysis of lungs following combination fimepinostat, platinum and etoposide treatment showed significant reduction in tumor mass but also revealed damage to the lung architecture. This is likely due to these regions of the lung previously containing tumor that was killed by combination therapy. Due to this, it is possible that the survival benefit observed from combination therapy is an underestimation of what may be observed in patients who receive far more sophisticated clinical care. The data in this manuscript suggests that the addition of fimepinostat to the upfront chemotherapy regime for the treatment of SCLC could improve outcomes and that whilst patients should not necessarily be stratified based on MYC expression those patients with MYC amplification are likely to achieve the benefit of the ability of fimepinostat to reduce MY C expression and prolong platinum response.

References

1. Zimmerman S, Das A, Wang S, Julian R, Gandhi L, Wolf J. 2017-2018 Scientific Advances in Thoracic Oncology: Small Cell Lung Cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2019;14(5):768-83 doi 10.1016/j.jtho.2019.01.022.

2. Pietanza MC, Byers LA, Minna JD, Rudin CM. Small cell lung cancer: will recent progress lead to improved outcomes? Clinical cancer research : an official journal of the American Association for Cancer Research 2015;21(10):2244-55 doi 10.1158/1078- 0432.CCR- 14-2958.

3. Wang S, Zimmermann S, Parikh K, Mansfield AS, Adjei AA. Current Diagnosis and Management of Small-Cell Lung Cancer. Mayo Clin Proc 2019;94(8): 1599-622 doi 10.1016/j.mayocp.2019.01.034.

4. Demedts IK, Vermaelen KY, van Meerbeeck JP. Treatment of extensive-stage small cell lung carcinoma: current status and future prospects. The European respiratory journal 2010;35(l):202-15 doi 10.1183/09031936.00105009.

5. Horn L, Mansfield AS, Szczesna A, Havel L, Krzakowski M, Hochmair MJ, et al. First-Line Atezolizumab plus Chemotherapy in Extensive-Stage Small-Cell Lung Cancer. The New England journal of medicine 2018;379(23):2220-9 doi 10.1056/NEJMoal809064.

6. Paz-Ares L, Dvorkin M, Chen Y, Reinmuth N, Hotta K, Trukhin D, et al. Durvalumab plus platinum-etoposide versus platinum-etoposide in first-line treatment of extensive-stage small-cell lung cancer (CASPIAN): a randomised, controlled, open-label, phase 3 trial. Lancet 2019;394(10212): 1929-39 doi 10.1016/S0140-6736(19)32222-6.

7. George J, Lim JS, Jang SJ, Cun Y, Ozretic L, Kong G, et al. Comprehensive genomic profiles of small cell lung cancer. Nature 2015;524(7563):47-53 doi 10.1038/naturel4664. 8. Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, Kasper LH, et al. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nature genetics 2012;44(10):l 104-10 doi 10.1038/ng.2396.

9. Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan Z, Shames DS, et al. Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer. Nature genetics 2012;44( 10): 1111-6 doi 10.1038/ng.2405.

10. Alves Rde C, Meurer RT, Roehe AV. MYC amplification is associated with poor survival in small cell lung cancer: a chromogenic in situ hybridization study. Journal of cancer research and clinical oncology 2014;140(12):2021-5 doi 10.1007/s00432-014-1769-l.

11. Johnson BE, Russell E, Simmons AM, Phelps R, Steinberg SM, Hide DC, et al. MYC family DNA amplification in 126 tumor cell lines from patients with small cell lung cancer. Journal of cellular biochemistry Supplement 1996;24:210-7.

12. Drapkin BJ, George J, Christensen CL, Mino-Kenudson M, Dries R, Sundaresan T, et al. Genomic and Functional Fidelity of Small Cell Lung Cancer Patient-Derived Xenografts. Cancer discovery 2018;8(5):600-15 doi 10.1158/2159-8290.CD-17-0935.

13. Grunblatt E, Wu N, Zhang H, Liu X, Norton JP, Ohol Y, et al. MYCN drives chemoresistance in small cell lung cancer while USP7 inhibition can restore chemosensitivity. Genes & development 2020;34(17-18): 1210-26 doi 10.1101/gad.340133.120.

14. Luo S, Sun M, Jiang R, Wang G, Zhang X. Establishment of primary mouse lung adenocarcinoma cell culture. Oncol Lett 2011;2(4):629-32 doi 10.3892/ol.2011.301.

15. Chen J, Guanizo A, Luong Q, Jayasekara WSN, Jayasinghe D, Inampudi C, et al. Lineage -restricted neoplasia driven by Myc defaults to small cell lung cancer when combined with loss of p53 and Rb in the airway epithelium. Oncogene 2022;41(l): 138-45 doi 10.1038/s41388-021-02070-3.

16. Sutherland KD, Proost N, Brouns I, Adriaensen D, Song JY, Berns A. Cell of origin of small cell lung cancer: inactivation of Trp53 and Rbl in distinct cell types of adult mouse lung. Cancer cell 2011;19(6):754-64 doi 10.1016/j.ccr.2011.04.019.

17. Rudin CM, Poirier JT, Byers LA, Dive C, Dowlati A, George J, et al. Molecular subtypes of small cell lung cancer: a synthesis of human and mouse model data. Nat Rev Cancer 2019;19(5):289-97 doi 10.1038/s41568-019-0133-9. 18. Leong TL, Marini KD, Rossello FJ, Jayasekara SN, Russell PA, Prodanovic Z, et al. Genomic characterisation of small cell lung cancer patient-derived xenografts generated from endobronchial ultrasound-guided transbronchial needle aspiration specimens. PloS one 2014;9(9):el06862 doi 10.1371/journal.pone.0106862.

19. Sun K, Atoyan R, Borek MA, Dellarocca S, Samson ME, Ma AW, et al. Dual HDAC and PI3K Inhibitor CUDC-907 Downregulates MYC and Suppresses Growth of MYC- dependent Cancers. Molecular cancer therapeutics 2017;16(2):285-99 doi 10.1158/1535- 7163.MCT-16-0390.

20. Escot C, Theillet C, Lidereau R, Spyratos F, Champeme MH, Gest J, et al. Genetic alteration of the c-myc protooncogene (MYC) in human primary breast carcinomas.

Proceedings of the National Academy of Sciences of the United States of America 1986;83(13):4834-8 doi 10.1073/pnas.83.13.4834.

21. Baker VV, Borst MP, Dixon D, Hatch KD, Shingleton HM, Miller D. c-myc amplification in ovarian cancer. Gynecol Oncol 1990;38(3):340-2 doi 10.1016/0090- 8258(90)90069-w.

22. Jenkins RB, Qian J, Lieber MM, Bostwick DG. Detection of c-myc oncogene amplification and chromosomal anomalies in metastatic prostatic carcinoma by fluorescence in situ hybridization. Cancer research 1997;57(3):524-31.

23. Takahashi Y, Kawate S, Watanabe M, Fukushima J, Mori S, Fukusato T. Amplification of c-myc and cyclin D 1 genes in primary and metastatic carcinomas of the liver. Pathol Int 2007 ;57(7) :437 -42 doi 10.1111 /j .1440- 1827.2007.02120.x.

24. Rochlitz CF, Herrmann R, de Kant E. Overexpression and amplification of c-myc during progression of human colorectal cancer. Oncology 1996;53(6):448-54 doi 10.1159/000227619.

25. Little CD, Nau MM, Carney DN, Gazdar AF, Minna JD. Amplification and expression of the c-myc oncogene in human lung cancer cell lines. Nature 1983;306(5939): 194-6 doi 10.1038/306194a0.

26. Mertz JA, Conery AR, Bryant BM, Sandy P, Balasubramanian S, Mele DA, et al. Targeting MYC dependence in cancer by inhibiting BET bromodomains. Proceedings of the National Academy of Sciences of the United States of America 20 l l;108(40): 16669-74 doi 10.1073/pnas.l 108190108. 27. Alqahtani A, Choucair K, Ashraf M, Hammouda DM, Alloghbi A, Khan T, et al.

Bromodomain and extra-terminal motif inhibitors: a review of preclinical and clinical advances in cancer therapy. Future Sci OA 2019;5(3):FSO372-FSO doi 10.4155/fsoa-2018- 0115.

28. Yang J, Nie J, Ma X, Wei Y, Peng Y, Wei X. Targeting PI3K in cancer: mechanisms and advances in clinical trials. Molecular Cancer 2019;18(l):26 doi 10.1186/s 12943 -019- 0954-x.

29. Autin P, Blanquart C, Fradin D. Epigenetic Drugs for Cancer and microRNAs: A Focus on Histone Deacetylase Inhibitors. Cancers 2019;ll(10) doi 10.3390/cancersl 1101530.

30. Rao E, Jiang C, Ji M, Huang X, Iqbal J, Lenz G, et al. The miRNA-17 approximately 92 cluster mediates chemoresistance and enhances tumor growth in mantle cell lymphoma via PI3K7AKT pathway activation. Leukemia 2012;26(5): 1064-72 doi 10.1038/leu.2011.305.

31. Gregory MA, Qi Y, Hann SR. Phosphorylation by glycogen synthase kinase-3 controls c-myc proteolysis and subnuclear localization. The Journal of biological chemistry 2003;278(51):51606-12 doi 10.1074/jbc.M310722200.

32. Kumar A, Marques M, Carrera AC. Phosphoinositide 3 -Kinase Activation in Late G&lt;sub&gt;l&lt;/sub&gt; Is Required for c-Myc Stabilization and S Phase Entry. Molecular and cellular biology 2006;26(23):9116 doi 10.1128/MCB.00783-06.

33. Nebbioso A, Carafa V, Conte M, Tambaro FP, Abbondanza C, Martens J, et al. c-Myc Modulation and Acetylation Is a Key HD AC Inhibitor Target in Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2017;23(10):2542-55 doi 10.1158/1078-0432.CCR-15-2388.

34. Mondello P, Derenzini E, Asgari Z, Philip J, Brea EJ, Seshan V, et al. Dual inhibition of histone deacetylases and phosphoinositide 3 -kinase enhances therapeutic activity against B cell lymphoma. Oncotarget 2017;8(8): 14017-28 doi 10.18632/oncotarget.14876.

35. Gui CY, Ngo L, Xu WS, Richon VM, Marks PA. Histone deacetylase (HDAC) inhibitor activation of p21WAFl involves changes in promoter-associated proteins, including HDAC1. Proceedings of the National Academy of Sciences of the United States of America 2004;101(5): 1241-6 doi 10.1073/pnas.0307708100.

36. Oki Y, Kelly KR, Flinn I, Patel MR, Gharavi R, Ma A, et al. CUDC-907 in relapsed/refractory diffuse large B-cell lymphoma, including patients with MYC-alterations: results from an expanded phase I trial. Haematologica 2017;102(l l):1923-30 doi 10.3324/haematol.2017.172882.

37. Simon GR, Turrisi A, American College of Chest P. Management of small cell lung cancer: ACCP evidence-based clinical practice guidelines (2nd edition). Chest 2007;132(3 Suppl):324S-39S doi 10.1378/chest.07-1385.

38. Ma L, Bian X, Lin W. Correction to: The dual HDAC-PI3K inhibitor CUDC-907 displays single-agent activity and synergizes with PARP inhibitor olaparib in small cell lung cancer. J Exp Clin Cancer Res 2021;40(l):7 doi 10.1186/sl3046-020-01805-6. The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.