Anticancer MTOR activator

Abstract

A method of treating cancer in a subject in need thereof is described. The method includes administering to the subject a therapeutically effective amount of an mTOR activator and a proteasome inhibitor to the subject. Tuberous sclerosis complex 2 (TSC2) inhibitors and methods of using them to inhibit TSC2 are also described.

Description

ANTICANCER MTOR ACTIVATORGOVERNMENT FUNDING

[0001] The present invention was made with government support under Grant No. 1R01CA299332 awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 694,338, filed on September 13, 2024, which is incorporated herein by reference.BACKGROUND

[0003] Acute myeloid leukemia (AML) is a consequence of transformed myeloid precursor cells in the bone marrow, resulting in an accumulation of abnormal, immature myeloid cells. AML is driven by leukemic stem cells (LSCs), which proliferate, gradually overtake the bone marrow, giving rise to AML blast cells that are released to the bloodstream. Inevitably, over time, an irreversible bone marrow failure ensues. AML affects both children and adults. While children respond well to allogeneic stem cell transplantation procedures and high dose chemotherapy, most adults do not. Hence, prognosis of AML in adults is dire with five-year survival of less than 5% in over 60-year-old patients. A major challenge in treatment is the inability of current therapy to reach a complete response. This results in a relapse of most patients within 3 years. The backbone of AML therapy in adults is cytarabine and daunorubicin. Additional drugs are given in a personalized manner. Finn et al., Ochsner J., 17(4):398-404 (2017). Thus, an urgent need exists for additional drugs that operate orthogonally to current therapeutics.

[0004] Proteasome inhibitors (Pls) have a unique mechanism of action. Hideshima et al., Blood, 101(4): 1530-1534 (2003). However, the clinical benefit of these drugs is limited to specific cancers, primarily multiple myeloma (MM) Ito S., Cancers (Basel), 12(2) (2020). Despite similarities between MM and AML with respect to protein synthesis and degradation, Pls fell short to significantly extend the overall survival in AML Csizmar et al., Blood Cancer J., 6(12):e503 (2016).

[0005] Mammalian target of rapamycin (mTOR) resides in either of two complexes: mTORd and mTORC2, which dictate specificity and regulation. mTORCl promotes anabolic programs, inhibits autophagy, and induces the biosynthesis of proteins, lipids and nucleic acids. These activities promote cell growth and survival and play a role in oncogenesis. However, despite promising preclinical data, the clinical effects of mTOR inhibitors have thus far been disappointing, in part due to prosurvival roles of mTORCl suppression in solid and hematological tumors in response to therapy. Liu et al., Nat Commun., 13(l):7047 (2022). mTORCl is negatively controlled by the tuberous sclerosis complex (TSC). TSC2 is the catalytic subunit of the complex, which operates as a GTPase activating protein (GAP) for Rheb, an essential G protein for mTORC 1 activation. Deletion of TSC2 leads to the most potent and direct hyper activation of mTORCl, and to a disconnection from upstream regulation. Darawshi et al., Cell Death Dis. 2022;13(l l):969 (2022).SUMMARY OF THE INVENTION

[0006] High mTORCl activity is oncogenic. However, in the presence of chemotherapy, the suppression of mTORCl promotes survival and endows tumors with drug resistance. While highly potent inhibitors of mTORCl were developed, small molecules that directly activate mTORCl are lacking. Activation of mTORCl is best achieved by suppression of its negative regulator, the tuberous sclerosis complex, composed of TSC 1, TSC2 and TBC1D7. In attempt to recapitulate this pharmacologically, the inventors employed in silico docking and additional chemical modifications to generate AcTor, a potential first of its kind TSC2 inhibitor. Deletion of TSC2 results in an increase sensitivity of multiple myeloma cells to proteasome inhibitors (Pls). When studied in combination with the PI ixazomib (IXZ), a strong potentiation of cytotoxic activity was observed in multiple myeloma cells. An even stronger synergism was observed across multiple acute myeloid leukemia (AML) cell lines. AcTor with IXZ induced a rapid loss of mitochondrial activity in AML, which was mapped to complex IV of the electron transport chain. In vivo, AcTor alone accelerated the growth of AML, consistent with the activation of mTORCl . When combined with IXZ for three weeks, AML growth was inhibited, and leukemic blasts and stem cells underwent apoptosis. AcTor / IXZ treatment maintained potency in a relapse model of AML. These results indicate that courses of treatment with AcTor / IXZ can have a durable anti- AML effect, before and after relapse.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0007] The present invention may be more readily understood by reference to the following figures, wherein:

[0008] Figures 1A-1D provide graphs and images showing the rationale design of AcTor as a TSC2 inhibitor. (A) In silico docking of cobicistat to the model of TSC2 and Rheb. Cobicistat mostly binds to TSC2. (B) A 2D projection of cobicistat binding demonstrates the proximity to the catalytic residue Arg 1749 of TSC2 and the protrusion of the phenyl group into Rheb, circled in black. (C) AcTor is a modified cobicistat. (D) RPMI8226 cells were treated for 24 h with the indicated concentrations of AcTor. Total cell lysates were analyzed by immunoblotting. mTORCl activity was assessed by P-S6K1, P-S6 and P-4EBP1 levels.

[0009] Figures 2A-2E provide graphs and images showing AcTor and IXZ synergize in AML cells. (A) MV4-11 cells were treated with 7 different concentrations of AcTor and 7 different concentrations of IXZ at all possible combinations. 24 h later, viability was assessed by TiterGlo. A synergy score was calculated using SynergyFinder tool. (B) MV4-11 cells were treated with DMSO, AcTor and / or IXZ at the indicated concentrations for 24 h. Live cells were separated by a lymphoprep gradient and total cell lysates were analyzed by immunoblotting for mTORCl activity. Shown is a typical result of three independent experiments. (C) Six different AML cell lines were treated with DMSO, AcTor (10 pM), IXZ (15 nM) or AcTor / IXZ combination for 24 h. Viability was measured by flow cytometry using Sytox Green as a viable dye. Shown is a representative result of three independent repetitions. (D) Quantification of three repetitions comparing IXZ to AcTor / IXZ. (E) Bone marrow aspirates of AML patients were grown ex vivo for a week and then treated with DMSO, AcTor, IXZ and combination for 72 h. Total viable cells counts were measured by ViCell and plotted as percentage than the DMSO control. Lines connect the individual samples across treatments.

[0010] Figures 3A-3G provide graphs and images showing the combination of AcTor / IXZ generates a mitochondrial damage. (A) MV4-11 cells were treated with DMSO, AcTor and / or IXZ at the indicated concentrations for 24 h and tested by Seahorse for oxygen consumption rate. Shown is the average + SD of three independent concentrations. (B) MV4-11 were treated for 24 h with 5 M of AcTor, 15 nM of IXZ or both. Then, cells were permeabilized with digitonin (0.02 mg / ml) and respiration was assessed in response to the indicated additions. Datanormalized to viable cell count. (C) KG-1 cells treated for 24hrs with DMSO or the combination of 10 pM of AcTor and 15 nM of IXZ. Then, cells were permeabilized with digitonin (0.02 mg / ml) and respiration was assessed in response to the indicated additions. Data normalized to viable cell count. (D, E) MV4-11 and KG- la cells were treated for 24 h with DMSO, AcTor (10 pM), IXZ (15 nM) or AcTor / IXZ and stained with JC-1 for mitochondrial membrane potential analysis. Shown is a representative experiment and the quantification of three independent experiments. (F) MV4-11 cells were treated with DMSO, AcTor (10 pM), DCZ (15 nM) or AcTor / IXZ, cells were washed and resuspended in MEMa without serum and stained with MitoSox (1 pM) for 20 min. (G) MV4-11 cells were treated with AcTor / IXZ for 24 h in the presence and absence of the caspase 9 inhibitor Z-LEHD-fmk (25 pM). Flow cytometry was performed with Annexin V / PI to determine the level of apoptosis. Shown is a representative result and the quantification of three independent repetitions.

[0011] Figures 4A-4E provide graphs and images showing AcTor / IXZ activates a stress program that is different from AcTor alone. (A) MV4-11 were treated with DMSO, AcTor (10 pM), IXZ (15 nM) or AcTor / IXZ for 24 h. Live cells were separated by a lymphoprep gradient and RNA was extracted and sequenced. Shown are Venn diagrams of the upregulated and downregulated genes between AcTor vs DMSO and AcTor / IXZ vs IXZ. (B) Analysis of differentially expressed genes of AcTor vs DMSO and AcTor / IXZ vs IXZ. (C, D) ADM2 is induced by AcTor / IXZ at the RNA level and at the protein level (E), assessed by immunofluorescence.

[0012] Figures 5 A-5F provide graphs and images showing treatment with AcTor / IXZ for three weeks improves survival in an aggressive in vivo model of AML. (A) NSG mice were engrafted with MV4- 11 -luciferase cells. Mice were treated for three weeks as indicated. (B) Measurement of body weight. (C) Typical spleen size after treatment. (D) Shown are two spleens of IXZ ( 1 mg / Kg) and the AcTor / IXZ cohorts analyzed at the end of the experiment by luminescence. Graph represents the average ± SD of three spleen samples. (E) Total body luminescence of the individual mice and the quantification of the signal. Statistical significance is between AcTor / IXZ and vehicle treated cohorts. (F) Relative levels of ADM2 in the serum of treated mice.

[0013] Figures 6A-6I provide graphs and images showing treatment with AcTor / IXZ for three weeks eradicates patient derived AML blasts and stem cells. (A) NSG mice were engraftedwith patient-derived AML cells. When more blood contained more than 75% of human CD45+ cells, treatment was initiated every other day for three weeks. (B) Measurement of body weight. (C) Typical spleen size after treatment. (D) Flow cytometry analyses of spleen cells for AML blasts and quantification. Only for AcTor / IXZ, mouse cells started to populate the spleen as evident by mCD45 staining, and quantification of the remaining. (E) Analysis of the CD45+ / CD33+ AML cells for apoptosis using 7-AAD / Annexin V staining. (F) Typical immunohistochemistry images of bone marrow for Ki67 from IXZ and AcTor / IXZ treated mice. (G) Flow cytometry analyses of bone marrow for AML stem cells and quantification. Bone marrow AML cells reduced expression of the stem cell marker CD34 following AcTor / IXZ treatment. (H) Analysis of CD34+ AML cells for apoptosis using propidium iodide / Annexin V staining. (I) Relative levels of ADM2 in the serum of treated mice.

[0014] Figures 7A-7G provide graphs and images showing treatment with AcTor / IXZ maintains potency following AML relapse. (A) NSG mice were engrafted with patient-derived AML cells. When more blood contained more than 75% of human CD45+ cells, treatment was initiated every other day for three weeks with AcTor / IXZ. Mice were left to recover for five weeks and then divided into two cohorts: untreated (termed “Before”) or treated again with AcTor / IXZ (termed “After”). (B) Shown is a representative spleen form each of the groups. (C) Flow cytometry analyses of spleen cells for AML blasts and quantification. Note that mouse hematopoietic cells populate the spleen for the second time. (D) Analysis of the CD45+ / CD33+ AML cells for apoptosis using 7-AAD / Annexin V staining. (E) Typical immunohistochemistry images of bone marrow for Ki67 from before and after a second treatment with AcTor / IXZ. (F) Flow cytometry analyses of bone marrow for AML stem cells and quantification. Bone marrow AML cells reduced expression of the stem cell marker CD34 following a second treatment with AcTor / IXZ. (G) Analysis of CD34+ AML cells for apoptosis using PI / Annexin V staining.

[0015] Figure 8 provides a schematic representation showing a working hypothesis. Under hypoxic conditions, TNBCs elevate the expression of CAIX, which prevents a drop in intracellular pH. In the presence of CAIX inhibitors (CAIXi), cellular pH mildly drops. Enhancement of cellular metabolism by AcTor exacerbates the acidic conditions, increases oxidative stress, and compromises DNA repair. Together, DNA damage leads to cell death regardless of BRCA1 / 2 function, which can be used for therapy.

[0016] Figures 9A-9E provide graphs and images showing the design of BS1 , a nonsulfonamide CAIX inhibitor. (A) Molecular structure of BS1. (B, C) Image of BS1 and SLC- 0111 dynamic simulations in the catalytic pocket of CAIX. The zinc ion is shown in cyan. (D) Dose response curve of BS1 using recombinant CAIX. IC50 was calculated to be approximately 15 nM. (E) Venn diagrams of the overlap between upregulated and downregulated genes (by 2 fold) of SLC-0111 or BS1 relative to DMSO control under hypoxic conditions.

[0017] Figures 10A-10D provide graphs and images showing AcTor promotes mTORCl activity under hypoxia and increases the metabolic effects of CAIX inhibitors. (A, B) HCC 1806 cells were subjected to hypoxia for 24 h where indicated. AcTor and / or BS1 were added prior to hypoxia. Cells that remained adhered to the plates were isolated and whole cells lysates were analyzed by immunoblotting for the indicated proteins, oc-tubulin was used as a loading control. Shown is a representative Western blot of three repetitions. (C) Flow cytometry analysis for cellular pH using BCECF-AM under hypoxic conditions. In black are unstained cells. A shift to the left corresponds to acidic conditions. AcTor (10 pM), BS1 (5 pM) and SLC-0111 (100 pM) and combinations were added. Live cells were gated. Shown is a representative histogram of three independent repetitions. (D) Flow cytometry analyses for ROS by DCF-DA staining of HCC 1806 cells following 24 h of hypoxia with the indicated treatments. Shown is a representative experiment of three independent repetitions.

[0018] Figures 11A-1 ID provide graphs and images showing AcTor promotes the anti-tumor effects of CAIX inhibitors in vitro and in vivo. (A) HCC 1806 cells were subjected to hypoxia for 24 h in the presence of the indicated drugs. Cells were analyzed by flow cytometry in the presence of propidium iodide (dead cells are shown in red), indicative of dead cells. Shown is a representative outcome of three independent repetitions and quantification. (B) Similar to A, under normoxic conditions. (C) Nude mice were inoculated with HCC1806 cells s.c. 10 days after inoculation mice were treated every other day with i.p. injections of vehicle, BS1 or BS 1+AcTor. AcTor alone was not used, as previous analyses in a wide variety of models show that it has no anti-tumor effect as a single drug. Shown are the bioluminescence images of mice 28 days after inoculation. (D) Tumor growth in the individual mice until termination at 45 days post inoculation.

[0019] Figures 12A-12C provide graphs and images showing AcTor / BSl sensitizes wt BRCA1 / 2 TNBC to PARP1 inhibitors. (A) HCC1806 cells were subjected to hypoxia for 24 h in the presence of AcTor (10 pM), BS1 (5 pM) and Olaparib (1 pM), as indicated. Cells were analyzed by flow cytometry in the presence of propidium iodide (dead cells are shown in red), indicative of dead cells. Shown is a representative outcome of three independent repetitions. (B) Average+SEM of four independent experiments. (C) Immunofluorescence analysis for y- H2AX (green). Shown is a representative cell.

[0020] Figures 13A and 13B provide a schematic representation of the synthetic procedure for preparing AcTor.DETAILED DESCRIPTION OF THE INVENTION

[0021] The invention is based on the discovery of the mechanisms of immediate adaptation to proteasome inhibitors (Pls): mTOR and the integrated stress response (ISR). The mTOR kinase resides in two complexes: mTORCl and mT0RC2. Of the two, mTORCl is a central coordinator of metabolism, as it obtains inputs on nutrient, oxygen, ATP, levels of free AAs and growth factor availability to adjust cellular metabolism, survival and growth. While mTORCl activity is frequently elevated in cancer, as it promotes anabolic metabolism and cancer cell growth, under stress conditions, such as AA starvation or in the presence of certain stress-inducing drugs, mTORCl suppression improves survival and, paradoxically, promotes tumor growth.

[0022] The ISR is a collective term for multiple adaptation pathways that converge on the phosphorylation of eIF2a on serine 51 . This includes, among several other conditions, the lack of AAs, the accumulation of unfolded proteins in the ER, response to viral infection, oxidative stress and hypoxia. When eIF2a is phosphorylated, by virtue of either of four eIF2a kinases, global protein synthesis is reduced and translation of a subset of mRNA is induced, of which the mRNA of ATF4 is the mostly studied. The reduction in protein synthesis and the transcriptional activities of ATF4 and other ISR-induced proteins promote adaptation to stress conditions, including to Pls.

[0023] The inventors found that within hours after the block of the proteasome, mTORCl and ISR respond in opposite manners. mTORCl is inhibited, while the ISR is induced. Both responses cooperate to minimize energy consumption and remodel cellular metabolism.Enforced activation of mTORCl or inhibition of the TSR strongly promoted the cytotoxicity of Pls and compromised the development of hypoxia-induced resistance, indicating that mTORCl suppression and ISR induction are adaptive responses required for the metabolic rewiring that mediate resistance. mTORCl and ISR overlap in many of their effector functions on cell metabolism, while using different and complementary mechanisms. Moreover, these two pathways regulate each other to optimize the response to stress. The inventors hypothesize that the cross talk between mTORCl and ISR in response to stress-inducing drugs, primarily Pls, involves the mitochondria and is part of the metabolic rewiring program under stress, which contributes to establish resistance. This can be targeted by small molecules that either enforce mTORCl activity and / or prevent the generation of the ISR.Definitions

[0024] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting of the invention as a whole. As used in the description of the invention and the appended claims, the singular forms “a”, “an”, and “the” are inclusive of their plural forms, unless contraindicated by the context surrounding such.

[0025] As used herein, the term "organic group" is used to mean a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups). In the context of the present invention, suitable organic groups for the compounds of this invention are those that do not interfere with the activity (e.g., anticancer activity) of the compounds. In the context of the present invention, the term "aliphatic group" means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example.

[0026] As used herein, the terms "alkyl", "alkenyl", and the prefix "alk-" are inclusive of straight chain groups and branched chain groups. Unless otherwise specified, these groups contain from 1 to 20 carbon atoms, with alkenyl groups containing from 2 to 20 carbon atoms. In some embodiments, these groups have a total of at most 10 carbon atoms, at most 8 carbon atoms, at most 6 carbon atoms, or at most 4 carbon atoms. Alkyl groups including 4 or fewer carbon atoms can also be referred to as lower alkyl groups. Alkyl groups can also be referred to by the number of carbon atoms that they includeCi - C4 alkyl groups are alky groups including 1 -4 carbon atoms).

[0027] Cycloalkyl, as used herein, refers to an alkyl group ( / .<?., an alkyl, alkenyl, or alkynyl group) that forms a ring structure. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 10 ring carbon atoms. A cycloalkyl group can be attached to the main structure via an alkyl group including 4 or less carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopropylmethyl, cyclopentyl, cyclohexyl, adamantyl, and substituted and unsubstituted bornyl, norbomyl, and norbomenyl.

[0028] The term "haloalkyl" is inclusive of groups that are substituted by one or more halogen atoms, including perfluorinated groups. This is also true of other groups that include the prefix "halo-". Examples of suitable haloalkyl groups are chloromethyl, trifluoromethyl, and the like. Halo moieties include chlorine, bromine, fluorine, and iodine.

[0029] The term "aryl" as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl. Aryl groups may be substituted or unsubstituted.

[0030] Unless otherwise indicated, the term "heteroatom" refers to the atoms O, S, or N. The term "heteroaryl" includes aromatic rings or ring systems that contain at least one ring heteroatom (e.g. , O, S, N). In some embodiments, the term "heteroaryl" includes a ring or ring system that contains 2 to 12 carbon atoms, 1 to 3 rings, 1 to 4 heteroatoms, and O, S, and / or N as the heteroatoms. Suitable heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl, pyrazinyl, 1 -oxidopyridyl, pyridazinyl, triazinyl, tetrazinyl, oxadiazolyl, thiadiazolyl, and so on.

[0031] When a group is present more than once in any formula or scheme described herein, each group (or substituent) is independently selected, whether explicitly stated or not. For example, for the formula -C(O)-NR2 each R group is independently selected.

[0032] As a means of simplifying the discussion and the recitation of certain terminology used throughout this application, the terms "group" and "moiety" are used to differentiate between chemical species that allow for substitution or that may be substituted and those that do not so allow for substitution or may not be so substituted. Thus, when the term "group" is used todescribe a chemical substituent, the described chemical material includes the unsubstituted group and that group with nonperoxidic O, N, S, Si, or F atoms, for example, in the chain as well as carbonyl groups or other conventional substituents. Where the term "moiety" is used to describe a chemical compound or substituent, only an unsubstituted chemical material is intended to be included. For example, the phrase "alkyl group" is intended to include not only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, tertbutyl, and the like, but also alkyl substituents bearing further substituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, "alkyl group" includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, cyanoalkyls, etc. On the other hand, the phrase "alkyl moiety" is limited to the inclusion of only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, tert-butyl, and the like.

[0033] The invention is inclusive of the compounds described herein in any of their pharmaceutically acceptable forms, including isomers (e.g., diastereomers and enantiomers), tautomers, salts, solvates, polymorphs, prodrugs, and the like. In particular, if a compound is optically active, the invention specifically includes each of the compound's enantiomers as well as racemic mixtures of the enantiomers. It should be understood that the term "compound" includes any or all of such forms, whether explicitly stated or not (although at times, "salts" are explicitly stated).

[0034] A subject, as defined herein, is an animal such as a vertebrate or invertebrate organism. In other embodiments, the subject is a mammal such as a domesticated farm animal (e.g., cow, horse, pig) or pet (e.g. , dog, cat). More preferably, the subject is a human. A subject at risk is a subject who has been determined to have an above-average risk that a subject will develop cancer, which can be determined, for example, through family history or the detection of genes causing a predisposition to developing cancer.

[0035] Treat", "treating", and "treatment", etc., as used herein, refer to any action providing a benefit to a subject at risk for or afflicted with a condition or disease such as cancer, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, prevention or delay in the onset of the disease, etc. The subject may be at risk due to exposure to carcinogenic agents, being genetically predisposed to disorders characterized by unwanted, rapid cell proliferation, and so on.

[0036] “Pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject for the methods described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0037] The terms “therapeutically effective” and “pharmacologically effective” are intended to qualify the amount of each agent which will achieve the goal of decreasing disease severity while avoiding adverse side effects such as those typically associated with alternative therapies. When multiple agents are being co-administered, it refers to the total amount of the two or more therapeutic agents being used. The therapeutically effective amount may be administered in one or more doses.Methods of Treating Cancer

[0038] In one aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an mTOR activator and a proteasome inhibitor to the subject.

[0039] An mTOR activator is a compound that increases the activity of the kinase mTOR. In some embodiments, the mammalian target of rapamycin (mTOR) effected by the mTOR activator is mTORC 1. mTORC l is a protein complex that functions as a nutrient / energy / redox sensor and controls protein synthesis, and plays a role in activating translation of proteins. mTOR activators includes a diverse range of chemicals that activate the activity of mTOR through both direct and indirect mechanisms. Examples of mTOR activators include rapamycin, everolimus, Torin 1, AZD8055, PP242, WYE-125132, sapanisertib, OSI-027, HRG-2149, and UPF-1069. See Hua et al., J Hematol Oncol., 12(1):71 (2019).

[0040] In some embodiments, the mTOR activator is a compound having a structure according to formula I:wherein R1is a phenyl or heteroaryl group, or a pharmaceutically acceptable salt thereof. In some embodiments, R1is an indole group, which can be substituted or unsubstituted.

[0041] In some embodiments the mTOR activator is the compound AcTor. AcTor has the structure shown below:

[0042] For cancer treatment, the mTOR activator is administered together with a proteasome inhibitor. As described herein, the inventors have shown that mTOR activators can have a strong potentiating effect, including synergistic effects, on proteasome inhibitors for cancer treatment. Proteasome inhibitors are a class of drugs that block the activity of proteasomes, typically by binding the proteasome and preventing it from degrading proteins. See Fricker, L., Annu Rev Pharmacol Toxicol, 60:457-476 (2020). Cotemporaneous administration together refers to administration of the two compounds close enough in time that their pharmaceutical effects overlap. Administration together includes simultaneous administration, administration first of the mTOR activator followed by administration of the proteasome inhibitor, or vice versa. In some embodiments, the proteasome inhibitor is bortezomib, carfilzomib, or ixazomib. A preferred proteasome inhibitor is ixazomib.

[0043] Cancer is generally named based on its tissue of origin. There are several main types of cancer. Carcinoma is cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Cancer which has metastasized will still retain traits associated with its tissue of origin. In some embodiments, the cancer is leukemia.

[0044] In some embodiments, the cancer being treated is leukemia. Leukemia is a group of blood cancers that usually begin in bone marrow and produce high numbers of abnormal blood cells. The main types of leukemia include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Leukemia is generally categorized between acute leukemia and chronic leukemia. The most common symptoms of leukemia include easy bruising, pale skin, fever, and an enlarged spleen or liver.

[0045] In some embodiments, the mTOR activator is administered together with an antileukemic agent. Anti-leukemic agents are anticancer agents known to be effective, or particularly effective, against leukemia. Examples of anti-leukemic agents include arsenic trioxide, azacitidine, cyclophosphamide, cytarabine, daunorubicin hydrochloride, doxorubicin hydrochloride, quizartinib, acalabrutinib, alemtuzumab, arzerra (ofatumumab), bendamustine hydrochloride, bendeka, 6-mercaptopurine, 6-thioguanine, asparaginase, cladribine, clofarabine, decitabine, etoposide, fludarabine, and gemtuzumab.|0046| In some embodiments, the cancer being treated is drug-resistant cancer. Drug-resistant cancer is cancer that has developed resistance to treatment with one or more anticancer agents. Vasan et al., Nature, 575, p. 299-309 (2019). Drug-resistance in cancer can occur through a variety of different mechanisms, including physical barriers, drug efflux, cancer size and cancer heterogeneity. In some embodiments, the cancer being treated has developed resistance to proteasome inhibitors.

[0047] In some embodiments, the cancer is recurrent cancer, which is cancer that has relapsed. Cancer relapse refers to the return of cancer after a period of remission after cancer treatment,during which no signs of cancer were detected. Cancer can relapse as a result of a variety of factors, such as the development of drug resistance, or the failure to eliminate microscopic cancer cells and / or cancer stem cells during the initial round of treatment.

[0048] The effectiveness of cancer treatment may be measured by evaluating a reduction in tumor load. The reduction in tumor load may be represent a direct decrease in mass, or it may be measured in terms of tumor growth delay, which is calculated by subtracting the average time for control tumors to grow over to a certain volume from the time required for treated tumors to grow to the same volume.

[0049] Candidate agents may be tested in animal models. Typically, the animal model is one for the study of cancer. The study of various cancers in animal models (for instance, mice) is a commonly accepted practice for the study of human cancers. For instance, the nude mouse model, where human tumor cells are injected into the animal, is commonly accepted as a general model useful for the study of a wide variety of cancers (see, for instance, Polin et al., Investig. New Drugs, 15:99-108 (1997)). Results are typically compared between control animals treated with candidate agents and the control littermates that did not receive treatment. Transgenic animal models are also available and are commonly accepted as models for human disease (see, for instance, Greenberg et al., Proc. Natl. Acad. Sci. USA, 92:3439-3443 (1995)). Candidate agents can be used in these animal models to determine if a candidate agent decreases one or more of the symptoms associated with the cancer, including, for instance, cancer metastasis, cancer cell motility, cancer cell invasiveness, or combinations thereof.AcTor Derivatives

[0050] In a further aspect, the present invention provides a tuberous sclerosis complex 2 (TSC2) inhibitor having a structure according to formula I:wherein R1is a phenyl or heteroaryl group, or a pharmaceutically acceptable salt thereof. In some embodiments, R1is positioned at the para position on the phenyl ring. These compounds can also be referred to herein as AcTor derivatives. In some embodiments, R1is an indole group. The indole group can be bound at any position on the indole ring, and can be substituted or unsubstituted. In some embodiments, the compound is AcTor.Methods of Inhibiting Tuberous Sclerosis Complex 2 (TSC2)

[0051] Another aspect of the invention provides a method of inhibiting tuberous sclerosis complex 2 (TSC2) by contacting it with an effective amount of the compound according to formula I:wherein R1is a phenyl or heteroaryl group, or a pharmaceutically acceptable salt thereof. In some embodiments, R1is an indole group, while in yet further embodiments the compound is AcTor. Inhibiting refers to a decrease in the activity of TSC2. Inhibition includes both partial and complete inhibition. As described herein, activation of mTORCl can be achieved by suppression of its negative regulator, the tuberous sclerosis complex, composed of TSC1, TSC2, and TBClD7.Formulation and Administration of Anticancer Compounds

[0052] The present invention provides a method for administering one or more anti-cancer compounds in a pharmaceutical composition. Examples of pharmaceutical compositions include those for oral, intravenous, intramuscular, subcutaneous, or intraperitoneal administration, or any other route known to those skilled in the art, and generally involves providing an anti-cancer compound formulated together with a pharmaceutically acceptable carrier. Accordingly, in some embodiments the mTOR activator is administered together with a pharmaceutically acceptable carrier.

[0053] In some embodiments, the mTOR activator is administered orally. When preparing the compounds described herein for oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are capsules, tablets, powders, granules or a suspension, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, com starch or gelatins; with disintegrators such as corn starch, potato starch or sodium carboxymethyl- cellulose; and with lubricants such as talc or magnesium stearate. The active ingredient may also be administered by injection as a composition wherein, for example, saline, dextrose or water may be used as a suitable carrier.

[0054] For intravenous, intramuscular, subcutaneous, or intraperitoneal administration, the compound may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient. Such formulations may be prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. The formulations may be present in unit or multi-dose containers such as sealed ampoules or vials.

[0055] Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably made isotonic. Preparations for injections may also be formulated by suspending or emulsifying the compounds in non-aqueous solvent, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol.

[0056] The dosage form and amount can be readily established by reference to known treatment or prophylactic regiments. The amount of therapeutically active compound that is administered and the dosage regimen for treating a disease condition with the compounds and / or compositions of this invention depends on a variety of factors, including the age, weight, sex, and medical condition of the subject, the severity of the disease, the route and frequency of administration, and the particular compound employed, the location of the unwanted proliferating cells, as well as the pharmacokinetic properties of the individual treated, and thus may vary widely. The dosage will generally be lower if the compounds are administered locally rather than systemically, and for prevention rather than for treatment. Such treatments may be administered as often as necessary and for the period of time judged necessary by the treating physician. One of skill in the art will appreciate that the dosage regime or therapeutically effective amount of the inhibitor to be administrated may need to be optimized for each individual. The pharmaceutical compositions may contain active ingredient in the range of about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mg and most preferably between about 1 and 200 mg. A daily dose of about 0.01 to 100 mg / kg body weight, preferably between about 0.1 and about 50 mg / kg body weight, may be appropriate. The daily dose can be administered in one to four doses per day.

[0057] For example, the maximum tolerated dose (MTD) for anti-cancer compounds can be determined in tumor-free athymic nude mice. Agents are prepared as suspensions in sterile water containing 0.5% methylcellulose (w / v) and 0.1% Tween 80 (v / v) and administered to mice (7 animals / group) by oral gavage at doses of 0, 25, 50, 100 and 200 mg / kg once daily for 14 days. Body weights, measured twice weekly, and direct daily observations of general health and behavior will serve as primary indicators of drug tolerance. MTD is defined as the highest dose that causes no more than 10% weight loss over the 14-day treatment period.

[0058] The active agents (e.g., mTOR activators and Pls) can also be provided as pharmaceutically acceptable salts. The phrase “pharmaceutically acceptable salts” connotes salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds may be preparedfrom an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic, methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, y-hydroxybutyric, galactaric, and galacturonic acids. Suitable pharmaceutically acceptable base addition salts of the compounds described herein include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. Alternatively, organic salts made from N,N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine may be used form base addition salts of the compounds described herein. All of these salts may be prepared by conventional means from the corresponding compounds described herein by reacting, for example, the appropriate acid or base with the compound.Preparation of Anticancer Compounds

[0059] Compounds of the invention may be synthesized by synthetic routes that include processes analogous to those well known in the chemical arts, particularly in light of the description contained herein. A specific method for synthesizing AcTor is provided in Example 3. The starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, Wisconsin, USA) or are readily prepared using methods well known to those skilled in the art (e.g., prepared by methods generally described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley, New York, (1967- 1999 ed.) and similar texts known to those skilled in the art.

[0060] An example has been included to more clearly describe a particular embodiment of the invention and its associated cost and operational advantages. However, there are a wide variety of other embodiments within the scope of the present invention, which should not be limited to the particular example provided herein.EXAMPLEExample 1: AcTor, a novel mTOR stimulator, potentiates proteasome inhibitor activity for the treatment of acute myeloid leukemia

[0061] We recently found that an early response to Pls is a strong suppression of mTORCl activity and MM deficient for TSC2 acquires sensitivity to Pls. Darawashi et al., Cell Death Dis., 13(11):969 (2022). Surprisingly, the mechanism of death was not associated with ER stress, but rather a mitochondrial dysfunction, which is orthogonal to DNA damage, the standard of care in AML. Here, we examined the activity of a new TSC2 inhibitor, AcTor. AcTor enhanced the cytotoxicity of IXZ by 15-fold across multiple acute myeloid leukemia (AML) cell lines. In vivo, AcTor / IXZ combination improved the survival of mice engrafted with AML and maintained potency after relapse. We propose AcTor as an enhancer of PI activity, which can be leveraged for AML therapy.Materials and methods

[0062] Computational screening of a potential TSC2 inhibitor

[0063] Interactions between TSC2 and Rheb were determined according to the crystal structures of human TSC2 (PDB ID 7DL2 Yang et al., Nat Commun., 12(1):339 (2021)) and GTP-bound Rheb (PDB ID 1XTS Yu et al., J Biol Chem., 280(17):17093-17100 (2005)), downloaded from the protein databank. The proteins were prepared using default settings in the Molecular Operating Environment (MOE) v. 2022.02 to determine appropriate protonation states and hydrogen bonding networks of amino acid residues and correct any missing loops. The AMBER10 force field was used for partial charges and other atomic parameters. Yan et al., Nat Protoc., 15(5): 1829- 1852 (2020). The protein docking program HDOCK was used to dock Rheb towards the GAP domain of TSC2 (Fig. 1 A), which confirmed the recognition site and catalytic site of TSC2, and the relevant residues in these. The structure obtained is in close agreement with the TSC2-Rheb construct by Yang et al., Nat Commun. 12( 1 ):339 (2021).

[0064] Further docking details and procedures for mouse xenografts, cell culture, immunoblotting, immunofluorescence, flow cytometry, immunohistochemistry, metabolic analyses, RNAseq, are detailed in the supplemental material.

[0065] Virtual docking

[0066] We virtually docked FDA-approved drugs to the recognition site using the 2021 database of FDA approved molecules. Molecules were downloaded in mol2 format from the ZINC 15 website and prepared using default settings in MOE to ensure relaxed structures and appropriate protonation states. Partial charges were assigned using the MMFF94 force field. Halgren, T.A., Comput Chem. 20(7):730-748 (1999). Docking was performed in two trenches, targeting either the recognition site or the catalytic site. The catalytic site was defined by residues K1638, R1639, H1640 and N1643, whereas for the recognition site located on the opposite wall of the ‘bowl-shaped’ GAP domain residues R1529, R1745, K1748 and R1749 of the GaN and GaC helices were selected as the key interaction region, in close agreement with the models explored by Yang et al., Nat Commun. 12( 1 ): 339 (2021). The initial screening was performed using rigid receptor docking, 50 poses per ligand using London free energy scoring and GBVI / WSA free energy refinement, keeping the binding pose with strongest interaction energy for each ligand. The ligands with docking score better (more negative) than -9 kcal / mol were retained, followed by induced fit docking and same settings as above. This screen yielded a single molecule with a potential to permeabilize the cell, cobicistat. Having identified Cobicistat as a potent binder from the docking simulations, attempts were made to further enhance the impact on Rheb binding. To this end, the solvent exposed phenyl ring showing no direct interactions with the TSC2 residues was targeted using the fragment library of MOE and R-group replacement, followed by redocking and evaluation of the docking score / free energy of interaction. Only compounds with docking cores better than -8.0 kcal / mol were retained. The aim of the enhancement is to further fill up the space of the binding region, thus blocking Rheb from fitting into the GAP domain.

[0067] Cell culture

[0068] OCI-AML3, MV4-11, THP-1, KG- la and HL60 AML cells were purchased from American Type Culture Collection, Manassas, VA. MV4-11 cells were stably transduced with firefly luciferase and used for in vitro and in vivo studies. Cells were maintained in RPMI 1640 medium, supplemented with 10% fetal bovine serum (Coming, MT35010CV), 2 mM L- glutamine (Thermo Fisher Scientific, 25030081), 1% penicillin-streptomycin solution (Thermo Fisher Scientific, 15070063), and 1 mM sodium pyruvate (Thermo Fisher Scientific,1 1360070) RPMI8226 were also cultured in supplemented RPMI 1640 medium. HEK293T were cultured in DMEM, supplemented with the same additives.

[0069] Primary AML culturing and survival

[0070] All patients provided informed consent prior to study enrollment (study ID: IRB00117251). Bone marrow aspirates were collected in sodium-heparinized Cell Preparation Tubes (CPT) (BD Biosciences #362761, Franklin Lakes, NJ) and centrifuged at 1,800 x g for 15 min. from patients undergoing confirmatory diagnosis for a range of hematological malignancies. PaOents with confirmed AML were enrolled in the study. Mononuclear cells were isolated and then washed in ACK lysis buffer to remove red blood cells and used immediately for experiments or banked in vapor phase of liquid nitrogen prior to experimentation. Primary AML cells of six different adult donors were subjected to short-term culture in IMDM-Glutamax, supplemented with 10% FBS, Kit ligand / SCF (lOOng / mL), GM- CSF (20ng / mL), and IL-3 (20ng / mL). Cells were exposed to Actor (lOpM), IXZ (10-30nM) or the combination of Actor plus IXZ for 48hrs. At the conclusion of the incubation period, viable cell count was determined using the trypan blue exclusion assay in a Vi-CELL BLU Cell Viability Analyzer (Beckman Coulter Life Sciences).

[0071] Mouse xenograft

[0072] Six-month-old female NOD / SCID / IL2-Ry- / _(NSG) mice (Jackson Laboratory, Bar Harbor, ME) were injected i.v. with 150 pl of Hank’s Balanced Salt Solution containing 1 million luciferase-expressing MV4-11 cells per mouse. All mice were randomly divided into groups of 12.

[0073] Patient derived xenograph model

[0074] Frozen patient derived cells (PDX) cells were thawed and expanded in NSG mice by injecting 1.5xl06cells though the tail vein. After 5 weeks, total splenocytes were isolated, washed twice in PBS, and suspended in Hank’s Balanced Salt Solution at a final concentration of 2 x 106cells per 150 pL, and immediately transferred to recipient mice. When 75% of peripheral blood cells were hCD45 positive (about 5 weeks later), mice were treated with vehicle, IXZ (Img / kg), AcTor (30mg / kg) and IXZ / AcTor every other day for 21 days. Cohortsof eight NSG mice per group were used. Mice were monitored for well-being and provided nutritional supplements as needed.

[0075] Drug treatment in vivo and monitoring of tumor progression by bioluminescence

[0076] Mice were randomly grouped into 4 cohorts. Group 1 received the control treatment, groups 2 and 3 were given IXZ at different concentrations, and groups 3 and 4 received a combination of AcTor / IXZ. AcTor was dissolved in 0.2% Tween 80 in sterile water at a concentration of 30 mg / kg. IXZ was dissolved in 2% 2-hydroxypropyl-[3-cyclodextrin in sterile water at a concentration of 0.625 mg / kg. Three days after challenge mice were treated i.p. with vehicle, 30 mg / kg of AcTor, 1 mg / kg or 0.5 mg / kg of IXZ. Treatment was given every other day for three weeks. AML engrafted mice underwent in vivo bioluminescence imaging at various times at least twice a week. Animals were monitored daily and were euthanized upon signs of leukemia onset (weight loss >15%, decreased activity, and / or hind limb paralysis). A group of 7 mice from each group was monitored for overall survival analysis. Progression of MV4-11 Luciferase cells was monitored over time using bioluminescence imaging with the IVIS Spectrum (PerkinElmer, USA). Before imaging, each mouse was injected intraperitoneally with D-luciferin (100 mg / kg). Mice were anesthetized with vaporized isoflurane (Abbott Laboratory, Abbott Park, IL) and placed in imaging chamber. After 7 min, each animal was imaged with an exposure time of 1 min for each position, bi-weekly for 3 weeks. All bioluminescent image data were provided by Living Image - Newton (for Spectrum data), Amira, Matlab, USA. Photons detected from leukemia models were converted to average radiance (photon / sec / cnr / sr). Average radiance values are quantitative data obtained from region of intensity (ROI) where photons emitted by bioluminescent cells of assigned rectangular area over the whole body of each mouse. Both luminescence and image data were analyzed using Living Image software.

[0077] Immunoblotting

[0078] Live cells were isolated by a centrifugation step over a lymphoprep gradient. Cells of the interphase were isolated and washed twice with ice cold PBS. Cell pellets were lysed in RIPA buffer supplemented with protease inhibitors (Bimake, bl4001) and phosphatase inhibitors (Bimake, bl5001). Following strong vortex for 10 min, lysates were cleared by centrifugation (4 °C, 16,000xg for 15 min). Supernatant was separated, protein content wasquantified and mixed with reducing sample buffer followed by boiling for 5 min. Samples were loaded on SDS-PAGE and resolved by electrophoresis (120V) and then transferred to PVDF membrane (4 °C, 100V, for 1.5h). Thereafter, membrane was blocked with 5% skim milk dissolved in TBST (at RT for Ih), washed (3 times, 5 min each), then incubated with primary antibody (at 4 °C for 16-24h), washed then incubated with anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (at RT for Ih), washed then detected by chemiluminescence using Immobilon®

[0079] Crescendo (Millipore, WBLUR0500) and imaged on Bio-RadChemiDoc™ XR. Antibodies were used according to the manufacturer’s instruction and listed here: anti-P-S6 (CST #5364) anti-S6 (CST #2217), anti-P-4EBPl (CST #9459), anti-4EBPl (CST #9644), anti-S6Kl (CST #9202), anti-P-S6Kl (CST #9205), anti-tubulin (CST #9099). Goat antimouse and goat anti-rabbit HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. Rheb»GTP levels were measured using a commercial kit of New East Biosciences (cat#26910). Assay

[0080] Immunofluorescence

[0081] Cells in suspension were seeded on slides by cytospin (Thermo Scientific) and then fixed with 4% paraformaldehyde (PFA). After 10 min of permeabilization by 0.5% Triton X- 100, the cells were incubated for 1 h with a mouse anti-ADM2 primary antibody (ThermoScientific, USA, 1 :200) and for another 1 h with the secondary antibody (Alexa-Fluor 647-conjugated, Jackson ImmunoResearch Inc., USA 1 :400) and with DAPI-mounting media (Vector Laboratories VECTASHIELD Antifade Mounting Medium with DAPI, Cole-Parmer North America, USA). The stained cells were observed under confocal laser scanning microscope Leica TCS SP8 LIGHTNING confocal microscope (Leica, USA).

[0082] Flow cytometry

[0083] Cells were passed through 70 pm cell strainers (Stellar Scientific, MD, USA). When isolated from mice, red blood cells were lysed prior to staining. Cell surface markers were stained for 30 minutes at room temperature. For the AML-PDX analysis to detect the AML blasts / LSCs & corresponding apoptosis cells, the following antibodies were used: Alexa Fluor® 700 anti-human CD34 (BioLegend, 343621); APC anti-human CD38 each (BioLegend, 356605); Alexa Fluor® 488 anti-human CD45RA (Biolegend, 304114); PE anti-human CD33 each (Biolegend, 303404); APC Annexin V (640920, BioLegend); Annexin V / FITC (185209000, Invitrogen) PerCP / Cyanine5.5 anti-mouse CD45.1 (110728, BioLegend). PE anti-human CDl lb (Biolegend, 379903); PE anti-human CD14 (Biolegend, 301805); PE anti-human CD15 (Biolegend, 376307); Isotype controls were purchased from Biolegend & BD Bioscience. Samples were analyzed by CytoFLEX S (Beckman Coulter) using CytExpert software. For viability measurements, cells were washed once with PBS, treated with propidium iodide (PI) (Thermo Fisher Scientific; P3566) or SYTOX-Green Nucleic acid stain (S7020, ThermoFisher Scientific) or 7-AAD viability Staining solution (420404, BioLegend) and immediately analyzed by flow cytometry.

[0084] For analyses of HSC we applied the protocol of Morcos et al., Nat Commun. 13( 1 ):4504 (2022). Bone marrow samples were collected from each group and processed into single-cell suspensions. After red blood cell lysis, the remaining cells were stained for Lin-Scal+c-Kit+ markers using the FITC anti-mouse Lineage Cocktail, BioLegend, USA; BD OptiBuild™ BV650 Rat Anti-Mouse Ly-6A / E(Scal) (BD Bioscience, USA); APC / Cyanine7 anti-mouse CD117 (c-kit), BioLegend, USA)]. HSCs were analyzed by gating on the LSKs (multipotent hematopoietic cells) for CD48-CD150+ (BD OptiBuildTM BV605 Hamster Anti-Mouse CD48, BD Bioscience, USA, BD Horizon™ BV510 Rat Anti-Mouse CD150, BD Bioscience, USA). Gating strategy is shown in Fig. 5.

[0085] Mitochondrial Superoxide (MitoSOX) & ROS Production: After treatment with the respective drugs 5xl06MV4-11 cells were resuspended in ImL preheated medium without serum and incubated with 1 pM MitoSOX™ Red (Thermo Fisher, cat#M36008) at 37°C for 20 min. After washing, the expression of MitoSOX (Ex / Em = 396 / 610) was quantified by flow cytometry. A similar protocol was followed for each treatment to quantify ROS (Ex / Em = 492 / 527) in MV4-11 cells, using 2',7'-dichlorodihydrolluorescein diacetate ( l .5pM H2DCFDA, D399, Invitrogen™, Thermofisher Scientific, USA) as an indicator.

[0086] Immunohistochemistry

[0087] For immunohistochemistry of Ki67, tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in wax, and sectioned at 5 pm. Paraffin-embedded sections were dewaxed, re-hydrated, and rinsed in PBS. After boiling for 10 min in 10 mmol / L sodium citrate buffer (pH 6.0), the sections were blocked in 1 % BS A in PBS for 1 h at room temperature, thenincubated overnight at 4 °C with Ki67 primary antibody. The stained slides were washed in 1 x PBS / 0.1% Tween-20 three times for 5 min each and incubated at room temperature for 1 h with the secondary antibody. Then, processed with the substrate reaction according to the instructions of Ki67 Kit (VitroView in situ Ki67 IHC / DAB Detection Kit, VB-4002-D, VitroVivo Biotech, USA)

[0088] Analysis of hypoxia in the bone marrow

[0089] The Hypoxyprobe™ (pimonidazole hydrochloride) was resuspended at a concentration of 30 mg / ml in 0.9% sterile saline. After the treatment period, mice were given an intravenous injection of PIMO solution (60 mg / kg) through the tail vein. Ninety minutes later, the mice in the PIMO-treated group were euthanized, and their hindlimb bones were collected. The samples were fixed in paraformaldehyde, decalcified, and cut into 8 pm sections using a Leica RM2255 Rotary Microtome (Leica Biosystems, USA). The sections were then stained with an FITC-conjugated anti-pimonidazole primary antibody (Pab27, 4.3.11.3, Hydroxyprobe Inc., Burlington, MA, USA) as described previously (Aguilera KY, Brekken RA, Bio Protoc. 4(19) (2014)), and counterstained with PE / Cyanine5 anti-human CD33 antibody (Cat no. 303406, BioLegend, USA). Imaging was performed using a Leica TCS SP8 confocal microscope (Leica Biosystems, USA).

[0090] Masson's trichrome staining

[0091] Bones from randomly selected animals in each group were fixed in 4% paraformaldehyde for 24 hours, then decalcified, embedded in paraffin, and cut into 5 pm sections. The tissue sections were stained using Masson's trichrome as per the manufacture instructions (75845, Trichrome Staining Kit, CP Lab Chemicals, CA, USA) and microscopically examined.

[0092] Measurement of mitochondrial respiratory activity

[0093] Oxygen consumption rate (OCR) measurements were performed in Seahorse XF HS Mini Analyzer (Agilent) by using Mito Stress test kit (Agilent, 103015-100) according to the manufacturer’s instructions. Cells were seeded at a density of 3xl04cells per well in a Seahorse XF RPMI assay medium into XF HS Mini FluxPak (103723, Agilent). Oligomycin and FCCP were added to a final concentration of 2 pM, rotenone and antimycin A were added to a finalconcentration of 0.5 pM. High resolution respirometry was performed using an Oroboros Oxgraph-2k (O2k; Oroboros Instruments, Innsbruck, Austria) in either a 0.5 or 1.0 mL reaction volume at 37°C. Approximately l-2xl06cells were harvested and centrifuged at 300 x g. After centrifugation, cells were washed in PBS and centrifuged again at 300 x g. Cells were then resuspended in either 0.5mL or ImL of Respiratory Buffer supplemented with creatine (105mM MES potassium salt, 30mM KC1, 8mM NaCl, ImM EGTA, lOmM KH2PO4, 5mM MgC12, 0.25% BSA, 5mM creatine monohydrate, pH 7.2). Assay additions were as follows: digitonin (0.015mg / mL; ‘Digi’); creatine kinase, phosphocreatine, ATP (20U / mL, ImM, 5mM; ‘AGATP-54’); cytochrome C (O.OlmM; ‘Cyt C’); pyruvate / malate (5mM / lmM; ‘Pyr / Mal’); glutamate (5mM; ‘Glut’); octanoyl-carnitine (0.2mM; ‘Oct-Cam’); rotenone (0.5pM; ‘Rot’); succinate (lOmM; ‘Succ’); antimycin a (0.5pM; ‘Ant A’); N,N,N',N'- Tetramethyl-p-phenylenediamine dihydrochloride / ascorbate (0.5mM / 2mM; ‘TMPD / Asc’). Data were normalized to viable cell count using Trypan Blue (0.4%) (Thermofisher; 15250- 061).

[0094] RNA Seq Analysis

[0095] Total RNA was obtained from live cells of each treatment group using TRIzol RNA Isolation Reagents (Thermo Fisher Scientific). For eliminating contaminating genomic DNA, we treated the RNA samples with the DNase I Kit (AMPD1, Sigma-Aldrich, MO, USA). Subsequent RNA purification was accomplished using the RNeasy Mini Kit (74104, Qiagen, USA). Following quality control, the RNA samples were subjected to RNA sequencing with PolyA selection and NEB Ultra II Directional RNA Library Prep Kit by Admera Health, LLC, NJ. DifferenOally expressed genes were then evaluated for all groups using DESeq2 package in Galaxy, EU and R. The cutoff to determine significant genes in all groups were false discovery rate-adjusted P value (q value) of <0.05. The Venn diagrams were calculated using Bioinformatics & Evolutionary Genomics, Belgium. The gene ontology and other genomic analysis were performed using online database from David Functional Annotation Bioinformatics Microarray Analysis, NIH, USA and SRplot, Chinese Academy of Sciences, Shanghai Jiaotong University, Xiangya School of Medicine. Enrichment scores were calculated using a chi-square test comparing the proportion of the gene list in a group to the proportion of the background genes and the gene set enrichment analysis online-tools from Broad Institute, Inc., Massachusetts Institute of Technology, and Regents of the University of California.

[0096] Statistical analyses

[0097] Statistical analysis was performed using R or GraphPad Prism software 10.0. The values are reported as mean ± SD. Data were compared between groups using one-way analysis of variance (ANOVA) followed by Tukey multiple comparisons post hoc test. The PDX-AML data for AML blasts and LSCs were visualized using a violin plot, showing the median value and the comparisons between the two groups were performed using an unpaired t-test. P values of less than 0.05 were considered significant.ResultsDesign of AcTor:

[0098] Protein-protein interactions rely on multiple contacts and small molecules are typically inefficient to dissociate protein complexes. However, binding of small molecules can modulate the configuration of the complex, affecting activity. We identified a gap within the interphase of TSC2 and Rheb that can accommodate a small molecule. Yang et al., Nat Commun., 12(1 ):339 (2021). The proximity to the catalytic site of TSC2 suggested that a small molecule could alter mTORCl activity. We docked in silico all clinically approved drugs to this interspace. Several of the top binders were chelating agents carrying several carboxylic acids, charged phosphates or quaternary amine groups. These molecules were discarded given their low cellular permeability. Other molecules, albeit obtaining a high free energy of binding, were found to interact deeper inside the binding area and thus not likely to impede TSC2 / Rheb interaction. The cytochrome P450 inhibitor cobicistat stood out as the only potential hit. We pursued it owing to drug-like structure and oral bioavailability (Fig. 1A). A two-dimensional projection of cobicistat binding onto the TSC2 binding pocket suggests proximity to Argininel749 in TSC2, a critical residue for TSC2 catalytic activity. The model shows that the morpholine group of cobicistat points to TSC2 and the phenyl group, circled, protrudes towards Rheb. Cobicistat formed multiple interactions with the key residues and covered / blocked essentially the entire recognition area (Fig. IB). However, when tested in RPMI 8226 cells, a reduction in mTORCl activity was observed, assessed by the phosphorylation level of S6. Reduction in activity was not observed in TSC2 KO RPMI8226 cells. Based on the in silico model, we predicted that modifications of the phenyl group of cobicistat with bulky moieties may convert it to an mTORCl activator. To identify potential modifications, the solventexposed phenyl ring was targeted using R-group replacement. Among the fragments explored, 5-indole bound in para position provided the best docking score, ~lkcal / mol better than the original compound itself. The indole moiety furthermore orients into the cavity of the GAP domain of TSC2, hindering Rheb from entering the binding site. Following optimization of a synthetic scheme, AcTor was prepared. In color are the modifications that were introduced to cobicistat (Fig. 1C).AcTor activates mTQRCl in the presence of IXZ and potentiates IXZ activity across multiple AML cell lines:

[0099] The similarity of AcTor to cobicistat suggested that it too may be orally available. We therefore studied its activity in the MM cell line RPMI8226 together with the orally available PI, ixazomib (IXZ). AcTor induced the activity of mTORCl in a concentration-dependent manner, plateauing around 10 uM (Fig. ID). At this concentration, AcTor prevented the suppression of mTORC 1 activity by IXZ, without affecting the levels of ubiquitinated proteins. AcTor alone mildly compromised the viability of RPMI8226 after prolonged incubation, an effect that was not observed with cobicistat. When combined with 20 nM of IXZ for 24 h, viability was compromised more than in the presence of IXZ alone. This was partially rescued by the addition of the mTOR inhibitor Torin- 1. Immunoprecipitation studies suggested that the interaction between TSC2 and Rheb was not prevented by AcTor, however Rheb»GTP levels increased. To assess binding of AcTor to TSC2 indirectly we performed thermostability assays. The addition of AcTor improved the solubility of TSC2 at 57 °C compared to 61 °C for the DMSO control. Utilizing this assay, the stabilization of TSC2 by AcTor was maximal at approximately 10 pM, in agreement with mTORCl activity. We conclude that AcTor most likely targets TSC2.

[0100] To test whether the enhanced activity of IXZ by AcTor is applicable also to AML cells, MV4-11 AML cells, which harbor the MLL-AF4 fusion gene, a FLT3 activation mutation and mutation of p53 (Zauli et al., Haematologica, 97(11):1722-1730 (2012)), were plated in a matrix of concentrations of AcTor and IXZ and viability was measured 24 h later by TiterGlo. To quantify synergism, we applied the SynergyFinder tool 24,25. A ZIP energy score of over 5 is indicative for synergism. We calculated a score of 24. The peak of the synergism map was at approximately 15 nM of IXZ and 10 ,uM of AcTor (Fig. 2A). When surviving cells were analyzed by immunoblotting following treatment at these concentrations, mTORCl output (P-S6K1 , P-S6, P-4EBP1) was higher in the presence of AcTor / IXZ compared to IXZ alone (Fig. 2B). We analyzed the activity of AcTor / IXZ at these concentrations in multiple AML cell lines by flow cytometry, using SYTOX green as a vital dye. A strong synergism was observed in all tested cells (Fig. 2C), quantified in Fig. 2D. Of note, in MV4-11 cells, a concentration as high as 250 nM of IXZ did not achieve a similar cytotoxic activity as 15 nM IXZ in the combination. These data indicate potentiation of IXZ activity by more than 15-fold. Primary AML cells ex vivo were sensitive to AcTor alone and more sensitive to the combination (Fig. 2E). KG-la cells are promyeloblast that express the stem cell marker CD34 and serves as a model of LSCs. In addition to the synergism of AcTor / IXZ in these cells, we noticed that the combined treatment resulted in a reduction in CD34 expression, a phenomenon previously documented for 500 nM of the PI bortezomib. Costa et al., J Cell Mol Med. 2024;28(8):el8333. Cobicistat, on the other hand, did not enhance the activity of IXZ, even at 30 pM. To exclude that AcTor operates through cytochrome P450 inhibition, we applied ketoconazole, a potent cytochrome P450 inhibitor with an IC50 of less than 1 pM 30. Ketoconazole did not enhance the activity of IXZ either at much higher than its IC50. Only when AcTor was added on top of ketoconazole / IXZ, enhanced activity was observed. We conclude that AcTor is a novel pharmacological potentiator of IXZ.AcTor / IXZ causes a mitochondrial dysfunction in AML:

[0101] TSC2 KO cells develop a mitochondrial dysfunction in the presence of IXZ. To examine whether AcTor exerts a similar function, we measured the effect of AcTor on cellular respiration in the presence and absence of IXZ. AcTor alone did not significantly affect the oxygen consumption rate of MV4-11 cells. A small reduction was observed by IXZ. When AcTor was combined with IXZ, a complete shutdown of mitochondrial respiration was recorded (Fig. 3A). To ensure that the lack of mitochondria activity is the cause of cell death, we applied a mitochondria diagnostic assay in which the cells are permeabilized with digitonin and the mitochondria are sequentially energized with carbon substrates. A 24 h exposure of MV4-11 cells to AcTor / IXZ was sufficient to nearly eliminate mitochondrial respiration regardless of added carbon sources (Fig. 3B). Sequential additions of substrates of the electron transport chain complexes did not restore oxygen consumption (Fig. 3C). This implies that the combination of AcTor and IXZ induces a bioenergetic crisis. Consistently, a complete loss of the mitochondrial membrane potential was measured by JC-1 for both MV4-11 (Fig. 3D) and KG-la cells (Fig. 3E). This was associated with induction of mitochondrial ROS, measured byMitoSOX (Fig. 3F). Total ROS levels were also increased but not to the same extent, suggesting that the source of ROS is primarily mitochondrial. The mitochondrial damage induced apoptosis, demonstrated by PARP1 cleavage and Annexin V / propidium iodide (PI). Viability of the MV4-11 and KG- la cells was improved by inclusion of the pan-caspase inhibitor zVAD-fmk. We then used two mitochondrial dyes to image mitochondria content and function. MitoTracker red accumulates in the mitochondria in a membrane potential-dependent manner, while MitoTracker green binding is insensitive to membrane potential, and serves as a readout of mitochondria content. MV4-11 cells treated for 24 h with AcTor, IXZ, or AcTor+lXZ displayed a similar mitochondria content (green signal), with a loss of mitochondrial activity (red signal). AcTor alone increased the red labeling, similarly to what has been seen in TSC2 silenced cells. Schieke et al., J Biol Chem. 2006;281(37):27643-27652. To assess whether the mitochondrial stress is the primary inducer of cell death, we blocked caspase 9 during AcTor / IXZ treatment with Z-LEHD-FMK. Percentage of apoptotic cells was significantly reduced (Fig. 3G). These data indicate that AcTor / IXZ combination creates irreparable damage to mitochondrial electron transport chain (ETC). Importantly, since induction of ROS induces differentiation of AML LSCs 32. Hence, the burst in ROS may be advantageous for AcTor / IXZ treatment.Gene expression induced by AcTor alone does not overlap with that of AcTor / IXZ:

[0102] We compared the transcriptome of MV4-11 cells treated with AcTor versus DMSO control to AcTor / IXZ versus IXZ. AcTor induced 2271 genes relative to DMSO. Only 343 of these overlapped with the induced genes of AcTor / IXZ versus IXZ. A similar small overlap was observed for the downregulated genes (Fig. 4A). Analysis of the differentially expressed genes indicated upregulation of Myc targets, genes of the oxidative phosphorylation pathway and mTORC 1 signaling by AcTor (Fig. 4B). These signatures were abolished when AcTor / IXZ was compared to IXZ. Instead, signatures of stress signaling and proapoptotic pathways emerged, such as a p53 signature, albeit p53 is mutated in MV4-11 cells. Comparisons of AcTor alone vs AcTor / IXZ demonstrate a diversion from the classical mTOR effect on anabolic programs and cell proliferation. Connectivity genes in AcTor vs DMSO were associated with proliferation and survival, such as tyrosine kinase receptor signaling and promotion of mitochondrial activity, while the connectivity genes in AcTor / IXZ vs IXZ were mostly stress-inducing genes. Taking together, although AcTor / IXZ elevates mTORC! activity, the downstream program is deviated into stress-induced cell death pathways. Whenanalyzing the volcano plots of AcTor / IXZ vs IXZ, we noticed that the induction of Adrenomedullin 2 (ADM2) by AcTor / IXZ combination (Fig. 4C). Since ADM2 is a secreted protein, we pursued it as a biomarker for the treatment. qPCR analysis confirmed the induction of ADM2 at the mRNA level (Fig. 4D) and immunofluorescence at the protein level (Fig. 4D).AcTor and IXZ synergize in vivo to eradicate AML blasts and AML stem cells:

[0103] Cobicistat is given to mice at a dose of 25 mg / Kg. With the adjustment of the higher mw of AcTor, we assessed 30 mg / Kg as a therapeutic dose. We conducted a thorough toxicity analysis that includes histology of the different organs, assessment of serum transaminase levels and complete blood counts. Repetitive daily doses of AcTor at 30 mg / Kg for three weeks did not show signs of toxicity. We did not observe overt toxicities following a single dose of AcTor of up to 300 mg / Kg. IXZ at doses above 2 mg / Kg i.p. was not tolerated. Daily doses of up to 1 mg / Kg did not show acute toxicity. We tested the combination of AcTor (30 mg / Kg) and IXZ (1 mg / Kg) for three weeks in C57BL / 6I mice. Mice continued to gain weight, splenic B cells and T cells were not significantly affected, blood counts were normal, and no pathologies were observed in the different organs. To assess concerns related to chemotherapy, we analyzed the effect of AcTor / IXZ on hematopoietic stem cells (HSCs) in C57BL / 6J mice. We observed a small reduction in the number of bone marrow HSCs, which indicate an intact HSC population.

[0104] Engraftment of NSG mice with MV4-11 cells is an aggressive AML model, which cripples the mice within 2-3 weeks. Huang et al., Mol Oncol., 14(10):2560-2573 (2020). One million MV4-11 cells that stably express luciferase were injected i.v. Three days after the challenge, we initiated treatment i.p. with vehicle, AcTor, IXZ or AcTor+IXZ every other day. Tumor burden was assessed by total body luminescence. Following three weeks, most mice in the vehicle control and the AcTor alone group succumbed. The best survival was obtained with the combination of 30 mg / Kg of AcTor with 1 mg / Kg of IXZ (Fig. 5A). Of note, AcTor alone seemed to modestly accelerate disease progression, consistent with the positive effect on mTORCl. Hence, AcTor should never be used alone. The positive effect of the combination was apparent in the mouse weight (Fig. 5B), the smaller spleen size (Fig. 5C) and the reduced tumor burden in the spleen (Fig. 5D). Total body luminescence indicates a significant reduction in tumor burden at day 16 and 22 after inoculation (Fig. 5E). ADM2 levels were increased inthe serum of the AcTor / IXZ treated mice, even though tumor burden gradually decreased with the combined treatment (Fig. 5F).

[0105] To address whether AcTor / IXZ is effective in eradicating AML stem cells, we challenged NSG mice with primary patient derived AML (PDX), which were isolated from a relapsed refractory patient that carried the FLT3-ITD mutation. Bhatt et al., Cancer Cell, 38(6):872-890 e876 (2020). When injected into NSG mice, these PDX cells generated LSCs in the bone marrow, while blast cells emigrate to the spleen. Treatment was initiated when human CD45RA+ cells exceeded 75% in the blood (Fig. 6A) and was limited to three weeks. At the endpoint, control mice were almost paralyzed. It was clear from mouse appearance and activity that the combined treatment improved their condition. Weight of AcTor / IXZ treated mice was increased within a week after treatment initiation (Fig. 6B) and spleen sizes were smaller (Fig. 6C). A strong reduction in the hCD33+ AML blast cells was observed with the emergence of a population of hCD33-negative, hCD45RA-negative, mouse CD45RA-positive cells (Fig. 6D). Analysis of the hCD45RA-positive spleen cellular for apoptosis using Annexin V and 7-AAD showed that approximately 30% of the AML cells were in late apoptosis following AcTor / IXZ treatment (Fig. 6E). Similar results were obtained for blasts cells in the bone marrow and peripheral blood.

[0106] Bone marrow AML cells at the late stages of the disease are positive for the proliferation marker Ki-67. Kaajik et al., Br J Cancer, 88(5):775-781 (2003). A reduction in the Ki-67 positive nuclei of AcTor / IXZ treated mice, compared to the other groups was seen (Fig. 6F). LSCs are enriched in the hCD34-positive, hCD38-negative population. AcTor / IXZ treatment reduced the hCD34-positive, hCD38-negative compartment (Fig. 6G), with an increase in apoptosis (Fig. 6H). We found no evidence of LSC maturation by CD1 lb, CD14 or CD15 markers. Masson's trichrome staining of the bone marrow (tibia section) shows engraftment and localization of AML cells (marked in yellow regions and arrows) at the trabecular and cortical regions in vehicle treated mice. These regions of the bone were cleared from AML cells in the AcTor / IXZ treated mice. Hypoxia is supporting the survival of LSCs stem cell. Cui et al., Blood Cells Mol Dis, 51(3): 177- 184 (2013). We assessed the level of bone marrow hypoxia by pimonidazole staining in the trabecular and cortical regions of the femur. In both locations, AcTor / IXZ treatment reduced hypoxic conditions. Analysis of serum ADM2 levels indicated an increase, providing further support for it as a marker for treatment (Fig. 61). We conclude that AcTor / IXZ combination causes apoptosis of AML blasts and LSCs.AcTor / IXZ treatment maintains efficacy after relapse:

[0107] Although the AML cells were barely detected after three weeks of treatment, five weeks after cessation of treatment, mice appeared sick again, indicating a relapse. Because PDX models of MM in mice show a rapid gain of resistance to proteasome inhibitors after relapse (Yue et al., Leuk Res., 122: 106949 (2022)), we examined whether AcTor / IXZ treatment maintains potency. Mice were challenged with PDX cells and when showing signs of sickness were treated for three weeks with AcTor / IXZ. Then, after relapse, a cohort of mice were sacrificed (marked “Before”) and a cohort of mice were treated for the second time with AcTor / IXZ for three weeks and sacrificed (marked “After”, Fig. 7A). Comparison of spleen size Before and After indicated a smaller size (Fig. 7B). hCD45RA+ cells disappeared and the mouse CD45RA+ population emerged in the spleen after the second treatment (Fig. 7C). Most hCD45RA+ cells were Annexin V positive with 15% at the late apoptotic stage (Fig. 7D). A smaller number of Ki-67-positive cells was seen after the second treatment (Fig. 7E) with a decrease in the hCD34+, hCD38- cells population (Fig. 7F), of those over 10% were apoptotic (Fig. 7G). We conclude that AcTor / IXZ maintains potency after relapse.Discussion

[0108] Pls exert their anti-cancer activity by perturbing proteostasis. Resistance to Pls employs cell intrinsic and tumor microenvironment-dependent mechanisms. Schwestermann et al., Front Oncol., 12:899272 (2022). The large number of mechanisms attributed to resistance to Pls make it a challenge to address therapeutically. However, an immediate adaptation to the proteostatic stress is essential. Based on our previous findings, the suppression of mTORCl is an immediate response to Pls and a gateway to resistance. We hypothesized that if high mTORCl activity is maintained during the initial exposures to Pls, resistance will be delayed if not prevented. Incentivized by this hypothesis we generated AcTor.

[0109] AcTor promotes the activity of IXZ in AML cells independently of specific mutations, including in TP53 (Fig. 2). Teoh et al., Biomed Res Int, 2014:717919 (2014). The combination of AcTor and IXZ elicited an irreparable damage to mitochondrial ETC, including to complex IV (Fig. 3). This mechanism was shown for IXZ in TSC2 KO MM cells, supporting that AcTor primary operates by blocking TSC2. Since all mitochondrial ETC complexes are embedded in the mitochondrial inner membrane, AcTor / IXZ may cause a defect in mitochondrial transport.A mild impairment in protein import to the mitochondri or low stability of one of the complex components can result in ETC insufficiency. Proteomic analyses suggested that mitochondrial components including components of the ETC are subjected to low level of ubiquitination. Sulkshane et al., J Proteomics, 229: 103949 (2020). It is therefore possible that in the presence of AcTor and IXZ, respiratory complexes accumulate ubiquitination to a level that perturbs proper function. Additionally, elevation of mitochondrial oxidative phosphorylation at the expense of glycolysis is one of the cellular strategies to adapt to Pls. Tsvetkov et al., Nat Chem Biol., 15(7):681-689 (2109). By suppressing oxidative phosphorylation, the insult to the mitochondria may invoke feed-forward feedback that results in a collapse of mitochondrial respiration.

[0110] AML cells rely on PI3K signaling for survival and inhibition of mTOR in the presence of chemotherapy impairs survival. Xu et al., Blood, 102(3):972-980 (2003). However, to achieve a durable anti-cancer effect, mTOR should be suppressed to a level that cannot be tolerated by most patients. A reporter mouse for mTOR activity showed a biphasic behavior of mTOR during AML progression. mTOR activity was reduced with the initial AML progression, while induced later even in the presence of treatment. Oki et al., Nat Commun., 12(1):245 (2021). We were therefore concerned that an mTOR inducer might accelerate AML growth. Transcriptome analyses indicate that on the background of IXZ, AcTor does not share the expression signature to when added alone. In fact, AcTor in the presence of IXZ fortified stress signaling pathways that are consistent with anti-cancer responses (Fig. 4). In support, when given alone, AcTor slightly accelerated disease progression (Fig. 5,6). The pharmacokinetic properties of IXZ are optimal for combination with AcTor. The half-life of IXZ is estimated in days, allowing its administration once a week to MM patients. While the pharmacokinetic parameters of AcTor have not been determined, cobicistat half-life is approximately 3 h. We therefore project that if given together on a once-a-day basis, IXZ will generate a stable steady state concentration, while AcTor will generate spikes in serum concentration, and be cleared within 12-24 h. This should ensure that tumor cells will never be exposed to AcTor alone.

[0111] All AML therapies compromise hematopoiesis. The major safety concern of AML patient is the ability to rapidly restore bone marrow functions following treatment. This relies on sparing enough HSCs. Encouraged by the fact that following treatment of PDX induced mice, murine hematopoietic cells repopulated the bone marrow, we addressed this in normalmice which displayed a normal blood count and an intact HSC compartment following AcTor / IXZ treatment. This is probably due to reliance on glycolysis by HSCs, making them less sensitive to mitochondrial damage. Papa et al., Stem Cells Int, 2019:4067162 (2019).

[0112] During remission, AML LSCs acquire mutation that result in a much aggressive disease upon relapse. In addition to the canonical resistance mechanisms, such as overexpression of P- glycoprotein, glutathione S-transferases, and activation mutations in key prosurvival pathways, AML adjusts cellular respiration, enhances autophagy and modify energy sources. We recently found that resistance of AML to venetoclax, a Bcl-2 inhibitor, is associated with ATP hydrolysis. Hagen et al., bioRxiv. 2024. Importantly, AcTor / IXZ maintained its potency after relapse obviating the need to adjust dosages (Fig. 7). It remains to be determined whether AcTor will be useful when combined with additional anti-AML drugs and how it affects LSC viability after a gain of resistance to chemotherapy.Example 2: mTOR activation creates a strong dependence on CAIX under hypoxia

[0113] Triple-negative breast cancers (TNBCs) are the deadliest type of breast cancers, characterized by aggressiveness and a poor response to therapy. A significant challenge to treating TNBCs is the hypoxic tumor microenvironment (TME), which causes an increase in tumor invasion, curtails the anti-tumor immune response and promotes drug resistance. A salient feature of hypoxic TMEs is a lower pH, which is a result of a direct enhancement of glycolysis. Damaghi et al., Proc Natl Acad Sci U S A, 118 (2021). Hypoxia drives the expression of surface carbonic anhydrases, isoforms IX (CAIX) and XII (CAXII) to buffer the acidic pH. Ivanov et al., Am J Pathol 158, 905-919 (2001). These enzymes convert CO2 at the surface of tumor cells into protons and bicarbonate anions. The bicarbonate anions are then carried into the cell by transporters, ensuring a physiological pH 6. Inhibition of CAIX results in acidification of cellular pH and an increase in cellular reactive oxygen species (ROS). Chafe et al., Sci Adv 7 (2021). CAIX is one of the most induced genes in TNBCs associated with poor survival and tumor progression. However, the effect of CAIX inhibitors on TNBC growth, in vitro and in vivo, as a single therapy is modest. Venkateswaran et al., Mol Cancer Ther 22, 1228-1242 (2023). We hypothesize that by enforcing an increase in production of cellular acids and ROS under hypoxic conditions, CAIX inhibitors will gain efficacy and their cytotoxic activity will be leveraged by magnifying cellular acidosis and oxidative stress.[001 14] The kinase mTOR is a key regulator of cell metabolism. In mammalian cells, mTOR resides in either of two complexes: mTORCl and mTORC2, which dictate mTOR specificity and regulation. mTORCl promotes the anabolic program of cells, inhibits autophagy, and induces the biosynthesis of proteins, lipids and nucleic acids. These activities promote cell growth and survival and drive oncogenesis. Consistent with pro-tumorigenic roles, the mTOR pathway is frequently activated across multiple cancer types. Bielska et al., Cancer Res 82, 3263-3274 (2022). TORC1 operates under a constant suppression of the tuberous sclerosis complex (TSC), of which TSC2 is the catalytic subunit. Since hypoxia suppresses mTORCl by promoting the activity of TSC2 (Brugarolas et al., Genes Dev 18, 2893-2904 (2004)), the deletion of TSC2 results in a strong activation of mTORCl under hypoxic conditions. Interestingly, the prominent mutations that activate mTOR in TNBCs and in most other cancers are upstream to TSC2, allowing the pathway to respond to stress including hypoxia. Inhibition of mTOR by rapamycin, rapalogs or direct mTOR inhibitors results in a strong antiproliferative effect in most tumors. However, despite promising preclinical data, the clinical effects of mTOR inhibitors have thus far been disappointing. The underlying reasons for the poor performance of mTOR inhibitors relate to the facts that under limited nutrient conditions and hypoxia, which simultaneously develop in the TME, some tumors grow faster in the presence of mTORCl inhibitors. Palm et al., Cell 162, 259-270 (2015). Furthermore, accumulating evidence indicates that in the presence of chemotherapy, suppression of mTOR is cytoprotective. Liu et al., Nat Commun 13, 7047 (2022). These paradoxical functions of mTOR as a tumor suppressive pathway were observed for solid and hematological tumors. Based on these findings, we hypothesized that the activation of mTORCl, specifically by inhibition of TSC2, should generate a metabolic liability for tumors that prosper in the presence of hypoxia. To investigate the validity of this hypothesis, we utilized in silico docking and medicinal chemistry to generate AcTor, a potential first of its kind inhibitor of TSC2. See Example 1. By activating mTORCl, AcTor promotes oxidative phosphorylation (OxPhos) and production of ROS under hypoxic conditions, resulting in TNBC dependence on CAIX activity for their survival. Therefore, we hope that the combination of AcTor with CAIX inhibitors will induce a pharmacological “synthetic lethality” within both developing and progressing TNBCs (Fig. 8).

[0115] CAIX is a zinc-containing metalloenzyme. Current inhibitors of CAIX target the zinc ion with sulfonamides or sulfamate moieties. SLC-0111 is a sulfonamide-based inhibitor that has been used in a Phase lb trial in a cohort of patients with various types of progressed cancers,including those of the breast. While a few patients showed no cancer progression for the trial duration, SLC-0111 did not show an overt response. Furthermore, many patients experienced severe adverse effects, which resulted in the discontinuation of SLC-0111 from clinical development. Evidence suggests that while the sulfonamide moiety provides an irreversible interaction with the CAIX active site, it may also be the source of the adverse effects due to off-target interactions with other metalloproteins. The objective of the proposed study is to overcome two hurdles in targeting CAIX for TNBC therapy: (i) Identification of combinations that improve the modest effect of CAIX inhibition alone in TNBC; and (ii) Generation potent non- sulfonamide CAIX inhibitors. If successfully addressed, the inhibition of CAIX can become a valid and effective strategy for TNBC therapy.

[0116] The current backbone of TNBC therapy consists of taxanes, anthracyclines and antimetabolites, which inhibit the cell cycle and generate DNA damage. Additional therapeutic modalities, such as biologicals and personalized therapies, are usually given as add-ons. Deficiency in DNA repair mechanisms sensitizes TNBCs for treatment, exemplified by the hypersensitivity of BRCAl / 2-deficient TNBCs to PARP1 inhibitors. Singh et al., Biomedicines 9(11):1512. (2021). However, BRCA 1 / 2 mutations only account for approximately 10-15% of TNBCs. DNA repair is exquisitely sensitive to cellular pH and a small reduction results in compromised nuclear excision repair (Yuan et al., Cancer Res 60, 4372-4376 (2000)), and inhibition of topoisomerase II activity. Xiao et al., Proc Natl Acad Sci U S A 100, 5205-5210 (2003). Accordingly, we hypothesize that if cellular pH drops below a certain threshold, chemotherapy should gain effectiveness irrespective of BRCA1 / 2 mutations, thereby sensitizing TNBCs that harbor normal BRCA1 / 2 to PARP1 inhibitors. Our work is significant because it seeks to provide pharmacodynamic understanding of a novel drug combination that has the potential to synergize with existing TNBC therapies that are largely ineffective, thereby improving survival outcomes for TNBC patients.INNOVATION

[0117] The proposed project plan is highly innovative in its approach to addressing scientific questions and developing novel therapies for effective treatment of TNBCs. Our project marks the first rigorous analysis of how mTOR activation creates a strong dependence on CAIX under hypoxia, resulting in a gain in the activity of CAIX inhibitors and sensitization of tumors to PARP1 inhibitors regardless of BRCA1 / 2 activity (Fig. 8). We will leverage uniquepharmacological tool, AcTor, a first of its kind TSC2 inhibitor, that allows testing this overarching hypothesis in a pharmacological relevant manner. To address the caveats of sulfonamides as drugs, we developed a novel thiazolidinone-based, non- sulfonamide inhibitor of CAIX, that we named BS 1. Our innovative findings will (i) identify metabolic activities that facilitate the acidification of TNBC cells, and (ii) provide the scientific foundation for combinations with DNA damage approaches as a general strategy for TNBC therapy.APPROACHPreliminary Data

[0118] BS 1 is a non- sulfonamide inhibitor of CAIX. The limitation of sulfonamides as CAIX inhibitors has instigated a search for alternatives. An unbiased high throughput chemical screen was unsuccessful to identify credible hits that do not contain sulfonamides. Tykvart et al., SLAS Discov 25, 1026-1037 (2020). A more targeted design identified a thiazolidinedione derivative as a potent non-sulfonamide CAIX inhibitor. Eldhena et al., J Enzyme Inhib Med Chem 37, 531-541 (2022). We built a virtual library of easily synthesizable thiazolidinedione derivatives, modified at two positions. Docking to the 6G9U crystal structure of the catalytic domain of human CAIX yielded BS1 (Fig. 9A), which exhibited a Molecular Operating Environment (MOE) score of -6.8, like the score of SLC-0111 (-6.9). The optimized docking configuration showed that the carbonyl moiety of the thiazolidinedione ring interacts with the zinc ion (Fig. 9B). Dynamic simulation for SLC-0111 yielded the expected interaction of the zinc molecule with the sulfonamide moiety (Fig. 9C). Of note, the carbonyl-zinc interaction is of lower energy than of sulfonamide, suggesting that the calculated affinity of BS 1 to CAIX is largely independent of zinc. We synthesized large quantities of BS 1 as a racemic mixture (the chiral carbon is marked with a *). Using a colorimetric assay on recombinant CAIX, the IC50 of BS1 was estimated to be 15 nM (Fig. 9D). Of note, SLC-0111 mildly compromises the viability of breast cancer cells at concentrations of 100-300 pM 23. Preliminary cell-based assays indicate that BS1 is affecting cell viability at concentrations of 5-10 pM. To compare the transcriptomic response of BS1 to that of SLC-0111, we conducted RNAseq analyses of HCC1806 TNBC cells subjected to 24 h of hypoxia in the presence of DMSO, BS1 (5 pM) or SLC-0111 (100 pM). Overall, we observed a larger number of suppressed genes than induced genes for both drugs. Of the suppressed genes relative to control, the vast majority was shared between BS1 and SLC-0111. A lower overlap was observed for the upregulated genes (Fig.9E). We conclude that BS1 inhibits CATX and probably affects additional pathways in a CAIX- independent manner.

[0119] AcTor elevates the dependence on CAIX to maintain homeostasis under hypoxia. Hypoxia attenuates mTORCl activity by mechanisms that converge on TSC 10. As a TSC2 inhibitor, AcTor treatment activated mTORCl activity in HCC1806 cells under hypoxic conditions. While it did not affect the expression of CAIX (Fig. 10A), it activated mTORCl even when combined with BS1 (Fig. 10B), determined by S6 and 4EBP1 phosphorylation. mTORC 1 activation in the presence of hypoxia and CAIX inhibition is expected to cause a metabolic liability. Accordingly, analysis of cellular pH by flow cytometry using BCECF-AM as an indicator, showed the expected reduction with BS 1. A further reduction in pH was observed when AcTor and BS 1 were combined (Fig. 10C). Although modest, the shift of the histogram to the left in the combined treatment may nonetheless be sufficient to cause irrevocable cellular damage. A similar effect of AcTor was observed when combined with SLC-0111 (Fig. 10C). Cellular ROS levels were increased by BS 1 alone under hypoxia and were further induced in the combined treatment (Fig. 10D). We conclude that AcTor exacerbates the metabolic stress that is caused by CAIX inhibitors under hypoxic conditions.

[0120] AcTor increases the cytotoxic activity of CAIX inhibitors. Flow cytometry analyses of viability under hypoxia indicated that SLC-0111 alone hardly compromises the viability of HCC1806 cells at concentrations up to 150 pM, neither AcTor at 10 pM. When combined with AcTor, enhanced cytotoxic activity was observed (Fig. 11 A). BS1 was active at 5 pM and a strong cooperation was observed when combined with AcTor (Fig. 11 A). Notably, this effect only occurred under hypoxia when CAIX is expressed, not under normoxia when CAIX expression is undetectable. These intriguing findings indicated that BS1 is functioning on an on-target effect (Fig. 1 IB). We then examined the effect of AcTor, BS1 and their combination on the growth of HCC1806 cells in nude mice. For this purpose, we stably transduced HCC1806 cells with firefly luciferase. Tumor growth was monitored by bioluminescence. BS1 reduced the growth of HCC1806 cells. When AcTor was added to the treatment, growth was further reduced (Fig. 11C, 11D). These data suggest that hypoxia is generated in situ during tumor progression, with AcTor enhancing BS1 activity in vivo.

[0121] AcTor / BSl promotes the activity of PARP1 inhibitors in BRCA1 / 2 active TNBC. The enhanced cellular acidosis in the presence of AcTor / BSl prompted us to examine the sensitivityto PARP1 inhibition in the absence of induction of DNA damage. Under hypoxic conditions, we analyzed whether the PARP1 inhibitor Olaparib gained cytotoxic activity in the presence of BS1, as well as whether such an effect was more pronounced when combined with AcTor / BSl (Fig. 12A, 12B). Similar outcomes were obtained for SLC-0111. Preliminary analysis of the cells for DNA damage using y-H2AX foci as readout, show little damage for all treatment. Large foci were observed only when the three drugs were combined (Fig. 12C). Of note, these experiments were performed in the absence of DNA damage-inducing drugs.

[0122] Working hypothesis: Hypoxia develops gradually in TNBCs as tumor burden increases. This provides time for tumor cells to undergo adaptation. Governed by hypoxia-inducible factors (HIFs), adaptation to hypoxia engages multiple mechanisms, cell intrinsic and TME- dependent. Part of the adaptation is mediated by induction of CAIX, which is a direct target of HIFla (Potter C., Harris AL., Cell Cycle 3, 164-167 (2004)), and suppression of mTOR by the HIFla-REDDl-TSC axis. A simultaneous inhibition of CAIX and activation of mTOR creates cellular acidification below tolerated values, a burst of ROS and a compromise of DNA repair. These stress conditions should intensify in correlation with the hypoxic conditions and by that restrict tumor growth. We will address this hypothesis in vitro using hypoxic chambers and in syngeneic animal models.

[0123] Aim 1: Define mechanisms by which mTOR activation sensitizes TNBC to CAIX inhibitors.

[0124] Hypoxia in the TME is a result of poor vascularization. This goes hand in hand with lower glucose levels and poor stimulation of tumor cells with serum proteins. All contribute to reduced cellular metabolism that is met with reduced activity of mTOR. We will assess the contribution of mTOR activation to metabolism and viability under stress conditions in vitro and in syngeneic mouse models of TNBC.

[0125] While AcTor induces mTORCl activity, it probably has off-target effects like all other drugs. To identify the specific effects of hyper mTOR, we will employ two syngeneic models of TNBC, in which an immune competent TME is generated. E0771 cells are syngeneic to C57BL / 6 mice, and 4T1 cells are syngeneic to BALB / c mice. We will engineer both cell lines with lentiviruses that encode an inducible shRNA to TSC2. We will refrain from using doxycycline or ecdysone because of their effect on cellular growth under hypoxia. As analternative, we will use the IPTG-induced shRNA Mission® system of Sigma (pLKO-puro- IPTG-3xLacO). When exposed to IPTG in vitro, or when IPTG is added to the drinking water of mice in vivo, mTOR activity should be induced in the presence of stress, including hypoxia. In vitro, we will subject the cells to hypoxia in hypoxic chambers for 24 h when cultured in normal media and in media that lacks glucose, serum and combinations thereof with and without IPTG. Cells will be studied for expression of CAIX, REDD1, HIFla and mTOR outputs by immunoblotting, mitochondrial activity by Seahorse, microscopy and flow cytometry for mitochondrial content and membrane potential. We will measure cellular pH by flow cytometry with the indicator BCECF. We will define the mechanisms of cell death, ferroptosis and apoptosis with specific inhibitors. To address the effect of mTOR induction on CAIX inhibition in vivo we will implant the IPTG-regulated shTSC2 E0771 and 4T1 cells in the mammary fat pad of their correspondent syngeneic hosts and allow the tumor to grow. Mouse cohorts will be divided between normal and IPTG-supplemented water and treated with BS 1 daily. If TSC2 silencing will show an enhanced anti-cancer effect in the presence of BS 1 , we will examine the effect on the TME. Tumors will be extracted and examined by histology and by single cell RNAseq. We will use IHC to examine the expression of CAIX, HIF1 a, and mTOR activity indicated by P-S6. Apoptosis will be assessed by IHC to cleaved caspase 3. Cellular pH will be assessed by flow cytometry on tumors dissociated into single cells stained with BCECF-AM 31. These analyses should provide initial proof of concept data on the implications of mTORj' / CAIXj, manipulation on tumor viability and the microenvironment. Positive results, i.e. lower pH, lower expression of HIFla, higher cell death, will be followed up by scRNAsec to define the cellular composition of the tumor, unravel the engagement of signaling pathways and identify potential biomarkers.

[0126] For most breast cancer patients, primary tumors are surgically removed, and chemotherapy is given to eradicate potential metastasis. Orthotopic E0771 tumors spontaneously metastasize to the lungs and peritoneal cavity. This spread accelerates after the primary tumor is removed. We generated E0771 that stably express firefly luciferase. The cells will be further transduced with the LacO shTSC2 lentiviruses. 8-12-week-old recipient C57BL / 6 female mice will be inoculated to the mammary fat pad with 1x103 E0771-ffLuc cells-shTSC2 (suspended in 50 pl of Matrigel® Matrix diluted 2-fold in PBS). We will surgically remove the tumor when it reaches approximately 1.5 cm. Mice will then be put on IPTG-supplemented water and treated daily with BS1. Control groups will include normal drinking water and vehicle treatments. 2-3 weeks after, mice will be monitored twice a weekby total body luminescence. Experiment will be terminated once a luminescence signal is observed in the lungs, indicative of metastases. Lungs will be removed upon euthanasia, and metastases will be examined by histology, immunohistochemistry for HIFla and CAIX as an indication of hypoxia, infiltrating immune cells (IHC for CD45, CD3, B220, CD14, Ly-6G etc.) and the tumor cells (luciferase). Positive results showing that TSC2 suppression enhances the activity of BS1 with respect to reducing tumorigenicity and metastases, will prompt us to analyze the effect of AcTor when given systemically. To this end, mice inoculated with E0771 or 4T1 will be treated with AcTor / BS 1 or AcTor / SLC-0111 and compared to the IPTG-induced shTSC2 with respect to the previously mentioned parameters. Taken together, we should be able to determine the effect of mTOR induction as a therapeutic strategy to enhance the effect of CAIX inhibitors in TNBC growth and spread. Expected Results, Potential Problems, and Alternative Approaches: We expect that in the presence of IPTG alone (TSC2 knockdown, mTOR activation), TNBC progression will be accelerated. However, when combined with CAIX inhibitors, the high mTOR activity will become a liability, and a strong anti-cancer effect will be observed, associated with immune cell infiltration and low cellular pH in the tumor cells. The effect on metastatic spread is dependent on multiple factors, all may be affected by mTOR. To avoid confounding factors, we will allow the lung metastasis to develop (verified by body luminescence) and only then initiate treatment. We do not expect technical difficulties in generating cellular models. While mTOR activation is not predicted to slow down cellular growth, if this happens in vitro it may confound the interpretation of the effects in vivo. In this case, we would need to focus on analyzing the short-term effects of mTOR activation. As an alternative, we will generate human TNBCs with the IPTG-regulated shTSC2 construct and perform the experiments in nude mice with the limitations of an immunocompromised TME. Our lab is experienced in all methodologies including microsurgery techniques. Bioinformatic analyses of scRNAseq data will be performed by the genomic center of CWRU.

[0127] Aim 2: Investigate the use of AcTor / CAIX inhibitors to actively disrupt DNA damage repair mechanisms.

[0128] DNA repair operates by various mechanisms, engaged in tandem. PARP1 is involved in base excision repair (BER), single-strand break repair (SSBR), homologous recombination (HR), and alternative non homologous end joining (a-NHEJ). Ray Chaudhuri A, Nussenzweig A, Nat Rev Mol Cell Biol 18, 610-621 (2017) BRCA1 / 2 is central for HR and when mutated, TNBC rely more on PARP1 for repair. Hence, PARP1 inhibitors gain efficacy when BRC Al / 2are mutated Jain A, Barge A, Parris CN, Oncogene 44, 193-207 (2025). However, most TNBCs carry intact BRCA1 / 2 genes. Since the DNA repair machinery is compromised at acidic pH, we reasoned that conditions of mTOR" / CAIX ( will increase DNA damage, creating a dependence on PARP1 also in wt BRCA1 / 2 TNBCs.

[0129] To assess the applicability of our pharmacological approach, we will analyze a broad range of TNBC cell lines. The cells will be treated with AcTor, SLC-0111, BS1 and combinations of AcTor / SLC-01 11 and AcTor / BSl under hypoxia. Normoxic conditions will be used to account for CAIX independent effects. Various DNA damage inducing agents will be added. We will use SCR7 which inhibits NHEJ, TRC102 which inhibits BER, topoisomerase inhibitors, cisplatin and PARP1 inhibitors. The cells will be treated for 24 h under normoxia and hypoxia and analyzed for viability by flow cytometry and DNA damage by immunofluorescence to 53BP1, yH2AX and single cell comet assay, all detect double strand breaks, and TUNEL assay for single strand breaks. Indications of synergism between AcTor / SLC-0111 or AcTor / BSl with any of the DNA damage repair inhibitors will be followed by immunoblotting for mTOR outputs, analysis of cellular pH, level of ROS. p53 is a key protein in the DNA damage response, and loss of function mutations in p53 occur in 80% of TNBC patients. Accordingly, most cell lines are p53-deficient (for instance HCC1806, HCC1937, MDA-MB-231, MDA-MB-468). In the absence of p53, alternative DNA damage pathways are activated, primarily the phosphorylation of MK2 39, Chk2 and ATM. These can be assessed by immunoblotting. For p53 wild type TNBC we will use DU4475 cells. In these cells we will analyze phospho-p53 levels and induction of its targets, primarily the cell cycle inhibitor p21. To assess if BRCA1 / 2 mutations enhance further AcTor / BSl -induced DNA damage, we will delete BRCA1 with CRISPR / Cas9 mutagenesis in the BRCA wt cells (MDA- MB-468, HCC1806 and MDA-MB-231 are BRCA wt), or use BRCA1 mutated TNBCs cells, such as HCC1937, MDA-MB-436. Analysis of DNA damage will be performed as above under normoxia and hypoxia. These extensive in vitro pharmacological analyses should allow us to determine the repair pathway that contributes most for survival, which TNBC genotypes are likely to respond (BRCA1 / 2, p53), and which drug combinations work best.

[0130] PARP1 inhibitors extend the overall survival of TNBC patients with BRCA mutations, when given in combination with carboplatin and paclitaxel and other drugs. Abraham et al., Nature 629, 1142-1 148 (2024). Our preliminary data suggests that a synergism exists between the PARPl inhibitor olaparib and BSl in BRCA wild type cells under hypoxia. We will addressin greater detail the potential synergism between Olaparib, BS1 and AcTor in BRCA1 / 2 wt TNBC tumors. We reasoned that this should be studied in immunocompetent vivo models, as inhibition of CAIX affects the tumor immune microenvironment. Chafe et al., Cancer Immunol Res 7, 1064-1078 (2019). The 4T1 mouse TNBC cells express wt BRCA1 / 2 and are highly invasive. Furthermore, expression of CAIX is induced in lung metastases of 4T1. To assess the effect of the treatments on 4T1 metastases, we will follow the protocol of Secondini et al., Oncoimmunology 6, el316437 (2017). BALB / c mice will be inoculated with 2.5X105 4T1 cells intravenously in the tail vein. Treatments with vehicle, Olaparib, BS1, Olaparib+BSl and Olaparib+BSl+AcTor will start daily from day 2 after inoculation until day 9, when mice will be killed for tumor analyses. We will quantify the lung weight and number of metastatic nodules and analyze by histology the tumor tissue for hypoxia (HIFla expression), pH, DNA damage by TUNEL and immunohistochemistry for yH2AX. Tumors tissues will be analyzed by flow cytometry and immunohistochemistry for immune cells, particularly T cells, NK, neutrophiles and macrophages. Significant differences in metastases size and / or infiltration of immune cells between the triple (Olaparib+BSl+AcTor) and the other treatments will prompt us to analyze the tumor tissues by scRNAseq, aimed at defining the different cell types in the TME and the signaling pathway that are engaged by tumor and immune cells. Specifically, we will look at the effect on Myc and AP-1 pathways known to induce resistance to Olaparib. Expected Results, Potential Problems, and Alternative Approaches: We expect a stronger effect of AcTor / BSl than BS1 alone on tumor size. We expect a lower pH in metastases that highly express CAIX, associated with a higher level of DNA damage. Olaparib+BSl+AcTor should compromise metastasis together with a higher number of tumor-infiltrated immune cells. A potential caveat is the timing of treatment. AcTor should be administrated after hypoxia develops and CAIX is already expressed. Induction of mTOR prior to hypoxia bears the risk of promoting tumor growth. We will therefore need to examine first the time needed for the development of hypoxia and CAIX expression and adjust the treatment accordingly. In case that hypoxia does not develop, or that CAIX expression is not induced in our hands, we will seek xenograft models in immunocompromised models that are more aggressive, such as MDA-MB-231 cells.

[0131] Scientific Rigor: For cell-based studies we will use appropriate controls (vehicle, mocktransfection, etc.). Western blotting, enzymatic assays, cell viability, qRT-PCR will be performed in biological and technical triplicates with appropriate normalizers and loading controls. In vivo studies will include cohorts of >7 mice for initial biological evaluation, and10 mice for final studies. Experiments will be performed in a semi-blinded fashion where the personnel that injected the mice will not be provided with the treatment details. Statistical significance will be defined as p < 0.05, determined with the Student t test, one-way ANOVA, or two-way ANOVA and student-Newman Keuls post-hoc analysis, as required for the experimental samples.Example 3: Synthesis of AcTor

[0132] Figure 13A shows the first half of the process involved in the preparation of AcTor, ending in the preparation of Compound 7. Figure 13B shows the second half of the process of preparing AcTor, designated as Al in Figure 13B.

[0133] The complete disclosure of all patents, patent applications, and publications, and electronically available materials cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. In particular, while various theories are presented describing possible mechanisms through with the compounds are effective, the compounds are effective regardless of the particular mechanism employed and the inventors are therefore not bound by theories described herein. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMSWhat is claimed is:

1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an mTOR activator and a proteasome inhibitor to the subject.

2. The method of claim 1, wherein the mTOR is mTORCl.

3. The method of claim 1, wherein the mTOR activator is a compound according to formula I:wherein R1is a phenyl or heteroaryl group, or a pharmaceutically acceptable salt thereof.

4. The method of claim 1, wherein R1is an indole group.

5. The method of claim 1, wherein the mTOR activator is AcTor.

6. The method of claim 1 , wherein the mTOR activator is administered together with a pharmaceutically acceptable carrier.

7. The method of claim 1, wherein the proteasome inhibitor is bortezomib, carfilzomib, or ixazomib.

8. The method of claim 1, wherein the proteasome inhibitor is ixazomib.

9. The method of claim 1 , wherein the cancer is leukemia.

10. The method of claim 9, wherein the mTOR activator is administered together with an anti-leukemic agent.

11. The method of claim 1 , wherein the cancer is drug-resistant cancer.

12. The method of claim 1 , wherein the cancer is recurrent cancer.

12. The method of claim 1, wherein the subject is human.

13. The method of claim 1, wherein the mTOR activator is administered orally.

14. The method of claim 1, wherein the mTOR activator and the proteasome inhibitor are administered contemporaneously.

14. A method of inhibiting tuberous sclerosis complex 2 (TSC2) by contacting it with an effective amount of a compound according to formula I:wherein R1is a phenyl or heteroaryl group, or a pharmaceutically acceptable salt thereof.

15. The method of claim 14, wherein R1is an indole group.

16. The method of claim 14, wherein the compound is AcTor.

17. A TSC2 inhibiting compound according to formula I:wherein R1is a phenyl or heteroaryl group, or a pharmaceutically acceptable salt thereof.

18. The compound of claim 17, wherein R1is an indole group.

19. The compound of claim 17, wherein the compound is AcTor.

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