Combination therapy with histone deacetylase inhibitors and geranylgeranyltransferase-1 inhibitors

Abstract

A method of treating cancer in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof and a geranylgeranyltransferase-1 (GGT-1) inhibitor or a pharmaceutically acceptable salt thereof. Dosage compositions and kits including the HDAC inhibitor and the GGT-1 inhibitor or pharmaceutically acceptable salts thereof are also provided.

Description

COMBINATION THERAPY WITH HISTONE DEACETYLASE INHIBITORS AND GERANYLGERANYLTRANSFERASE- 1 INHIBITORS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of United States provisional patent application 63 / 747,923, filed January 22, 2025, the contents of which are incorporated herein by reference.STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENTThis invention was made with government support under grant number R35 CA197731, R01 CA186644 and P30 CA16059 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.FIELD OF THE INVENTIONThe invention is generally related to a combination of a histone deacetylase (HDAC) inhibitor and a geranylgeranyltransferase- 1 (GGT-1) inhibitor for the treatment of cancer.BACKGROUND OF THE INVENTIONT-cell lymphomas (TCL) encompass a heterogeneous group of poor prognosis T-cell hematological malignancies and comprise approximately 10-15% of all non-Hodgkin lymphomas (NHL) (1, 2). They are subdivided into multiple subsets (leukemic, nodal, extranodal) according to the recent 2017 WHO classification (3). Despite treatment advances, TCL presents a formidable challenge due to its limited curative therapeutic options. TCL incidence has risen over the past decades (2, 4), underscoring the need for innovative therapeutic strategies. TCL mortality remains high, with a 5-year progression-free survival of only 20% (5) and median survival for relapsed / refractory patients of only 3-6 months (6).TCL is divided into multiple subtypes, including T-cell lymphoblastic lymphoma, cutaneous T-cell lymphomas e.g., CTCL (further subdivided into mycoses fungoides and Sezary syndrome), and members of the peripheral T-cell lymphoma family (PTCL). Current treatment options, aside from standard anthracyclinc-bascd chemotherapy, include recently FDA-approved agents e.g., the histone deacetylase inhibitors (HDACIs) belinostat and romidepsin (7), the anti-metabolite praletrexate, and the CD30 conjugate brentuximab vedotin (8), which provide response rates of 25-30%, and overall 5-year survival of 30% (9, 10).Patients experiencing relapse or resistance (7, 11), have particularly poor prognoses (12). HDACIs modify chromatin by inducing de-condensation, promoting gene expression implicated in tumor cell differentiation and death (27). Despite extensive investigation, mechanisms underlying the sensitivity of CTCL and PTCL to HDACIs remain unknown. Attention has focused on perturbations in survival signaling pathways e.g., AKT, MAPK, STAT3, and NF-KB (12, 31-34).Therapies that can enhance the efficacy of HDAC inhibitors are needed.SUMMARYAs described herein, geranylgeranyltransferase- 1 (GGT-1) inhibitors overcome resistance and enhance histone deacetylase (HDAC) inhibitor anti-tumor activity by inhibiting phosphorylated Akt (p-Akt) providing a synergistic anti-cancer therapy.An aspect of the disclosure provides a method of treating a T-cell lymphoma in a subject in need thereof, comprising administering to the subject i) a therapeutically effective amount of a HDAC inhibitor or a pharmaceutically acceptable salt thereof; and ii) a therapeutically effective amount of a GGT-1 inhibitor or a pharmaceutically acceptable salt thereof, wherein the administration of the HDAC inhibitor and the GGT-1 inhibitor provides a synergistic enhancement of anti-tumor activity compared to administration of either agent alone. In some embodiments, the HDAC inhibitor is romidepsin or belinostat. In some embodiments, the GGT-1 inhibitor is GGTI-2417 or GGTI-2418. In some embodiments, the T-cell lymphoma is cutaneous T-cell lymphoma (CTCL) or peripheral T-cell lymphoma (PTCL). In some embodiments, the T-cell lymphoma is resistant to treatment with the HDAC inhibitor alone. In some embodiments, the HDAC inhibitor or pharmaceutically acceptable salt thereof and the GGT-1 inhibitor or pharmaceutically acceptable salt thereof are administered simultaneously or sequentially. In some embodiments, the synergistic anti -tumor activity is mediated by inhibition of p-Akt.Another aspect of the disclosure provides a method of inhibiting tumor cell viability of or inducing apoptosis in T-cell lymphoma cells, comprising exposing the T-cell lymphoma cells to i) a HDAC inhibitor or a pharmaceutically acceptable salt thereof; and ii) a GGT-1 inhibitor or a pharmaceutically acceptable salt thereof. In some embodiments, the HDAC inhibitor is romidepsin or belinostat. In some embodiments, the GGT-1 inhibitor is GGTI-2417 or GGTI-2418. In some embodiments, the T-cell lymphoma cells are cutaneous T-celllymphoma (CTCL) cells or peripheral T-cell lymphoma (PTCL) cells. In some embodiments, the T-cell lymphoma cells are resistant to exposure with the HDAC inhibitor alone.Another aspect of the disclosure provides a dosage composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt thereof, and a GGT-1 inhibitor or a pharmaceutically acceptable salt thereof, wherein the HDAC inhibitor and GGT-1 inhibitor or pharmaceutically acceptable salts thereof are present together in a single dosage form. In some embodiments, the single dosage form is selected from the group consisting of a tablet, dragee, liquid, drop, capsule, caplet, and gelcap.Another aspect of the disclosure provides a kit, comprising a first dosage composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt thereof; and a second dosage composition comprising a GGT-1 inhibitor or a pharmaceutically acceptable salt thereof.BRIEF DESCRIPTION OF THE DRAWINGSFigures 1A-E. GGTI2417 interacts synergistically with Romidepsin (FK228) to inhibit viability and to induce cell death in the T-cell lymphoma cell line H9. A, H9 cells were exposed to the indicated concentrations of GGTI2417 + / - Romidepsin for 24 h, followed by CellTiter-Glo® Luminescent Cell Viability Assay to monitor cell viability. Data were normalized to percentage of UT. Values represent the mean % ± standard deviations (SD) for five separate experiments. *P < 0.05, ****P < 0.0001. B, H9 cells were exposed (48 h) to varying concentrations of GGTI2417 + / - Romidepsin at a fixed ratio (1000:1). Median doseeffect analysis was then employed to characterize the nature of the interaction between these agents. Combination index values < 1.0 denote a synergistic interaction. C, H9 cells were incubated with GGTI2417 + / - Romidepsin for 24 h, after which PARP, cleaved-PARP, Caspase 3, cleavage of Caspase-3, and yH2A.X were monitored by immunoblotting analysis. P-actin was assayed to ensure equivalent loading and transfer. D, H9 cells were treated for 24 hours with 10 pM GGTI2417, 20 nM Romidepsin or the combination. Cells were stained with Annexin V-FITC and DAPI. Images were obtained with an 1X71 -Olympus inverted system microscope at x 200 magnification. Scale bar = 150 pm. E, Cell death was assessed by flow cytometric analysis after staining with 7-AAD.Figures 2A-D. Co-administration of Romidepsin (FK288) or Belinostat (PDX101) potentiates p-AKT down- regulation by GGTI in H9 and HH TCL cells. A, B, H9 cellswere treated with the indicated concentrations of GGTI2417 + / - Romidepsin or Belinostat for 24 h. Western blot analysis of p-AKT (S473), AKT, and RAP1A were then performed. GAPDH and P-actin were assayed in parallel to ensure equivalent loading and transfer. C, D, Assays were performed as in A-B in HH cells.Figures 3A-E. Expression of CA AKT limits the ability of the HDACIs / GGTI-2417 combination to kill TCL H9 cells. A, H9 cells ectopically expressing constitutively active (myristolated) AKT. B, D, H9 AKT CA cells were treated with GGTI2417 + / -Romidepsin or Belinostat for 24 hours after which CellTiter-Glo® Luminescent Cell Viability Assay was performed to monitor cell viability. Data were normalized to percentage of UT. Error bars = SD for at least 3 independent experiments; * P < 0.05, **** P < 0.0001 vs single drug EV;####p < 0.0001 vs combination-treated EV. C, E, Western blot analysis for the indicated proteins in H9 AKT CA following exposure to GGTI2417 + / - Romidepsin or Belinostat for 24 hours. GAPDH and P-actin were assayed in parallel to ensure equivalent loading and transfer.Figures 4A-G. TCL H9 cells highly resistant to Romidepsin and Belinostat express high p-AKT levels accompanied by diminished apoptosis induction following treatments with Romidepsin and Belinostat. A, D, Assessment of cell viability using CellTiter-Glo® Luminescent Cell Viability Assay after treating sensitive H9 cells and acquired Romidepsin and Belinostat resistant H9 cells with Romidepsin 10 nM and a higher concentration of Belinostat 400nM for 48 hours. Data were normalized to percentage of UT. Error bars = SD for at least 3 independent experiments. ****P < 0.0001. B, E, Assessment of cell viability using CellTiter-Glo® Luminescent Cell Viability Assay after treating sensitive H9 cells and acquired Romidepsin and Belinostat resistant H9 cells with Romidepsin 2.5, 5, 10, 20, 30 nM and Belinostat 0.25, 0.5, 1, 2, 4 pM for 48 hours. *P < 0.05; ***P < 0.001. C, F, Western blot analysis in sensitive H9 cells and acquired Romidepsin and Belinostat-resistant H9 cells following exposure to Romidepsin at 5, 10, 20 and Belinostat 0.1, 0.3, 0.5 pM for 24 hours. G, Western blot analysis of sensitive H9 and 2 Romidepsin and Belinostat-resistant H9 cells without treatment.Figures 5A-F. GGTI-2417 overcomes TCL cells resistance to Romidepsin and Belinostat. A, D, Acquired Romidepsin- and Belinostat-resistant H9 cells were exposed to the indicated concentrations of GGTI2417 + / - Romidepsin and Belinostat for 24 or 48 h, followed by CellTiter-Glo® Luminescent Cell Viability Assay to monitor cell viability. Datawere normalized to percentage of UT. Values represent the means ± standard deviations (SD) for at least 4 independent experiments; *P < 0.05; ****P < 0.0001. B, E, Median Dose Effect Analysis in acquired Romidepsin and Belinostat resistant H9 cells displaying Combination Index consistently below 1.0 which indicates synergistic effect at dose ratio GGTI2417: Belinostat = 40: 1 and GGTI2417: Romidepsin = 1000: 1 for 24 hours. C, F, Western blot analysis in 2 acquired Romidepsin and Belinostat resistant H9 cells following exposure to GGTI2417 + / - Romidepsin and Belinostat for 24 hours.Figures 6A-E. AKT depletion sensitizes HDACI-resistant TCL cells to HDACIs, and GGTI-2417 further sensitizes these cells to HDACIs. A, H9 cells and acquired Belinostat-resistant H9 cells ectopically expressing shAKT. B, C, H9 cells and acquired Belinostat resistant H9 cells ectopically expressing shAKT were treated with Belinostat for 48 hours, after which CellTiter-Glo® Luminescent Cell Viability Assay was performed to monitor cell viability. Values represent the mean % ± SD for four separate experiments. * P < 0.05, ** P < 0.01; **** P < 0.0001 vs UT;####P < 0.0001 vs Belinostat-treated sensitive cells; & P < 0.05; && P < 0.01 vs Belinostat-treated EV. D, H9 cells and acquired Belinostat resistant H9 cells ectopically expressing shAKT were treated with GGTI2417 + / - Belinostat for 48 hours after which CellTiter-Glo® Luminescent Cell Viability Assay was performed to monitor cell viability. Error bars = SD for 6 independent experiments; * P < 0.05; ** P<0.01; **** P < 0.0001 vs single drug; #### P < 0.0001 vs combination-treated sensitive cells; &&<0.01 vs combination-treated EV cells. B and D, Data were normalized to percentage of UT. E, Western blot analysis in 119 cells and acquired Belinostat-resistant 119 cells ectopically expressing shAKT following exposure to GGTI2417 + / -Belinostat for 48 hours.Figures 7A-C. The GGTI-2418 and Romidepsin combination but not single-agent treatment of mice inhibits tumor growth in vivo. NOD / SCID-y (NSG) mice (4 mice / group) were inoculated in the right flank with 5 x 106HH cells. Treatment was initiated after 7 days. GGTI2418 (100 mg / kg , i.p„ 5 day / week) ± Romidepsin (1 mg / kg, i.p., twice / week) were administrated for 3 weeks. A, Tumor size were monitored every other day. *P < 0.05. B, Body weight were monitored every other day. No significant weight loss was observed among the treatment groups. C, At day 21, tumors were harvested and weighted.Figures 8A-D. Chemical structures of (A) GGTI-2418, (B) GGTI-2417, (C) romidepsin, and (D) belinostat.DETAILED DESCRIPTIONEmbodiments of the disclosure provide compositions and methods for a synergistic anticancer treatment comprising a combination of a HDAC inhibitor or a pharmaceutically acceptable salt thereof and a GGT-1 inhibitor or a pharmaceutically acceptable salt thereof.HDACs are enzymes that remove the acetyl group from histones and thus are regulators of gene expression. HDAC inhibitors block these enzymes leading to increased histone acetylation and altered gene expression which causes tumor cell death. The HDAC inhibitors described herein may target any class of HDAC, i.e. class I, II, lib, III, or IV, or may be a pan HDAC inhibitor. Exemplary HDAC inhibitors include, but are not limited to, romidepsin (depsipeptide or FK-228), belinostat (PXD101), vorinostat (SAHA), panobinostat, valproic acid, mocetinostat (MGCD0103), abrexinostat, SB939, resminostat, givinostat (ITF2357), CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, LAQ824, ACY-1215, kevetrin, sodium butyrate, trichostatin A, MS-275 (Entinostat), trapoxin, apicidin, chlamydocin, phenylbutyrate, AN-93, pimelic diphenylamide, N-acetyldinaline, N-2-aminophenyl-3-|4-(4-methylbenzenesulfonylamino)-phenyl]-2-propenamide, LBH-589, SK7041, SK7068, tubacin, depudecin, C1994, Quisinostat (JN.T-26481585), ME-344, sulforaphane, BML-210, PCI-3405, PCI- 24781, luteolin, VAHA, chidamide, PTACH, Oxamflatin, biphenyl-4-sulfonyl chloride, HC toxin, (S)-HDAC-42, 4-iodo-SAHA, cambinol, splitomycin, SBHA, scriptaid, and resveratrol.Protein geranylgeranylation is a fundamental post-translational modification catalyzed by GGT-1 that attaches covalently the lipid geranylgeranyl to cysteine residues at the C-terminus of proteins that end with CAAX (C: cysteine, A: aliphatic amino acid, X: any amino acid) motif. This lipid modification is required for membrane association, participation in signaling pathways, and cancer-causing activity of proteins such as those of the RHO family of GTPases. GGT-1 inhibitors induce G1 arrest by inducing CDK inhibitors like p27, inhibiting CDK2 and CDK4, and inducing retinoblastoma hypophosphorylation. Exemplary GGT-1 inhibitors include, but are not limited to, GGTI-2418, GGTI-2417, GGTI-298, GGTI-DIJ40, and P61-E7.Chemical structures of exemplary inhibitors as described herein are provided in Figures 8A-D.Embodiments provide methods for treating T-cell lymphoma by the administration of a combination as described herein. As used herein “treating” or “treatment” means any mannerof managing the cancer by medicinal or other therapies, such that the cancer no longer increases in size, metastasizes, or otherwise progresses in severity on a diagnosis scale. In some embodiments, the treatment ameliorates the disease through a reduction in cell proliferation or otherwise beneficially improves the severity on a diagnosis scale. The T-cell lymphoma or leukaemia may be selected from cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), angio-immunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma nasal type, primary cutaneous ALCL, T cell prolymphocytic leukaemia, and T-cell acute lymphoblastic leukaemia.In some embodiments, the T-cell lymphoma is resistant to treatment with a HDAC inhibitor or GGT-1 inhibitor alone. In some embodiments, the subject has previously been administered a HDAC inhibitor or GGT-1 inhibitor. In some embodiments, the subject has not previously been administered a HDAC inhibitor or GGT-1 inhibitor.As described in the Example, GGT-1 inhibitors synergistically enhance the anti -tumor activity of HDAC inhibitors. In some embodiments, the synergistic anti-tumor activity is mediated by inhibition of p-Akt.In some embodiments, the HDAC inhibitor or pharmaceutically acceptable salt thereof and the GGT-1 inhibitor or pharmaceutically acceptable salt thereof are administered simultaneously. In some embodiments, the HDAC inhibitor or pharmaceutically acceptable salt thereof and the GGT-1 inhibitor or pharmaceutically acceptable salt thereof are administered sequentially, i.e. the HDAC inhibitor is administered prior to the GGT-1 inhibitor or vice versa.The anti-cancer agents described herein may be administered in vivo by any suitable route (e.g. parenterally or enterally) including but not limited to: inoculation or injection (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular, intramammary, and the like), topical application, and by absorption through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal, rectal, gastrointestinal mucosa, and the like). Other suitable means include but are not limited to: inhalation (e.g. as a mist or spray), orally (e.g. as a pill, capsule, liquid, etc.), intravaginally, intranasally, rectally, by ingestion of a food or probiotic product containing the compound, as eye drops, etc. In preferred embodiments, the mode of administration is oral or by injection. The anti-cancer agents described herein may be administered simultaneously or sequentially. The present disclosure also provides amethod of treatment comprising administering to a subject a formulation as described herein, with or without an additional biological active agent or anti-cancer agent, e.g. an immunotherapy agent, chemotherapeutic agent, anti-angiogenesis agent, signal transduction inhibitor, antiproliferative agent, glycolysis inhibitor, or autophagy inhibitor. In some embodiments, the additional therapeutic agent is selected from an anti-PD-1 antibody, a chemotherapeutic agent, a MEK inhibitor, an EGFR inhibitor, a TOR inhibitor, a SHP2 inhibitor, PI3K inhibitor, and an AKT inhibitor. In some embodiments, the treatment described herein is administered with or without radiation therapy.A patient or subject to be treated by any of the compositions or methods of the present disclosure can mean either a human or a non-human animal including, but not limited to mammals, dogs, horses, cats, rabbits, gerbils, hamsters, rodents, birds, aquatic mammals, cattle, pigs, camelids, and other zoological animals.In some embodiments, the formulation or active agent is administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" or an “effective amount” is meant a sufficient amount to treat the disease or disorder at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compositions of the present disclosure 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; 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 coincidental with the specific active agent employed; and like factors well known in the medical arts. In the case of cancer, the effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer. It is well within the skill of the art to start doses of the compound at levels or frequencies lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage or frequency until the desired effect is achieved. However,the daily dosage of the active agent may be varied over a wide range from 1 to 3000 mg per adult per day. In particular, the compositions contain at least or up to 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, or 3000 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 3000 mg of the active ingredient, in particular from 100 mg to about 3000 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level at least or up to 1 mg / kg to 100 mg / kg of body weight per day, e.g. about 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, or 100 mg / kg of body weight per day. Such doses may be administered in a single dose or it may be divided into multiple doses.Further embodiments provide a method of inhibiting tumor cell viability of or inducing apoptosis in cancer cells in vitro or in vivo comprising the step of exposing the cancer cells to a combination therapy as described herein.Embodiments of the disclosure also provide compositions comprising the anti-cancer agents described herein. For example, the HD AC inhibitor and GGT-1 inhibitor, or the pharmaceutically acceptable salts thereof, may be present together in a single dosage form. In some embodiments, the single dosage form is selected from the group consisting of a tablet, dragee, liquid, drop, capsule, caplet and gelcap.Embodiments of the disclosure further provide combining separate pharmaceutical compositions in kit form. The kit comprises two separate pharmaceutical compositions: HD AC inhibitor and GGT-1 inhibitor. The kit comprises a container for containing the separate compositions such as a divided bottle or a divided foil packet. Additional examples of containers include syringes, boxes, and bags. In some embodiments, the kit comprises directions for the use of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing health care professional.An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet ofrelatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.The pharmaceutical compositions can be formulated according to known methods for preparing pharmaceutically useful compositions. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, e.g. a human, as appropriate. As used herein, the phrase “pharmaceutically acceptable carrier’’ means any of the standard pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as implant carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil / water emulsions. The carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Formulations are described in a number of sources that are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Sciences (Martin E W

[1995] Easton Pa., Mack Publishing Company, 19thed.) describes formulations which can be used in connection with the subject invention. The final amount of the compounds in the formulations may vary. However, in general, the amount in the formulations will be from about 0.01-99%, weight / volume.Compositions as described herein may be prepared either as liquid solutions or suspensions, or as solid forms such as tablets, pills, granules, capsules, powders, ampoules, and the like. The liquid may be an aqueous liquid. Solid forms suitable for solution in, or suspension-Ilin, liquids prior to administration may also be prepared.Formulations suitable for parenteral administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the subject invention can include other agents conventional in the art having regard to the type of formulation in question. The pharmaceutical composition can be adapted for various forms of administration. Administration can be continuous or at distinct intervals as can be determined by a person skilled in the art.The compositions of the present disclosure may also contain other components such as, but not limited to, additives, adjuvants, buffers, tonicity agents, bioadhesive polymers, and preservatives. In any of the compositions of this disclosure, the mixtures are preferably formulated at about pH 5 to about pH 8. This pH range may be achieved by the addition of buffers to the composition. It should be appreciated that the compositions of the present disclosure may be buffered by any common buffer system such as phosphate, borate, acetate, citrate, carbonate and borate-polyol complexes, with the pH and osmolality adjusted in accordance with well-known techniques to proper physiological values.An additive such as a sugar, a glycerol, and other sugar alcohols, can be included in the compositions of the present disclosure. Pharmaceutical additives can be added to increase the efficacy or potency of other ingredients in the composition. For example, a pharmaceutical additive can be added to a composition of the present disclosure to improve the stability of the bioactive agent, to adjust the osmolality of the composition, to adjust the viscosity of the composition, or for another reason, such as effecting drug delivery. Non-limiting examples of pharmaceutical additives of the present disclosure include sugars, such as, trehalose, mannose, D-galactose, and lactose.In an embodiment, if a preservative is desired, the compositions may optionally bepreserved with any well-known system such as benzyl alcohol with / without EDTA, benzalkonium chloride, chlorhexidine, Cosmocil® CQ, or Dowicil 200.“Salts” or “pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present disclosure. These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-.beta.-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, 1-19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use such as ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, anddicyclohexylamine, and the like.The compounds of the present disclosure may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water (hydrate), ethanol, and the like. A solvate is the result of solvation which is an interaction of a solute (i.e. compound of the disclosure) with a solvent. Solvation leads to stabilization of the solute species in the solution. A solvate refers to the solvated state, whereby an ion in a solution is surrounded or complexed by solvent molecules. Exemplary solvents include, but are not limited to, propylene glycol; polypropylene glycol; polyethylene glycol (for example, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 900, polyethylene glycol 540 (all available from Union Carbide) and the like); pharmaceutically acceptable alcohols (for example, ethanol or 2-(2-ethoxyethoxy)ethanol (Transcutol®, Gattefosse, Westwood, N.J. 07675) and the like); polyoxyethylene castor oil derivatives (for example, polyoxyethyleneglycerol triricinoleate or polyoxyl 35 castor oil (Cremophor®EL, BASE Corp.), polyoxyethyleneglycerol oxystearate (Cremophor®RH 40 (polyethyleneglycol 40 hydrogenated castor oil) or Cremophor®RH 60 (polyethyleneglycol 60 hydrogenated castor oil), BASF Corp.) and the like); fractionated coconut oil (for example, mixed triglycerides with caprylic acid and capric acid (Miglyol®812, available from Huis AG, Witten, Germany) and the like); Tween®80; isopropyl palmitate; isopropyl myristate; pharmaceutically acceptable silicon fluids; and the like.Before exemplary embodiments of the present invention are described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention.EXAMPLEMaterials and MethodsCell lines and reagentsH9 (Cat# HTB-176, RRID: CVCL_1240), HH (Cat# CRL-2105, RRID: CVCL_1280), and HuT-78 (Cat# TIB- 161, RRID: CVCL_0337) TCL cell lines were obtained from the ATCC. They were maintained in RPMI-1640 supplemented with 10% fetal bovine serum and penicillin-streptomycin. All experiments utilized logarithmically growing cells (3-4xl05cells / ml). MycoAlert® (Lonza, Allendale, NJ) assays were performed, demonstrating that all cell lines were free of mycoplasma contamination. Belinostat or Romidepsin-resistant H9 and HH cells were obtained by culturing cells in the presence of increasing Belinostat concentrations (from 50 nM to 300 nM) or Romidepsin (from InM to 8nM) over a period of 3 months.GGTI-2417 and GGTI-2418 were synthesized as previously described (1). Belinostat (PXD101) was from Spectrum Pharmaceuticals, Irvine, CA; Romidepsin (FK228) was purchased from MedChemExpress (cat# HY-15149, Monmouth Junction, NJ, USA). Copanlisib (BAY 80-6946) was purchased from AdooQ (cat# HY-11766, Irvine, CA, USA); MK-2206 was purchased from MedChemExpress (cat# HY-10358). All drugs were dissolved in DMSO, aliquoted, and stored at -80°C. In all experiments, final DMSO concentrations did not exceed 0.1%.Plasmid transfection and virus infectionLentiviruses were generated in 293T cells (RRID: CVCL_0063) by transfecting cells with plasmids pCDH-puro-myr-HA-Aktl (Addgene #46969, RRID: Addgene_46969) (2), psPAX2 (Addgene #12260, RRID: Addgene_12260), and pMD2-VSVG (Addgene #12259, RRID: Addgene_12259). PEI transfection reagents were used. Viral supernatant was collected two and three days after transfection, filtered through 0.45 pm membranes, and added to HH and H9 cells in the presence of polybrene (8 pg / ml, Millipore). Puromycin (1.5 pg / ml) was used to treat cells for two days for selection, which eliminated all cells in the uninfected control group.AKT knockdown in H9-Bel-res cells was achieved via lentiviral transduction using pLKO.I-based shRNA constructs targeting AKT.Analysis of cell deathApoptosis was evaluated by flow cytometry and microscopy utilizing Annexin V-EITC / DAPI staining as before (3). Loss of mitochondrial membrane potential and cell death were assessed by a 7-AAD staining assay as previously described (4).Cell viability assayCell proliferation was determined by CellTiter-Glo® luminescence cell viability assay (G7570; Promega, Madison, WI, US) in accordance to the manufacturer’s instructions. ImmunoblottingSamples were prepared from whole-cell pellets followed by lysis with M-PERTM Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IE). Total protein or nuclear protein were quantified using Coomassie Protein Assay Reagent (Pierce ThermoFisher Scientific, Rockford, IL). Equal amounts of protein (20 pg) were separated by SDS-PAGE and electro-transferred onto nitrocellulose membrane. Primary antibodies used were: AKT (Cat#4691, RRID:AB_915783), phospho-AKT (Ser 473) (Cat#4060, RRID:AB_2315049), cleaved PARP (Cat#9541, RRID:AB_331426), PARP (Cat# 9532, RRID:AB_659884), yH2A.X (Cat# 2577, RRID:AB_2118010), BCL-XL(Cat# 2762, RRID:AB_10694844), cleaved Caspase-3 (Cat# 9661, RRID:AB_2341188), MCL-1 (Cat# 94296, RRID:AB_2722740), GAPDH (Cat# 97166, RRID:AB_2756824) are from Cell Signaling Technology. P-actin (Cat# A2066, RRID: AB_476693) is purchased from Sigma-Aldric. Unprenylated RaplA (Goat) is from Santa Cruz (Cat# sc-1482, RRID: AB_2177124). The secondary antibodies used were goat anti-mouse (SeraCare KPL Cat# 5450-0011, RRID: AB_2687537) and anti-rabbit (SeraCare Cat# 5220-0458, RRID: AB_2920567) IgG-peroxidase labeled. Primary antibodies were used at 1:1000 dilutions (Cell Signaling Technology, Abeam), 1:1,000 dilutions (Sigma- Aldrich), and 1:500 dilutions (Santa Cruz). Secondary antibodies were used at 1:5000 dilutions (sera care). Images were captured with the Odyssey® Fc Imaging System (LLCOR). Images were quantified and analyzed by using Image.! software (RRID:SCR_003070).Animal studiesAll animal studies were performed under protocol AM 10204, approved by our local IACUC, and regulated by VCU’s Animal Care and Use Program, in accordance with AAALAC, USDA, and PHS guidelines. NOD-SCID IL2Rgammanu11mice (Jackson Laboratories Bar Harbor, ME, RRID: IMSR_.TAX:005557) were subcutaneously injected with 5 x 106HH for flank model (4 mice / group). The HD AC inhibitor Romidepsin was prepared in a 5% / 95% (v / v / v) mixture of DMSO and com oil at a concentration of 0.25 mg / ml. GGTI-2418 was formulated in 20% DMSO / 40% PEG300 / 40% com oil (v / v / v) at a concentration of 40 mg / ml. When tumors grew to 5 mm (length), mice were subjected to treatment witheither Romidepsin (1 mg / kg, twice per week, i.p.), GGTI-2418 (100 mg / kg, 5 days per week, i.p.), or a combination of both treatments for 3 weeks. Control animals received an equal volume of vehicle. Tumor growth and body weight were monitored every other day; when tumor size reached 17mm, mice were euthanized. Tumor volumes were calculated using the formula (length x widthA2) / 2.Statistical analysisValues represent the means ± standard deviation (SD) for three separate experiments. The significance of differences between experimental variables was determined using the Student’s z-test or one-way analysis of variance with the Tukey-Kramer multiple comparisons test. For cell viability assays, data were normalized to percentage of UT. Values were considered statistically significant at *P < 0.05; **P < 0.01; ***P < 0.001. Analysis of synergism was performed by Median Dose Effect analysis using the software Calcusyn (Biosoft) as before (5). Additionally, the SynergyFinder tool (6) was employed to calculate synergy scores and generate the corresponding 3D response surface plots.References for Materials and Methods:1. Peng H, Carrico D, Thai V, Blaskovich M, Bucher C, Pusateri EE, et al. Synthesis and evaluation of potent, highly-selective, 3-aryl-piperazinone inhibitors of protein geranylgeranyltransferase-I. Org Biomol Chem. 2006;4(9): 1768-84.2. Cheng Z, Gong Y, Ma Y, Lu K, Lu X, Pierce LA, et al. Inhibition of BET bromodomain targets genetically diverse glioblastoma. Clin Cancer Res. 2013;19(7):1748-59.3. Hu X, Li L, Nkwocha J, Kmieciak M, Shang S, Cowart LA, et al. Src inhibition potentiates MCL-1 antagonist activity in acute myeloid leukemia. Signal Transduct Target Ther. 2025; 10(1 ):50.4. Chen S, Dai Y, Harada H, Dent P, Grant S. McLl down-regulation potentiates ABT-737 lethality by cooperatively inducing Bak activation and Bax translocation. Cancer Res.2007;67(2):782-91.5. Hu X, Li L, Nkwocha J, Sharma K, Zhou L, Grant S. Synergistic Interactions between the Hypomethylating Agent Thio-Deoxycytidine and Venetoclax in Myelodysplastic Syndrome Cells. Hematol Rep. 2023;15(l):91-100.6. Kazi A, Vasiyani H, Ghosh D, Bandyopadhyay D, Shah RD, Vudatha V, et al. FGTI-2734 Inhibits ERK Reactivation to Overcome Sotorasib Resistance in KRAS G12C Lung Cancer. J Thorac Oncol. 2025 ;20(3): 331-44.ResultsThe combination of GGTI-2417 and HDAC inhibitors (romidepsin and belinostat) interact synergistically in inhibiting the viability and inducing apoptosis of T cell lymphoma (TCL) cells.The GGTI-2417 prodrug is more potent than GGTI-2418 in cultured cells due to superior cellular uptake. Therefore, the TCL line H9 (40-42) was exposed for 24 hr to GGTI-2417 (10 |iM) ± romidepsin (10 nM). Figure 1A shows that whereas single agents exerted little effect, the combination sharply increased cell death by CellTiter-Glo® assay. Median Dose Effect analysis revealed Combination Index (CI) values markedly below 1.0, indicating highly synergistic interactions (Figure IB). GGTI-2417 also interacted synergistically with romidepsin in apoptosis induction reflected by a) sharp increase in PARP, caspase-3 cleavage, and yH2A.X formation (Figure 1C), b) enhanced Annexin V staining (light microscopy;Figure ID), and c) pronounced hypodiploid population (Figure IE). Similar interactions occurred in H9 cells exposed to GGTI-2417 and belinostat and in the TCL line HH (42, 43) exposed to GGTI-2417 and romidepsin or belinostat (data not shown). Moreover, both drug combinations induced increased cell death in H9 and HH cells across a range of concentrations after 24 hours and exhibited significant synergistic activity across multiple concentrations after 72 hours (data not shown). Finally, comparable results occurred in the TCL line Hut78 exposed to GGTI-2417 and romidepsin or belinostat (data not shown), indicating that GGTI-2417 interacts synergistically with HDACIs to induce cell death in multiple TCL lines. Synergistic Interactions between GGTI-2417 and HDAC inhibitors (romidepsin and belinostat) in TCL cells are mediated in part by AKT inactivation.The GGTI-2417 and HDACI combination is effective at suppressing P-AKT levels in TCL cellsWe first examined the impact of HDACi / GGTI-2417 exposure on AKT activation in TCL cells by examining responses to the agents alone or together. Treatment (24 hr) of H9 and HH cells with GGTI-2417 alone or in combination with belinostat or romidepsin induced RAP1A geranylgeranylation inhibition, reflected by increased levels of non-geranylgeamylated RAP1A (the antibody used recognizes only non-geranylgeranylated RAP1A; see Methods), demonstrating GGTI-2417 on-target effects (Figures 2A-D).Interestingly, whereas GGTI alone modestly reduced p-AKT(S473) in H9 and HH cells in a dose-dependent manner, combination with belinostat markedly suppressed P-AKT levels (Figures 2A and 2C). Similar results were observed with romidepsin + GGTI-2417 in H9 and HH cells (Figures 2B and 2D), suggesting that AKT inactivation may contribute to GGTI-2417 / HDACi synergism in TCL.AKT inhibition plays a functional role in GGTI / HDACI synergism in TCL cells.To evaluate the functional significance of AKT inactivation, we postulated that ectopic expression of constitutively active myristoylated AKT would diminish HDACI / GGTI-2417 lethality. We generated H9 cells expressing empty-vector or constitutively active (myristoylated membrane -bound) AKT (46) as previously described (47) (Figure 3A) and exposed cells to GGTI-2417 ± HDACIs, after which viability loss was determined. In contrast to empty-vector cells, viability of H9 cells expressing constitutively active AKT was not inhibited by GGTI-2417 and belinostat alone or in combination (Figure 3B). Note that here and elsewhere, EV cell values may vary somewhat from responses of un-transfected cells, presumably the consequence of the transfection and selection processes. Similarly, constitutively active AKT significantly reduced apoptosis induction by these agents (cleaved caspase 3 and PARP, generation of yH2A.X; Figure 3C). Similar results were obtained in H9 cells expressing CA-AKT and exposed to GGTI-2417 / romidepsin (Figure 3D-E).Comparable responses were seen in HH cells expressing CA-AKT and exposed to GGTI-2417 + romidepsin (data not shown). Lastly, the PI3K inhibitor BAY80-6946 (48) and the AKT inhibitor MK2206 (47) mimicked the ability of GGTI to potentiate HDACI killing in H9 cells (data not shown). Collectively, these findings indicate that sub-lethal concentrations of GGTI-2417 synergistically increase cell death induced by minimally toxic concentrations of HDACIs in TCL cells. They also suggest that GGTI inactivation of the AKT survival signaling pathway, implicated in the MOA of HDACIs (44), contributes to synergistic interactions between HDAC inhibitors and GGTI-2417 in TCL cells.TCL cells with acquired HDACI resistance exhibit AKT activation: GGTI / HDACI decreases P-AKT levels and sensitizes resistant TCL cells to HDACIs.Our results support the notion that inactivation of the PI3K / AKT survival signaling pathway contributes to synergistic interactions between HDACIs and GGTIs. We thenpostulated that further support for this concept could be provided by generation of HDACI-resistant cells exhibiting PI3K / AKT signaling activation as a compensatory survival mechanism, and that HDACI / GGTI-2417 co-administration could suppress this pathway, overcoming HDACI resistance. Accordingly, we generated highly HDACI-resistant cells by culturing H9 cells in progressively higher romidepsin concentrations. These cells displayed 5-10-fold resistance to romidepsin compared to parental counterparts (Figure 4A-B) and diminished apoptosis (decreased cleaved caspase-3, cleaved PARP, and generation of yf A.X (Fig 4C). Similar results occurred in H9 cells resistant to belinostat (Figure 4D-F). Notably, resistant cells were highly resistant to high concentrations of belinostat e.g., 400 nM (Figure 4D). BCL-2 family protein analysis revealed that romidepsin or belinostat-resistant cells failed to exhibit major changes in the expression of BCL-2 family members e.g., MCL-1, BCL-2, BCL-xL, or NOXA (Figure 4G). In marked contrast, both resistant cell lines exhibited marked up-regulation of activated p-AKT(S473) (Fig 4G). Virtually identical results were obtained in romidepsin-resistant HH cells (data not shown), consistent with previous results supporting a functional role for AKT activation in conferring GGTI / HDACI resistance.We then determined whether GGTL2417 co-administration restored the sensitivity of resistant cells to HDACIs, accompanied by recapitulation of pharmacodynamic events exhibited by sensitive cells e.g., AKT inactivation. In belinostat-resistant H9 cells, combined GGTI-2417 / belinostat treatment synergistically triggered cell death (Figure 5A-B) and induced increased killing over a range of concentrations (data not shown). Furthermore, combined treatment effectively down-regulated p-AKT(S473) in both sensitive and resistant cells, accompanied by increased PARP / caspase 3 cleavage (Figure 5C). GGTI-2417 equally increased unprenylated RAP1A in both cell lines. Similar results occurred in romidepsin-resistant H9 cells (Figure 5D-F), consistent with evidence that AKT activation confers HDACI resistance in TCL cells and that AKT inactivation by the GGTI-2417 / HDACI regimen overcomes resistance, at least in part.AKT inactivation plays a functional role in potentiation of HDACI activity in resistant TCL cells.Genetic approaches were then employed to confirm the role of AKT inactivation in circumventing HDACI resistance as well as GGTI-2417 / HDACI synergism in HDACI-resistant TCL cells. Empty-vector and shAKT clones were generated from belinostat-resistantcells (Figure 6A). Parallel studies were performed in untransfected sensitive or resistant cells. Notably, following 24 hr exposure to 150 nM, belinostat-resistant shAKT H9 cells were modestly but significantly more susceptible to cell death than empty-vector counterparts (Figure 6B). Belinostat dose-response studies confirmed increased cell death in shRNA AKT knock-down lines (Figure 6C).Additionally, when belinostat-resistant H9 cells exhibiting AKT knock-down were exposed to 150 nM belinostat ± 10 pM GGTI-2417, combined exposure induced significantly greater lethality compared to either single-agent- treated or empty- vector cells (Figure 6D), results confirmed with western blot analysis of AKT expression, caspase-3 cleavage, and yH2A.X generation (Figure 6E). Finally, the AKT inhibitor MK2206 and the PI3 kinase inhibitor BAY 80-6946 increased HDACI lethality in resistant H9 cells (data not shown), arguing that in HDACI-resistant TCL cells exhibiting increased AKT expression, GGTI-2417 co-administration restores HDACI sensitivity, at least in part, through an AKT-dependent process.GGTI enhances HDACI sensitivity in T-cell lymphoma cells in vivo.Finally, we determined whether the in vitro findings could be recapitulated in vivo. Both parent and pro-drug are equipotent in vivo because of high serum esterases that convert GGTI-2417 to GGTI-2418. Consequently, in mouse studies GGTI-2418 is used whereas GGTI-2417 is used in cell culture studies. NSG mice were inoculated in the flank with 5 x 106HH cells, after which treatment was begun when tumors became palpable (after 7 days). Mice were treated with GGTI-2418 (100 mg / kg ip 5 days / wk) ± romidepsin (1 mg / kg ip twice per week). Treatment was continued for 24 days, during which tumor volumes were monitored twice / week. As shown in Figure 7A, neither FGTI-2418 nor romidepsin treatment alone significantly affected tumor growth. In contrast, mice treated with the GGTI-2418 / romidepsin combination displayed a significant reduction in tumor volume (Figures 7A and 7C) without a significant loss in animal weights (Figure 7B), arguing that the GGTI-2418 / romidepsin combination is significantly more effective at inhibiting tumor growth in vivo compared to single-agent treatment, recapitulating the combination’s superior effectiveness in vitro. DiscussionThe findings that GGTIs and HDACIs interact synergistically to kill TCL cells in vitro and that the clinically relevant agent GGTI-2418 potentiates HDACI activity in TCL xenografts in vivo prompted us to develop a novel strategy for T-cell malignancies such asPTCL and CTCL, e.g., by targeting two distinct cellular pathways — prenylation (via GGTI-2418) and histone deacetylation (via HDACIs). To date, GGTIs have been minimally explored clinically in solid tumors (25) and in hematologic malignancies (26). In contrast, HDACIs including belinostat and romidepsin have been FDA-approved in these disorders (35, 39), although remissions tend to be transient due to resistant disease (7, 11) (12) (13). Notably, GGTI / HDACI interactions have never previously been investigated in any malignancy. The present findings therefore provide novel insights into mechanisms underlying GGTI / HDACI synergism in T-cell malignancies, focusing on the interplay between prenylation- and chromatin-related survival signaling pathways e.g., AKT. Such information could help optimize GGTI / HDACI regimens and identify reliable pharmacodynamic response determinants in individual patients.While it is assumed that HDACIs act by modifying chromatin structure and derepressing genes encoding cell death and / or differentiation (35, 49), mechanisms responsible for their pronounced activity in TCL remain unknown, nor is information available concerning the basis for GGTI / HDACI synergism. One plausible possibility is that GGTI-mediated perturbations in diverse survival signaling / cell cycle regulatory pathways may be implicated in these events (20, 21, 23). The present results provide evidence that interference with the PI3K / AKT pathway contributes functionally to GGTI / HDACI synergism in TCL cells. They also raise the possibility that this strategy may be effective in circumventing HDACI resistance, which ultimately supervenes in the large majority of TCL patients (50-52).GGTIs regulate the activity of diverse proteins implicated in survival signaling pathways. For example, GGTIs induce CDK inhibitors such as p21 and p27, inhibiting CDK2 and CDK4, and inducing hypophosphorylation of Rb, leading to G1 phase cell cycle arrest (18) (20). However, in contrast to such early studies, we did not see perturbations in these pathways in T-cell lymphoma cell lines, arguing against their contribution to GGTI / HDACI synergism. Significantly, GGTIs induce apoptosis by inhibiting the PI3K / AKT pathway (21, 53). Thus, GGTIs may potentiate HDACI anti-tumor activity in TCL cells by disabling the AKT pathway.In the present study, co-administration of GGTI with HDACIs induced pronounced AKT inactivation. Furthermore, it is noteworthy that GGTIs overcame HDACI resistance in part by interfering with the AKT pathway. This is supported by the following evidence: a) HDACLresistant TCL cells expressed high levels of p-AKT that were down-regulated byGGTI / HDACI co-administration, triggering HDACI sensitization, b) ectopic expression of constitutively active AKT conferred resistance of cells to the GGTFHDACI regimen, and c) shRNA knock-down of AKT in HDACI-resistant cells significantly restored HDACI / GGTI sensitivity. These results are consistent with our previous demonstration that GGTIs induce apoptosis in cisplatin-resistant human ovarian cancer cells by inhibition of PI3K / AKT (21). Indeed, GGTI treatment inhibited the kinase levels of PI3K and AKT, and ectopic expression of constitutively active AKT significantly rescued cells from GGTI-induced apoptosis (21, 54). While it remains likely that GGTIs act by disrupting multiple pro-survival pathways, our findings argue strongly that AKT inactivation in TCL cells plays a significant functional role in potentiating HDACI activity and circumventing resistance.Given the ability of AKT and PIK3 inhibitors to enhance HDACI activity in T-cell lines, the question arises whether GGTI offers unique advantages over these agents. There are at least two reasons why GGTI might be superior in this setting. First, in addition to the P13K / AKT pathway, disruption of prenylation may disable alternative pro-survival pathways e.g., MAPK. In addition, results of our phase I GGTI study revealed remarkably little toxicity (26), in contrast to PI3K and AKT inhibitors. The very limited toxicity profile of GGTI might be particularly useful in the context of combination regimens.In summary, the present findings demonstrate that the GGT-1 inhibitor GGTI-2417 interacts synergistically with HDACIs to induce cell death in different T-cell lymphoma lines through an AKT-dependent mechanism. They also suggest that this mechanism contributes to GGTI-mediated circumvention of HDACI resistance in these cells. This is consistent with the finding that the clinically relevant agent GGTI-2418 enhanced the anti -tumor of HDACIs in TCL xenografts in vivo.References1. Satou A, Bennani NN, Feldman AL. Update on the classification of T-cell lymphomas, Hodgkin lymphomas, and histiocytic / dendritic cell neoplasms. Expert Rev Hematol.2019;12(10):833-43.2. Phan A, Veldman R, Lechowicz MJ. T-cell Lymphoma Epidemiology: the Known and Unknown. Cun-Hematol Malig Rep. 2016;ll(6):492-503.3. Jiang M, Bennani NN, Feldman AL. 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J Invest Dermatol. 2019;139(9):I975-84 e2.While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modificationsand equivalents thereof within the spirit and scope of the description provided herein.

Claims

We claim:

1. A method of treating a T-cell lymphoma in a subject in need thereof, comprising administering to the subjecti) a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt thereof; andii) a therapeutically effective amount of a geranylgeranyltransferase- 1 (GGT-1) inhibitor or a pharmaceutically acceptable salt thereof,wherein the administration of the HDAC inhibitor and the GGT-1 inhibitor provides a synergistic enhancement of anti-tumor activity compared to administration of either agent alone.

2. The method of claim 1 , wherein the HDAC inhibitor is romidepsin or belinostat.

3. The method of claim 1, wherein the GGT-1 inhibitor is GGTI-2417 or GGTI-2418.

4. The method of claim 1, wherein the T-cell lymphoma is cutaneous T-cell lymphoma (CTCL).

5. The method of claim 1, wherein the T-cell lymphoma is peripheral T-cell lymphoma (PTCL).

6. The method of claim 1, wherein the T-cell lymphoma is resistant to treatment with the HDAC inhibitor alone.

7. The method of claim 1, wherein the HDAC inhibitor or pharmaceutically acceptable salt thereof and the GGT-1 inhibitor or pharmaceutically acceptable salt thereof are administered simultaneously.

8. The method of claim 1, wherein the HD AC inhibitor or pharmaceutically acceptable salt thereof and the GGT-1 inhibitor or pharmaceutically acceptable salt thereof are administered sequentially.

9. The method of claim 1 , wherein the synergistic anti -tumor activity is mediated by inhibition of phosphorylated Akt (p-Akt).

10. A method of inhibiting tumor cell viability of or inducing apoptosis in T-cell lymphoma cells, comprising exposing the T-cell lymphoma cells toi) a HD AC inhibitor or a pharmaceutically acceptable salt thereof; andii) a GGT-1 inhibitor or a pharmaceutically acceptable salt thereof.

11. The method of claim 10, wherein the HD AC inhibitor is romidepsin or belinostat.

12. The method of claim 10, wherein the GGT-1 inhibitor is GGTI-2417 or GGTI-2418.

13. The method of claim 10, wherein the T-cell lymphoma cells are cutaneous T-cell lymphoma (CTCL) cells.

14. The method of claim 10, wherein the T-cell lymphoma cells are peripheral T-cell lymphoma (PTCL) cells.

15. The method of claim 10, wherein the T-cell lymphoma cells arc resistant to exposure with the HD AC inhibitor alone.

16. A dosage composition comprising:a HD AC inhibitor or a pharmaceutically acceptable salt thereof, anda GGT-1 inhibitor or a pharmaceutically acceptable salt thereof,wherein the HDAC inhibitor and GGT-1 inhibitor or pharmaceutically acceptable salts thereof are present together in a single dosage form.

17. The dosage composition of claim 16, wherein said single dosage form is selected from the group consisting of a tablet, dragee, liquid, drop, capsule, caplet, and gelcap.

18. A kit, comprising:a first dosage composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt thereof; anda second dosage composition comprising a GGT-1 inhibitor or a pharmaceutically acceptable salt thereof.

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