Methods for enhancing immunotherapy in the treatment of glioblastoma

Abstract

Disclosed herein are methods of treating glioblastoma in a subject comprising, among others, administration of sub-maximal tolerance doses and / or intermittent doses of phosphoinositide 3-kinase (PI3K) inhibitors, autophagy inhibitors and / or checkpoint inhibitors.

Description

[0001] METHODS FOR ENHANCING IMMUNOTHERAPY IN THE TREATMENT OF GLIOBLASTOMA

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Patent Application No. 63 / 715,481, filed November 1, 2024 and U. S. Provisional Patent Application No. 63 / 805,869, filed May 14, 2025 each of which is hereby incorporated by reference in its entirety.

[0004] BACKGROUND

[0005] Glioblastoma (GBM) is the most common primary brain malignancy with over 10,000 new diagnoses every year in the United States. Despite surgery, radiation, and adjuvant treatments, most patients with GBM survive less than 2 years, highlighting the need for more effective therapeutics. A promising treatment strategy for many cancers over the past decade has been the use of Immune Checkpoint Inhibitors (ICIs). By disabling the molecules that keep the immune response in check, ICIs empower the body’s own immune system to fight cancer, yielding prolonged remission in nearly one half of patients with advanced melanoma and a significant portion of patients with other cancers. In GBM, however, clinical trials of ICIs, alone or in combination with other treatments, have failed to improve the median survival. While a small number of GBM patients may benefit from ICI treatment, efforts to improve the response rate have proven elusive, possibly due to the multifaceted challenge of mounting an effective immune response in GBM.

[0006] While the blood-brain barrier restricts entry of circulating immune cells and therapeutics in a healthy brain, the inflammatory environment of GBM permits entry of peripheral leukocytes of both myeloid and lymphoid lineages. Entry of immune cells into the GBM microenvironment does not, however, assure an effective response against the tumor. GBM is often heavily infiltrated by immunosuppressive cell populations such as myeloid-derived suppressor cells (MDSC), tumor-associated macrophages and microglia (TAM), and regulatory T cells (Treg), which collectively dampen the anti-tumor immune response by cytotoxic (CD8+) and helper (CD4+) T cells. Furthermore, in the absence of proper antigen presentation and T cell recognition, tumor-infiltrating T lymphocytes can frequently exhibit an exhausted phenotype, compounding the immune system’s difficulty in mounting an effective response against GBM.

[0007] SUMMARY

[0008] In some aspects, methods of treating glioblastoma (GBM) are disclosed herein. In some embodiments, the method comprises treating glioblastoma (GBM) in a subject using a sub-maximum tolerated dose (sub-MTD) of a pharmaceutical composition comprising administering to a subject having GBM a checkpoint inhibitor and a pharmaceutical composition comprising a compound of Formula (I) administered in a sub-MTD to treat the GBM in the subject:

[0009]

[0010] wherein

[0011] R1is -(CRaRb)m-aryl, -CH=CH-aryl, -(CRcRd)n-heteroaryl, -(CReRf)o-heterocycloalkyl, or -(CRgRh)P-cycloalkyl; wherein

[0012] m, n, o, and p are each independently 0, 1, or 2;

[0013] Ra, Rb, Rc, Rd, Re, Rf, Rg, and Rhare each independently H, halo, or Ci-4alkyl, or Raand Rbare taken together with the carbon to which they are attached to form a cycloalkyl ring,

[0014] or Raand Rbare taken together to form =CH2 or =0;

[0015] each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl present in R1is unsubstituted or substituted with one or two Rxsubstituents;

[0016] wherein each Rxsubstituent is independently halo, Ci-4alkyl, cycloalkyl, -C1-2- haloalkyl, -OH, -OCi-4alkyl, -O-Ci-2-haloalkyl, cyano, -C(O)Ci-4alkyl, - C(O)NRiRj, -SO2Ci-4alkyl, -SO2NRkRl, -NRqRr, -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -CO2Ci-4alkyl, -CO2aryl, - C(O)CH2-aryl (optionally substituted with methyl or halo), -CH2-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)Ci-4alkyl, or -CO2Ci-4alkyl); wherein Ri, Rj, Rk, and Rlare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, or -Ci-4alkyl-O-Ci-4alkyl,

[0017] wherein Rqand Rrare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, -Ci- 4alkyl-O-Ci-4alkyl, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, or -SO2Ci-4alkyl;

[0018] L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or-CRsRt-;

[0019] wherein Rsand Rtare independently H or alkyl, or Rsand Rtare taken together with the carbon atom to which they are attached to form a cycloalkyl ring;

[0020] X is O, S, NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), N(SO2cycloalkyl), or CH2;

[0021] Yi, Y2, and Y3 are each independently CH or N; wherein when L is other than -S(O)2-, Y2and Y3 are each CH;

[0022] G2is N or CR2;

[0023] G3 is N or CR3;

[0024] G4is N, NR4b, or CR4a;

[0025] Gs is N or CR5; and

[0026] G6is N or CR6;

[0027] wherein R2, R3, R4a, R5, and R6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NRURV;

[0028] or R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring; wherein the heteroaryl ring comprising R4band R6comprises no more than one N and is optionally substituted with alkyl, and the heterocyclic ring comprising R4band R6is optionally substituted with oxo,

[0029] Ruis H or Ci-4alkyl;

[0030] Rvis H, Ci-4alkyl, monocyclic cycloalkyl, -C(O)Ci-4alkyl, or -C(O)NRwRy;

[0031] wherein each alkyl present in Rvis unsubstituted or substituted with -OH, -NH2, - NH(Ci-4alkyl), or -N(Ci-4alkyl)2,

[0032] Rwand Ryare each independently H or Ci-4alkyl;

[0033] wherein

[0034]

[0035] not unsubstituted phenyl; and

[0036] R7and R8are each independently hydrogen or Ci-4alkyl, or R7and R8are taken together to form -CH2CH2-;

[0037] or a pharmaceutically acceptable salt thereof.

[0038] In some embodiments, the sub-MTD of a compound of Formula (I) is less than 30 mg / day; optionally, the sub-MTD is 0.1 - 30, 1 - 25, 1 - 10, 1 - 5 or 5 - 10 mg / day.

[0039] In some embodiments, the sub-MTD of a compound of Formula (I) is 1 - 10 mg / day.

[0040] In some embodiments, the compound comprises the structure:

[0041]

[0042] salts or combinations thereof.

[0043] In some embodiments, the compound is GCT-007

[0044]

[0045] In some embodiments, methods of treating glioblastoma (GBM) in a subject using intermittent doses of a pharmaceutical composition are disclosed herein. In some embodiments, the method comprises administering to a subject having GBM a checkpoint inhibitor, and intermittent doses of a pharmaceutical composition comprising a compound of Formula (I) for at least 2 consecutive days, followed by no administration of the pharmaceutical composition comprising a compound of Formula (I) for at least 2 consecutive days, to treat the GBM in the subject:

[0046]

[0047] wherein

[0048] R1is -(CRaRb)m-aryl, -CH=CH-aryl, -(CRcRd)n-heteroaryl, -(CReRf)o-heterocycloalkyl, or -(CRgRh)P-cycloalkyl; wherein

[0049] m, n, o, and p are each independently 0, 1, or 2;

[0050] Ra, Rb, Rc, Rd, Re, Rf, Rg, and Rhare each independently H, halo, or Ci-4alkyl, or Raand Rbare taken together with the carbon to which they are attached to form a cycloalkyl ring,

[0051] or Raand Rbare taken together to form =CH2 or =0;

[0052] each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl present in R1is unsubstituted or substituted with one or two Rxsubstituents;

[0053] wherein each Rxsubstituent is independently halo, Ci-4alkyl, cycloalkyl, -C1-2- haloalkyl, -OH, -0Ci-4alkyl, -O-Ci-2-haloalkyl, cyano, -C(O)Ci-4alkyl, - C(O)NRiRj, -SO2Ci-4alkyl, -SO2NRkRl, -NRqRr, -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -CO2Ci-4alkyl, -CO2aryl, - C(O)CH2-aryl (optionally substituted with methyl or halo), -CH2-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)Ci-4alkyl, or -CO2Ci-4alkyl); wherein Ri, Rj, Rk, and Rlare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, or -Ci-4alkyl-O-Ci-4alkyl, wherein Rqand Rrare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, -Ci- 4alkyl-O-Ci-4alkyl, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, or -SO2Ci-4alkyl;

[0054] L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or CRsRt-;

[0055] wherein Rsand Rtare independently H or alkyl, or Rsand Rtare taken together with the carbon atom to which they are attached to form a cycloalkyl ring;

[0056] X is O, S, NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), N(SO2cycloalkyl), or CH2;

[0057] Yi, Y2, and Y3are each independently CH or N; wherein when L is other than -S(O)2-, Y2and Y3are each CH;

[0058] G2is N or CR2;

[0059] G3is N or CR3;

[0060] G4is N, NR4b, or CR4a;

[0061] Gs is N or CR5; and

[0062] Ge is N or CR6;

[0063] wherein R2, R3, R4a, R5, and R6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NRURV;

[0064] or R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring; wherein the heteroaryl ring comprising R4band R6comprises no more than one N and is optionally substituted with alkyl, and the heterocyclic ring comprising R4band R6is optionally substituted with oxo,

[0065] Ruis H or Ci-4alkyl;

[0066] Rvis H, Ci-4alkyl, monocyclic cycloalkyl, -C(O)Ci-4alkyl, or -C(O)NRwRy;

[0067] wherein each alkyl present in Rvis unsubstituted or substituted with -OH, -NH2, - NH(Ci-4alkyl), or -N(Ci-4alkyl)2,

[0068] Rwand Ryare each independently H or Ci-4alkyl;

[0069] wherein

[0070]

[0071] not unsubstituted phenyl; and

[0072] R7and R8are each independently hydrogen or Ci-4alkyl,

[0073] or R7and R8are taken together to form -CH2CH2-;

[0074] or a pharmaceutically acceptable salt thereof.

[0075] In some embodiments, the intermittent doses of a pharmaceutical composition comprising a compound of Formula (I) is for at least 4 consecutive days, followed by no administration of the pharmaceutical composition comprising a compound of Formula (I) for at least 3 consecutive days; optionally, the intermittent doses of a pharmaceutical composition comprising a compound of Formula (I) is for at least 5 consecutive days, followed by no administration of the pharmaceutical composition comprising a compound of Formula (I) for at least 4 consecutive days.

[0076] In some embodiments, methods of treating glioblastoma (GBM) in a female subject by rendering the GBM susceptible to a programmed cell death protein 1 (PD1) inhibitor and / or a CD96 inhibitor is disclosed herein.

[0077] In some embodiments, the method comprises (i) measuring a first PD1 level in a first sample from a female subject and determining if the first PD1 level is below a predetermined PD1 level and / or measuring a first CD96 level in a first sample from a female subject and determining if the first CD96 level is below a pre-determined CD96 level,

[0078] wherein (a) if the first PD1 level and / or the first CD96 level is below a predetermined PD1 level and / or a pre-determined CD96 level, administering to the female subject in (i) a phosphoinositide 3 -kinase (PI3K) inhibitor for a sufficient time to increase the first PD1 level to or above the pre-determined PD1 level and / or the first CD96 level to or above the pre-determined CD96 level; or (b) if the first PD1 level and / or the first CD96 level is at or above a pre-determined PD1 level and / or a predetermined CD96 level, administering to the female subject in (i) a first PD1 inhibitor and / or a first CD96 inhibitor to treat the GBM in the female subject.

[0079] In some embodiments, the method further comprises (ii) measuring a second PD1 level in a second sample from the female subject in (a) and determining the second PD1 level and / or measuring a second CD96 level in the second sample from the female subject in (a), and (iii) if the second PD1 level in the second sample from the female subject in (ii) is at or above the pre-determined PD1 level and / or the second CD96 level in the second sample from the female subject in (ii) is at or above the pre-determined CD96 level, administering a second PD1 inhibitor and / or a second CD96 inhibitor to treat the GBM in the female subject.

[0080] In some embodiments, the method further comprises repeating (i)(a) - (ii) to produce in the subject a third PD1 level at or above the pre-determined PD1 level and / or a third CD96 level at or above the pre-determined CD96 level. In some embodiments, the first sample, the second sample or both the first sample and the second sample is a population of GBM tumor cells.

[0081] In some embodiments, the compound comprises the structure:

[0082]

[0083] In some embodiments, the PI3K inhibitor or the pharmaceutical composition is administered to the subject systemically.

[0084] In some embodiments, the subject is treated with the checkpoint inhibitor within 8 hours of the PI3K inhibitor or the pharmaceutical composition.

[0085] In some embodiments, the subject is treated with the checkpoint inhibitor within 24 hours of the PI3K inhibitor or the pharmaceutical composition. In some embodiments, the subject is treated with the checkpoint inhibitor within 1 week of the PI3K inhibitor or the pharmaceutical composition.

[0086] In some embodiments, the subject is treated with the checkpoint inhibitor within 1 month of the PI3K inhibitor or the pharmaceutical composition.

[0087] In some embodiments, the subject is treated with the checkpoint inhibitor within 6 months of the PI3K inhibitor or the pharmaceutical composition.

[0088] In some embodiments, the subject is administered at least 2 doses of PI3K inhibitor or the pharmaceutical composition.

[0089] In some embodiments, the checkpoint inhibitor is an antibody selected from an anti-PDl antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-Ll antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof.

[0090] In some embodiments, the checkpoint inhibitor or PD1 inhibitor is pembrolizumab, BMS-936558 (nivolumab), REGN-2810 (cemiplimab), orPDROOl (spartalizumab).

[0091] In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody selected from atezolizumab, avelumab, or durvalumab.

[0092] In some embodiments, the CD96 inhibitor is an antibody.

[0093] In some embodiments, the GBM is an isocitrate dehydrogenase (IDH) mutant GBM.

[0094] In some embodiments, the GBM is an IDH-wildtype GBM.

[0095] In some embodiments, the GBM is characterized by one or more of increased epidermal growth factor receptor (EGFR) amplification relative to a GBM without EGFR amplification, a telomerase reverse transcriptase (TERT)-promoter mutation, or a combined gain of chromosome7 / loss of chromosome 10 copy number changes.

[0096] In some embodiments, the GBM is characterized by hypermethylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter.

[0097] In some embodiments, the GBM is characterized by a BRAFv600 mutation, a fibroblast growth factor receptor (FGFR) mutation, a FGFR-TACC gene fusion, a H3K27M mutation, dysregulated CDK4 / 6 and / or dysregulated tumor protein p53 (TP53). In some embodiments, the method further comprises administering at least one additional treatment; optionally, wherein the at least one additional treatment is chemotherapy, radiation, surgery, administration of temozolomide, or combinations thereof.

[0098] In some embodiments, the subject is a mammal.

[0099] In some embodiments, the subject is a human.

[0100] In some aspects a method for treating glioblastoma (GBM) in a subject is provided. The method involves administering to a subject having GBM an effective amount to treat the GBM in the subject an autophagy inhibitor and a PI3K inhibitor comprising a compound of Formula (I):

[0101]

[0102] wherein

[0103] R1is -(CRaRb)m-aryl, -CH=CH-aryl, -(CRcRd)n-heteroaryl, -(CReRf)o-heterocycloalkyl, or -(CRgRh)P-cycloalkyl; wherein

[0104] m, n, o, and p are each independently 0, 1, or 2;

[0105] Ra, Rb, Rc, Rd, Re, Rf, Rg, and Rhare each independently H, halo, or Ci-4alkyl, or Raand Rbare taken together with the carbon to which they are attached to form a cycloalkyl ring,

[0106] or Raand Rbare taken together to form =CH2 or =0;

[0107] each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl present in R1is unsubstituted or substituted with one or two Rxsubstituents;

[0108] wherein each Rxsubstituent is independently halo, Ci-4alkyl, cycloalkyl, -C1-2- haloalkyl, -OH, -OCi-4alkyl, -O-Ci-2-haloalkyl, cyano, -C(O)Ci-4alkyl, - C(O)NRiRj, -SO2Ci-4alkyl, -SO2NRkRl, -NRqRr, -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -CO2Ci-4alkyl, -CO2aryl, - C(O)CH2-aryl (optionally substituted with methyl or halo), -CH2-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)Ci-4alkyl, or -CO2Ci-4alkyl); wherein Ri, Rj, Rk, and Rlare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, or -Ci-4alkyl-O-Ci-4alkyl,

[0109] wherein Rqand Rrare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, -Ci- 4alkyl-O-Ci-4alkyl, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, or -SO2Ci-4alkyl;

[0110] L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or-CRsRt-;

[0111] wherein Rsand Rtare independently H or alkyl, or Rsand Rtare taken together with the carbon atom to which they are attached to form a cycloalkyl ring;

[0112] X is O, S, NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), N(SO2cycloalkyl), or CH2;

[0113] Yi, Y2, and Y3are each independently CH or N; wherein when L is other than -S(O)2-, Y2and Y3are each CH;

[0114] G2is N or CR2;

[0115] G3is N or CR3;

[0116] G4is N, NR4b, or CR4a;

[0117] Gs is N or CR5; and

[0118] G6is N or CR6;

[0119] wherein R2, R3, R4a, R5, and R6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NRURV;

[0120] or R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring; wherein the heteroaryl ring comprising R4band R6comprises no more than one N and is optionally substituted with alkyl, and the heterocyclic ring comprising R4band R6is optionally substituted with oxo,

[0121] Ruis H or Ci-4alkyl;

[0122] Rvis H, Ci-4alkyl, monocyclic cycloalkyl, -C(O)Ci-4alkyl, or -C(O)NRwRy;

[0123] wherein each alkyl present in Rvis unsubstituted or substituted with -OH, -NH2, - NH(Ci-4alkyl), or -N(Ci-4alkyl)2,

[0124] Rwand Ryare each independently H or Ci-4alkyl;

[0125] wherein

[0126]

[0127] not unsubstituted phenyl; and

[0128] R7and R8are each independently hydrogen or Ci-4alkyl, or R7and R8are taken together to form -CH2CH2-;

[0129] or a pharmaceutically acceptable salt thereof.

[0130] In some embodiments the PI3K inhibitor comprises the structure:

[0131]

[0132] salts or combinations thereof.

[0133] In some embodiments the compound is GCT-007:

[0134]

[0135] salts thereof.

[0136] In some embodiments the autophagy inhibitor is a Vps34 inhibitor. In some embodiments the autophagy inhibitor is GCT-008:

[0137]

[0138] In some embodiments the Vps34 inhibitor is selected from nucleic acid inhibitors such as antisense oligonucleotides or siRNA targeting exon 4 of Vps34, Spautin-1 (4-[[3,4-(methylenedioxy)benzyl]amino]-6-chloroquinazoline, MBCQ), pyrimidinone catalytic inhibitors of Vps34 and bisaminopyrimidine catalytic inhibitors of Vps34.

[0139] In some embodiments the autophagy inhibitor is a fatty acid oxidation inhibitor, ranolazine, di chloroacetate, chloroquine (CQ), hydroxychloroquine (HCQ), or 3-methyladenine (3-MA). In some embodiments the fatty acid oxidation inhibitor is selected from etomoxir, oxfenicine, perhexiline, mildronate (a carnitine biosynthesis inhibitor), trimetazidine, and pFOX.

[0140] In some embodiments the PI3K inhibitor is administered to the subject prior to the autophagy inhibitor.

[0141] In some embodiments the method further comprises administering to the subject a checkpoint inhibitor.

[0142] In some embodiments the PI3K inhibitor is administered to the subject prior to the autophagy inhibitor and the checkpoint inhibitor. In some embodiments the autophagy inhibitor is administered to the subject prior to the checkpoint inhibitor. In some embodiments the checkpoint inhibitor is administered to the subject prior to the autophagy inhibitor. In some embodiments the autophagy inhibitor is administered to the subject at the same time as the checkpoint inhibitor.

[0143] In some embodiments 1 - 10 mg / day of the PI3K inhibitor is administered to the subject.

[0144] In some embodiments the PI3K inhibitor and / or the autophagy inhibitor is administered to the subject systemically. In some embodiments the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 8 hours of the PI3K inhibitor. In some embodiments the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 24 hours of the PI3K inhibitor. In some embodiments the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 1 week of the PI3K inhibitor. In some embodiments the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 1 month of the PI3K inhibitor. In some embodiments the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 6 months of the PI3K inhibitor. In some embodiments the subject is administered at least 2 doses of PI3K inhibitor.

[0145] In some embodiments the checkpoint inhibitor is an antibody selected from an anti-PDl antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-Ll antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof.

[0146] In some embodiments the checkpoint inhibitor or PD1 inhibitor is pembrolizumab, BMS-936558 (nivolumab), REGN-2810 (cemiplimab), orPDROOl (spartalizumab).

[0147] In some embodiments the checkpoint inhibitor is an anti-PD-Ll antibody selected from atezolizumab, avelumab, or durvalumab.

[0148] In some embodiments the GBM is an isocitrate dehydrogenase (IDH) mutant GBM.

[0149] 59. The method of any one of claims 33-57, wherein the GBM is an IDH-wildtype GBM. In some embodiments the GBM is characterized by one or more of increased epidermal growth factor receptor (EGFR) amplification relative to a GBM without EGFR amplification, a telomerase reverse transcriptase (TERT)-promoter mutation, or a combined gain of chromosome7 / loss of chromosome 10 copy number changes. In some embodiments the GBM is characterized by hypermethylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter. In some embodiments the GBM is characterized by a BRAFv600 mutation, a fibroblast growth factor receptor (FGFR) mutation, a FGFR-TACC gene fusion, a H3K27M mutation, dysregulated CDK4 / 6 and / or dysregulated tumor protein p53 (TP53).

[0150] A pharmaceutical composition comprising a checkpoint inhibitor and an autophagy inhibitor comprising:

[0151]

[0152] rovided in some aspects.

[0153] This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0154] BRIEF DESCRIPTION OF DRAWINGS

[0155] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0156] FIGs. 1A-1D show that GCT-007 inhibits GL261 cell growth and enhances surface expression of immune response molecules. Values: mean ± s.d. of 3 replicates.

[0157] FIG. 1A and FIG. IB show that GCT-007 enhances GL261 cell surface expression of MHC-II, CD80, CD74, and PD-L1, but not CD86. * p <0.05 vs. control (0). FIG. 1C and FIG. ID show that GCT-007 (GCT, aka 520-337) is superior to Paxalisib (Pax) in enhancing surface expression of MHC-II and PD-L1. * Student’s t test p <0.05.

[0158] FIG. 2 shows that GCT-007 synergizes with anti-PD-1 in the treatment of GL261 syngeneic GBM model. Mice having intracranial implantation of GL261 cells were treated with GCT-007 and / or anti-PD-1 and resulting tumor growth was observed with MRI. FIG. 2 is a graph showing quantification of the tumor size based on the MRI analysis, * ANOVAp <0.05.

[0159] FIGs. 3A-3D show that GCT-007 synergizes with anti-PD-1 to improve response in the native GBM mouse model. FIG. 3A and FIG. 3B show Kaplan-Meier survival plots comparing GCT-007 or Paxalisib treatment against Control, using different treatment paradigms as indicated. FIG. 3C shows Kaplan-Meier survival analysis combining the treatment paradigms in FIG. 3B comparing GCT-007 vs Control. FIG.

[0160] 3D shows Kaplan-Meier survival plots comparing 4 different treatment arms as indicated, with durable responses in the combination arm.

[0161] FIGs. 4A-4G show sex differences in response to anti-PD-1 treatment. FIG. 4A shows Kaplan-Meier survival plot showing prolonged remission only in female mice treated with anti-PD-1. Addition of GCT-007 increases the durable response rate in both sexes. FIG. 4B shows sex differences in untreated tumor checkpoint molecules by RNAseq. FIG. 4C shows sex differences in tumor size after anti-PD-1 treatment. FIG.

[0162] 4D and FIG. 4E show H& E of brain slices of female mice in remission at 6 months. * cavitation with a small residual tumor nodule. FIG. 4F shows magnified IHC of tumor nodule in FIG. 4E shows sparse CD8+cells (arrowheads). FIG. 4G shows IHC of a second female brain in remission shows a cavity without any visible tumor nodules and only background staining with a higher anti-CD8 titer.

[0163] FIGs. 5A-5B show representative co-culture of mouse GBM and T cells. FIG.

[0164] 5A shows brightfield image of T cells (arrowheads) in mono-culture. FIG. 5B shows brightfield and GFP channels showing T cell:GBM cell co-culture. In the mouse model, GBM cells are GFP+. T cells are GFP'.

[0165] FIGs. 6A-6F show effects of GCT-007 on human GBM cells. FIG. 6A and FIG.

[0166] 6B show GCT-007 inhibits the proliferation of U251 human GBM cells and increases cell death. FIG. 6C and FIG. 6D show that GCT-007 enhances U251 cell surface expression of PD-L1 and MHC-II. FIG. 6E and FIG. 6F show that addition of 4 Gy radiation treatment (XRT) to GCT-007 further enhances its effect on U251 cell surface expression of PD-L1 and MHC-II. * p < 0.05.

[0167] FIG. 7 shows the results of an assay showing selectivity profile of several compounds on vsp34, PI3Ka, mTor, pAKT and cell viability. The compounds tested included NPT520-322 (GCT-008), NPT520-319, NPT520-318, PIK-93 and NVP-19. FIGs. 8A-8B show the results of a titration assay of GCT-007 alone, GCT-008 alone, and various combinations of GCT-007 and GCT-008 on the percent change in cellular viability relative to a vehicle control.

[0168] DETAILED DESCRIPTION

[0169] For many years glioblastoma (GBM) has been a disease with very high mortality. The advent of check-point inhibitor (ICI) therapies for GBM has provided for an avenue of therapy based on immunotherapy, increasing cancer free survival rates. Checkpoint inhibitors unlock the “brakes” that are placed on an effective anti-tumor immune response by certain molecular interactions between the immune system and the cancer. However, in some patients those brakes cannot be unlocked. It has been found that phosphatidylinositol 3 -kinase (PI3K) inhibitors can effectively modulate immune cells in order to enhance the reaction to checkpoint inhibitors in some subsets of patients and under certain conditions to enhance therapy.

[0170] The PI3K pathway, or more broadly the RTK-RAS-PI3K-AKT-mT0R pathway, is the growth and proliferation cascade most frequently activated in GBM. The pathway is also frequently activated in other cancers, promoting cell cycle and migration as well as resistance to apoptosis. The catalytic subunit of PI3K has several isoforms, and the alpha subunit gene, PIK3CA, is frequently mutated in many cancers including GBM.

[0171] Notably, various PIK3CA mutations exert differential roles in GBM cell proliferation and microenvironment interaction. Among the isoforms-specific inhibitors, PI3Ka and PI3KP inhibitors are thought to more directly inhibit tumor cell growth and proliferation. Several PI3K inhibitors have been approved for the treatment of hematologic malignancies as well as PIK3CA-mutant solid tumors. In GBM, among several PI3K inhibitors tried to date, Paxalisib has been shown to have brain penetrance and low toxicity. Aside from its effects on cell cycle, PI3K pathway inhibition augments the surface expression of immune response molecules such as MHC class I and II and CD80 on tumor cells, enhancing their ability to present antigens and promoting the conversion of immunologically “cold” tumors into “hot” tumors. This, in addition to other immunomodulatory effects discussed below, contributes to the anti-tumor activity of PI3K inhibitors and is thought to sensitize tumors to the effects of ICI treatment. While the blood-brain barrier restricts entry of circulating immune cells and therapeutics in a healthy brain, the inflammatory environment of GBM permits entry of peripheral leukocytes of both myeloid and lymphoid lineages. Entry of immune cells into the GBM microenvironment does not, however, assure an effective response against the tumor. GBM is often heavily infiltrated by immunosuppressive cell populations such as myeloid-derived suppressor cells (MDSC), tumor-associated macrophages and microglia (TAM), and regulatory T cells (Treg), which collectively dampen the anti-tumor immune response by cytotoxic (CD8+) and helper (CD4+) T cells. Furthermore, in the absence of proper antigen presentation and T cell recognition, tumor-infiltrating T lymphocytes can frequently exhibit an exhausted phenotype, compounding the immune system’s difficulty in mounting an effective response against GBM.

[0172] T cells, key cellular players in an immune response, become activated by recognizing antigens bound to Major Histocompatibility Complex (MHC) molecules on the surface of an antigen presenting cell (APC). CD8+and CD4+T cells utilize their T cell receptors (TCRs) to interact with MHC class I and class II bound antigens, respectively, and require costimulatory interactions with other APC molecules such CD80 for full activation. In contrast, interactions with immune checkpoint molecules such as PD-1, PD-L1, and CTLA-4 inhibit T cell activation, blunting their response against tumors. Another mechanism thought to contribute to immune evasion by tumors is downregulation or loss of MHC class I and possibly MHC class II molecules. On the other hand, MHC class II expression by tumor cells is associated with ICI response and improved patient survival in a number of cancers including melanoma, supporting the role of helper T cell recognition in cytotoxic T cell activation and ICI response in tumors. Specifically in glioblastoma, MHC class II antigen presentation and CD4+T cell activation is essential for proper CD8+T cell function. Therefore, a therapeutic approach that enhances the surface expression of MHC class II as well as costimulatory molecules on tumor and immune cells, as disclosed herein, improves the GBM immunotherapy response.

[0173] GBM incidence in the United States is higher in men, with a male to female ratio of 1.60. The excess male cancer incidence and mortality seen in GBM is also seen in a majority of other cancers across the world. While this discrepancy is likely multifactorial, a heightened baseline immune activation and surveillance in females is one of the reasons commonly proposed for their lower cancer burden. Assessing GBM survival in a native mouse model, a significant sexual dimorphism has been observed in response to genetic manipulations that corresponds closely to TAM and T cell changes in the tumor microenvironment. Other studies in mouse models and human GBM tissue have demonstrated intrinsic sex differences in MDSC populations and T cell exhaustion that contribute to sexual dimorphism in GBM. Whether these sex differences influence the outcome of immunotherapy in various cancers has been the subject of a number of studies. In GBM, a recent meta-analysis of clinical trial data demonstrated a survival advantage for female patients undergoing immunotherapy, correlating with elevated baseline immunological signatures in female GBM tumors. Given the complex interplay between human GBM development, tumor immune surveillance and response, and their sex-based determinants, it is critical for animal models of GBM to capture and be sensitive to these complexities.

[0174] Disclosed herein are therapeutic approaches that enable more effective anti-tumor responses in GBM, which improve the durable response rate and enhance patient survival. A native, immune-competent GBM mouse model can be used to test the synergy between highly brain-penetrant PI3K inhibitors and a variety of ICIs in promoting an effective anti-tumor response. A GBM mouse model can be used to investigate the cellular and biological determinants of the response including any sex differences. The durability of GBM immune memory as well as the potential to transfer GBM immunity to other mice via adoptive cell transfer can also be examined.

[0175] Additionally, to determine the translational potential to human GBM, co-culture tumor cells and autologous immune cells are harvested from human GBM and blood. The synergistic effects of PI3K inhibitors and ICIs on immune recognition of tumor cells and their cytotoxic interaction is analyzed. Thus, disclosed herein, are combinations of brainpenetrant PI3K inhibitors (e.g., GCT-007) and ICIs at optimal dosing regimens which improve GBM survival in a subject (e.g., mammal, human, etc.).

[0176] In some embodiments, a highly brain-penetrant PI3K inhibitor to treat GBM is disclosed herein. Disclosed herein is the use of PI3K inhibitors, such as Paxalisib and GCT-007, a PI3K inhibitor with anti -turn or properties (see Examples). Similarly to Paxalisib, GCT-007 is highly brain-penetrant and a pan-inhibitor of PI3K isoforms and mTOR (downstream mediator of PI3K signaling), albeit with varying affinities and pharmacokinetics (Table 1). Remarkably, among their differences, GCT-007 is a stronger PI3KP and mTOR inhibitor and has a longer half-life than Paxalisib.

[0177] Surprisingly, at lower concentrations, where toxicities are minimized, GCT-007 is more efficient than Paxalisib in upregulating the surface expression of MHC class II and other immune response molecules on GBM cells (see Examples). Without wishing to be bound by theory, this phenomenon is thought to promote the conversion of immunologically “cold” tumors into “hot” tumors, sensitizing them to the effects of immune checkpoint inhibition. In some embodiments, the improved therapeutic efficacy of GCT-007 and / or reduced toxicities, compared to, for instance, Paxalisib is disclosed herein.

[0178] B7P Ki^. (sM

[0179]

[0180] ratio PBKn PI3K(3 PISKv PI3KS mT0R GCT-007 3.6 15 4.9 7.1 0.33 3.5 24 5.2 3 43 Paxalisib 1.2 17 1.7 18 0.38 2 46 3 10 70 Table 1. Pharmacokinetic and pharmacodynamic properties of PI3K inhibitors GCT-007 and Paxalisib in mice, tm: half-life; CR: clearance rate; Va: volume of distribution; AUC: area under the curve; B / P ratio: brain / plasma ratio of unbound fraction; Kiapp: apparent inhibition constant.

[0181] In some embodiments, combining a brain-penetrant PI3K inhibitor (e.g., GCT-007 and derivatives thereof) with ICIs improves GBM immunotherapy response. As disclosed herein, PI3K inhibitors in combination with ICIs demonstrate analogous functions in promoting T cell response against tumors. Their combined use in clinical trials has shown therapeutic synergy and durable responses in heavily pretreated patients with solid tumors. Without wishing to be bound by theory, in GBM, this synergy is likely to be achieved using a combination of highly brain-penetrant PI3K inhibitors and ICIs, an approach not previously explored in patients or preclinical models. A native GBM mouse model disclosed herein shows an improved durable response rate when GCT-007 is added to anti-PD-1 treatment (see Examples). This is highly remarkable in GBM, which helps provide effective treatment to GBM patients.

[0182] A GBM mouse model disclosed herein incorporates the main oncogenic events of human GBM, including PI3K pathway activation, into embryonic glial progenitors in mice. This allows for autochthonous development of a high-grade IDH-wildtype diffuse glioma that parallels human GBM in histology and behavior. Specifically, the model involves in utero electroporation of CRISPR constructs targeting Trp53, Pten, and Nfl tumor suppressor genes along with a piggyBac transposon expression and reporter system selective for glial progenitor cells. This system has been modified as needed to introduce these and other genetic changes at desired embryonic stages to reliably obtain the intended tumors. What makes this GBM model uniquely suitable for immunotherapy studies is that the GBM tumors develop spontaneously and at a natural pace in fully immune-competent mice. The mice ultimately develop tumor symptoms and are euthanized with a median survival of about 100 days depending on the model. Mouse models that involve syngeneic GBM cell line injection in an adult mouse brain are often suboptimal for immunotherapy studies. Reasons include introduction of injection trauma and iatrogenic inflammation as well as an aggressive non-diffuse tumor growth and a short survival, which makes therapeutic studies challenging given the short window. The native GBM mouse model disclosed herein does not have these limitations, demonstrates sexual dimorphism similar to human GBM, and is thus highly suitable for the proposed studies.

[0183] Remarkable sexual dimorphism was observed in response to ICI treatment (see Examples). In some embodiments, sex is an independent variable in the mouse studies discussed herein. GBM mice starting treatment at different ages (4 weeks vs. 8 weeks) is disclosed herein and age, in some embodiments, is considered a variable. For experiments utilizing GBM patient samples, sex and age and prior treatment status such as radiation are considered.

[0184] The methods, in some aspects, involve a combination therapy. The combination involves the use of a PI3K inhibitor to increase the numbers of patients that respond to checkpoint inhibitors by combining the activity of checkpoint inhibitors with a PI3K inhibitor under ideal conditions. The combination therapy involves the treatment of a subject with both therapies. The treatment may occur at the same times or at different times. The compositions may be delivered in one formulation or, preferably in separate formulations.

[0185] Thus, the invention, in aspects, involves methods of treating cancer (e.g., glioblastoma). In some embodiments, a method of treating GBM in a subject using a submaximum tolerated dose (sub-MTD) of a pharmaceutical composition comprising administering to a subject having GBM a checkpoint inhibitor and a pharmaceutical composition comprising a compound of Formula (I) administered in a sub-MTD to treat the GBM in the subject:

[0186]

[0187] wherein

[0188] R1is -(CRaRb)m-aryl, -CH=CH-aryl, -(CRcRd)n-heteroaryl, -(CReRf)o-heterocycloalkyl, or -(CRgRh)P-cycloalkyl; wherein

[0189] m, n, o, and p are each independently 0, 1, or 2;

[0190] Ra, Rb, Rc, Rd, Re, Rf, Rg, and Rhare each independently H, halo, or Ci-4alkyl, or Raand Rbare taken together with the carbon to which they are attached to form a cycloalkyl ring,

[0191] or Raand Rbare taken together to form =CH2 or =0;

[0192] each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl present in R1is unsubstituted or substituted with one or two Rxsubstituents;

[0193] wherein each Rxsubstituent is independently halo, Ci-4alkyl, cycloalkyl, -C1-2- haloalkyl, -OH, -OCi-4alkyl, -O-Ci-2-haloalkyl, cyano, -C(O)Ci-4alkyl, - C(O)NRiRj, -SO2Ci-4alkyl, -SO2NRkRl, -NRqRr, -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -CO2Ci-4alkyl, -CO2aryl, - C(O)CH2-aryl (optionally substituted with methyl or halo), -CH2-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)Ci-4alkyl, or -CO2Ci-4alkyl); wherein Ri, Rj, Rk, and Rlare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, or -Ci-4alkyl-O-Ci-4alkyl, wherein Rqand Rrare each independently H, Ci-4alkyl, -Ci-4alkyl-0H, -Ci- 4alkyl-O-Ci-4alkyl, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, or -SO2Ci-4alkyl;

[0194] L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or CRsRt-;

[0195] wherein Rsand Rtare independently H or alkyl, or Rsand Rtare taken together with the carbon atom to which they are attached to form a cycloalkyl ring;

[0196] X is O, S, NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), N(SO2cycloalkyl), or CH2;

[0197] Yi, Y2, and Y3 are each independently CH or N; wherein when L is other than -S(O)2-, Y2and Y3 are each CH;

[0198] G2is N or CR2;

[0199] G3 is N or CR3;

[0200] G4is N, NR4b, or CR4a;

[0201] Gs is N or CR5; and

[0202] Ge is N or CR6;

[0203] wherein R2, R3, R4a, R5, and R6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NRURV;

[0204] or R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring; wherein the heteroaryl ring comprising R4band R6comprises no more than one N and is optionally substituted with alkyl, and the heterocyclic ring comprising R4band R6is optionally substituted with oxo,

[0205] Ruis H or Ci-4alkyl;

[0206] Rvis H, Ci-4alkyl, monocyclic cycloalkyl, -C(O)Ci-4alkyl, or -C(O)NRwRy;

[0207] wherein each alkyl present in Rvis unsubstituted or substituted with -OH, -NH2, - NH(Ci-4alkyl), or -N(Ci-4alkyl)2,

[0208] Rwand Ryare each independently H or Ci-4alkyl;

[0209] wherein

[0210]

[0211] not unsubstituted phenyl; and

[0212] R7and R8are each independently hydrogen or Ci-4alkyl,

[0213] or R7and R8are taken together to form -CH2CH2-;

[0214] or a pharmaceutically acceptable salt thereof,

[0215] is disclosed herein. A sub-maximum tolerated dose (sub-MTD) refers to a dose in which an effect is achieved below a maximum tolerated dose (MTD). An MTD refers to the highest dose of one or more compounds or pharmaceutical composition disclosed herein (e.g., PI3K inhibitor, CPI, or combination thereof, etc.) with acceptable side effects in a subject. An MTD can be determined, for instance, in a clinical trial by testing increasing doses on different subject groups until the highest dose with acceptable side effects is found.

[0216] In some embodiments, a sub-MTD administered to a subject is less than 30 mg / day of a compound or pharmaceutical composition disclosed herein (e.g., PI3K inhibitor or pharmaceutical composition comprising a PI3K inhibitor). In some embodiments, a sub-MTD is 0.1 - 30 mg / day of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 1 - 25, 1 - 10, 1 - 5 or 5 - 10 mg / day of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 1 - 25 mg / day of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 1 - 10 mg / day of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 1 - 5 mg / day of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 5 - 10 mg / day of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 0.1 or less than 0.1, 1 or less than 1, 2 or less than 2, 3 or less than 3, 4 or less than 4, 5 or less than 5, 6 or less than 6, 7 or less than 7, 8 or less than 8, 9 or less than 9, 10 or less than 10, 11 or less than 11, 12 or less than 12, 13 or less than 13, 14 or less than 14, 15 or less than 15, 16 or less than 16, 17 or less than 17, 18 or less than 18, 19 or less than 19, 20 or less than 20, 21 or less than 21, 22 or less than 22, 23 or less than 23, 24 or less than 24, 25 or less than 25 mg / day of a compound or pharmaceutical composition disclosed herein.

[0217] In some embodiments, a sub-MTD administered to a subject is less than 30 mg of a compound or pharmaceutical composition disclosed herein (e.g., PI3K inhibitor or pharmaceutical composition comprising a PI3K inhibitor). In some embodiments, a sub-MTD is 0.1 - 30 mg of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 1 - 25, 1 - 10, 1 - 5 or 5 - 10 mg of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 1 -25 mg of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 1 - 10 mg of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 1 - 5 mg of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 5 -10 mg of a compound or pharmaceutical composition disclosed herein. In some embodiments, a sub-MTD is 0.1 or less than 0.1, 1 or less than 1, 2 or less than 2, 3 or less than 3, 4 or less than 4, 5 or less than 5, 6 or less than 6, 7 or less than 7, 8 or less than 8, 9 or less than 9, 10 or less than 10, 11 or less than 11, 12 or less than 12, 13 or less than 13, 14 or less than 14, 15 or less than 15, 16 or less than 16, 17 or less than 17, 18 or less than 18, 19 or less than 19, 20 or less than 20, 21 or less than 21, 22 or less than 22, 23 or less than 23, 24 or less than 24, 25 or less than 25 mg of a compound or pharmaceutical composition disclosed herein.

[0218] In some embodiments, the sub-MTD is administered 1, 2, 3, 4, or 5 times per day. In some embodiments, the sub-MTD is divided among 1, 2, 3, 4 or 5 doses per day. In some embodiments, administration of a compound or pharmaceutical composition (e.g., PI3K inhibitor) disclosed herein minimizes or avoids the occurrence of an adverse event (AE) and / or minimizes toxicity. An AE refers to an untoward or unfavorable medical occurrence in a subject (e.g., human) including any abnormal sign (e.g. abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with the method of treatment.

[0219] In some embodiments, an AE is a serious AE (sAE). A sAE refers to an AE that results in death, is life-threatening or places the participant at immediate risk of death from the event as it occurred, requires or prolongs hospitalization, causes persistent or significant disability or incapacity, results in congenital anomalies or birth defects, or a condition which a healthcare provider (e.g., medical doctor, nurse, etc.) judges to represent significant hazard. In some embodiments, an AE is a non-serious AE (nsAE).

[0220] In some embodiments, the AE is mild (i.e., awareness of signs or symptoms, but easily tolerated and are of minor irritant type causing no loss of time from normal activities; symptoms do not require therapy or a medical evaluation; signs and symptoms are transient), moderate (i.e., introduces a low level of inconvenience or concern to the subject and may interfere with daily activities, but are usually improved by simple therapeutic measures; moderate experiences may cause some interference with functioning), or severe (i.e., events interrupt the subject’s normal daily activities and generally require systemic drug therapy or other treatment; they are usually incapacitating).

[0221] In some embodiments, methods of prolonging survival of a subject having glioblastoma (GBM) are disclosed herein. In some embodiments, the method comprises administering to a subject having GBM a checkpoint inhibitor and a pharmaceutical composition comprising a compound of Formula (I) to prolong survival in the subject, wherein survival in the subject is prolonged by at least 50 days relative to a baseline. In some embodiments, the baseline is a subject having GBM who has been administered the checkpoint inhibitor alone (monotherapy) or a subject having GBM who has not been administered the checkpoint inhibitor nor the pharmaceutical composition. In some embodiments, survival in the subject is prolonged by at least 100 days; optionally, survival in the subject is prolonged by 100 - 1000, 100 - 900, 100 -800, 100 - 700, 100 - 600, 100 - 500, 100 - 400, 100 - 300, 100 - 200 or 100 days.

[0222] In some embodiments, methods of treating GBM in a female subject by rendering the GBM susceptible to a programmed cell death protein 1 (PD1) inhibitor and / or a CD96 inhibitor comprises (i) measuring a first PD1 level in a first sample from a female subject and determining if the first PD1 level is below a pre-determined PD1 level and / or measuring a first CD96 level in a first sample from a female subject and determining if the first CD96 level is below a pre-determined CD96 level, wherein (a) if the first PD1 level and / or the first CD96 level is below a pre-determined PD1 level and / or a predetermined CD96 level, administering to the female subject in (i) a phosphoinositide 3-kinase (PI3K) inhibitor for a sufficient time to increase the first PD1 level to or above the pre-determined PD1 level and / or the first CD96 level to or above the pre-determined CD96 level; or (b) if the first PD1 level and / or the first CD96 level is at or above a predetermined PD1 level and / or a pre-determined CD96 level, administering to the female subject in (i) a first PD1 inhibitor and / or a first CD96 inhibitor to treat the GBM in the female subject.

[0223] In some embodiments, the method further comprises: (ii) measuring a second PD1 level in a second sample from the female subject in (a) and determining the second PD1 level and / or measuring a second CD96 level in the second sample from the female subject in (a), and (iii) if the second PD1 level in the second sample from the female subject in (ii) is at or above the pre-determined PD1 level and / or the second CD96 level in the second sample from the female subject in (ii) is at or above the pre-determined CD96 level, administering a second PD1 inhibitor and / or a second CD96 inhibitor to treat the GBM in the female subject.

[0224] In some embodiments, a pre-determined PD1 level is a PD1 level in a sample from a subject in the absence of administration of a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein) which increases the PD1 level in the subject to or above a predetermined PD1 level. In some embodiments, a pre-determined CD96 level is a CD96 level in a sample from a subject in the absence of administration of a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein) which increases the CD96 level in the subject to or above a pre-determined CD96 level.

[0225] In some embodiments, a pre-determined PD1 level is a 1.5-fold or a two-fold increase in a PD1 level measured in a sample from the subject relative to a PD1 level measured in a sample from the subject before administering a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein). In some embodiments, a pre-determined CD96 level is a 1.5-fold or a two-fold increase in a CD96 level measured in a sample from the subject relative to a CD96 level measured in a sample from the subject before administering a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein).

[0226] In some embodiments, a pre-determined PD1 level is a 3-fold, is a 4-fold, is a 5-fold, is a 6-fold, is a 7-fold, is an 8-fold, is a 9-fold or is a 10-fold, increase in a PD1 level measured in a sample from the subject relative to a PD1 level measured in a sample from the subject before administering a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein). In some embodiments, a pre-determined CD96 level is a 3-fold, is a 4-fold, is a 5-fold, is a 6-fold, is a 7-fold, is an 8-fold, is a 9-fold or is a 10-fold, increase in a CD96 level measured in a sample from the subject relative to a CD96 level measured in a sample from the subject before administering a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein). In some embodiments, a pre-determined PD1 level is a clinically relevant PD1 level. In some embodiments, a pre-determined PD1 level is a clinically relevant plasma PD1 level, immune cell PD1 level or tumor PD1 level. In some embodiments, a predetermined PD1 level is the lowest PD1 level at which a beneficial effect (e.g., partial or complete tumor regression) is observed in the subject. In some embodiments, a predetermined CD96 level is a clinically relevant CD96 level. In some embodiments, a predetermined CD96 level is a clinically relevant plasma CD96 level, immune cell CD96 level or tumor CD96 level. In some embodiments, a pre-determined CD96 level is the lowest CD96 level at which a beneficial effect (e.g., partial or complete tumor regression) is observed in the subject.

[0227] In some embodiments, methods of treating GBM in a female subject by rendering the GBM susceptible to a programmed cell death protein 1 (MHC-II) inhibitor and / or a PD-L1 inhibitor comprises (i) measuring a first MHC-II level in a first sample from a female subject and determining if the first MHC-II level is below a pre-determined MHC-II level and / or measuring a first PD-L1 level in a first sample from a female subject and determining if the first PD-L1 level is below a pre-determined PD-L1 level, wherein (a) if the first MHC-II level and / or the first PD-L1 level is below a predetermined MHC-II level and / or a pre-determined PD-L1 level, administering to the female subject in (i) a phosphoinositide 3 -kinase (PI3K) inhibitor for a sufficient time to increase the first MHC-II level to or above the pre-determined MHC-II level and / or the first PD-L1 level to or above the pre-determined PD-L1 level; or (b) if the first MHC-II level and / or the first PD-L1 level is at or above a pre-determined MHC-II level and / or a pre-determined PD-L1 level, administering to the female subject in (i) a first MHC-II inhibitor and / or a first PD-L1 inhibitor to treat the GBM in the female subject.

[0228] In some embodiments, the method further comprises: (ii) measuring a second MHC-II level in a second sample from the female subject in (a) and determining the second MHC-II level and / or measuring a second PD-L1 level in the second sample from the female subject in (a), and (iii) if the second MHC-II level in the second sample from the female subject in (ii) is at or above the pre-determined MHC-II level and / or the second PD-L1 level in the second sample from the female subject in (ii) is at or above the pre-determined PD-L1 level, administering a second MHC-II inhibitor and / or a second PD-L1 inhibitor to treat the GBM in the female subject. In some embodiments, a pre-determined MHC-II level is a MHC-II level in a sample from a subject in the absence of administration of a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein) which increases the MHC-II level in the subject to or above a pre-determined MHC-II level. In some embodiments, a pre-determined PD-L1 level is a PD-L1 level in a sample from a subject in the absence of administration of a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein) which increases the PD-L1 level in the subject to or above a pre-determined PD-L1 level.

[0229] In some embodiments, a pre-determined MHC-II level is a 1.5-fold or a two-fold increase in a MHC-II level measured in a sample from the subject relative to a MHC-II level measured in a sample from the subject before administering a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein). In some embodiments, a pre-determined PD-L1 level is a 1.5-fold or a two-fold increase in a PD-L1 level measured in a sample from the subject relative to a PD-L1 level measured in a sample from the subject before administering a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein).

[0230] In some embodiments, a pre-determined MHC-II level is a 3-fold, is a 4-fold, is a 5-fold, is a 6-fold, is a 7-fold, is an 8-fold, is a 9-fold or is a 10-fold, increase in a MHC-II level measured in a sample from the subject relative to a MHC-II level measured in a sample from the subject before administering a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein). In some embodiments, a pre-determined PD-L1 level is a 3-fold, is a 4-fold, is a 5-fold, is a 6-fold, is a 7-fold, is an 8-fold, is a 9-fold or is a 10-fold, increase in a PD-L1 level measured in a sample from the subject relative to a PD-L1 level measured in a sample from the subject before administering a compound (e.g., GCT-007 and / or its derivatives) or composition (e.g., pharmaceutical composition comprising a compound disclosed herein).

[0231] In some embodiments, a pre-determined MHC-II level is a clinically relevant MHC-II level. In some embodiments, a pre-determined MHC-II level is a clinically relevant plasma MHC-II level, immune cell MHC-II level or tumor MHC-II level. In some embodiments, a pre-determined MHC-II level is the lowest MHC-II level at which a beneficial effect (e.g., partial or complete tumor regression) is observed in the subject. In some embodiments, a pre-determined PD-L1 level is a clinically relevant PD-L1 level. In some embodiments, a pre-determined PD-L1 level is a clinically relevant plasma PD-L1 level, immune cell PD-L1 level or tumor PD-L1 level. In some embodiments, a predetermined PD-L1 level is the lowest PD-L1 level at which a beneficial effect (e.g., partial or complete tumor regression) is observed in the subject.

[0232] In some embodiments, a compound (e.g., PI3K inhibitor, GCT-007, etc.) or pharmaceutical composition disclosed herein can also be administered alone or in combination with other compounds. In some embodiment, any PI3K inhibitor can be used in the present invention either alone or in combination with GCT-007 and / or a checkpoint inhibitor. Many are disclosed herein, and additional PI3K inhibitors are disclosed in U. S. Published Patent Application No. 2021 / 0163462; U. S. Patent No.

[0233] 11,492,348; WO 2009 / 066087; WO 2007 / 084786; U. S. Patent No. 8,921,361; each of which is incorporated by reference herein in its entirety.

[0234] In some embodiments, the disclosure provides a compound of Formula (I):

[0235]

[0236] wherein

[0237] R1is -(CRaRb)m-aryl, -CH=CH-aryl, -(CRcRd)n-heteroaryl, -(CReRf)o-heterocycloalkyl, or -(CRgRh)P-cycloalkyl; wherein

[0238] m, n, o, and p are each independently 0, 1, or 2;

[0239] Ra, Rb, Rc, Rd, Re, Rf, Rg, and Rhare each independently H, halo, or Ci-4alkyl, or Raand Rbare taken together with the carbon to which they are attached to form a cycloalkyl ring,

[0240] or Raand Rbare taken together to form =CH2 or =0; each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl present in R1is unsubstituted or substituted with one or two Rxsubstituents;

[0241] wherein each Rxsubstituent is independently halo, Ci-4alkyl, cycloalkyl, -C1-2- haloalkyl, -OH, -OCi-4alkyl, -O-Ci-2-haloalkyl, cyano, -C(O)Ci-4alkyl, - C(O)NRiRj, -SO2Ci-4alkyl, -SO2NRkRl, -NRqRr, -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -CO2Ci-4alkyl, -CO2aryl, - C(O)CH2-aryl (optionally substituted with methyl or halo), -CH2-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)Ci-4alkyl, or -CO2Ci-4alkyl); wherein Ri, Rj, Rk, and Rlare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, or -Ci-4alkyl-O-Ci-4alkyl,

[0242] wherein Rqand Rrare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, -Ci- 4alkyl-O-Ci-4alkyl, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, or -SO2Ci-4alkyl;

[0243] L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or-CRsRt-;

[0244] wherein Rsand Rtare independently H or alkyl, or Rsand Rtare taken together with the carbon atom to which they are attached to form a cycloalkyl ring;

[0245] X is O, S, NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), N(SO2cycloalkyl), or CH2;

[0246] Yi, Y2, and Y3 are each independently CH or N; wherein when L is other than -S(O)2-, Y2 and Y3 are each CH;

[0247] G2 is N or CR2;

[0248] G3 is N or CR3;

[0249] G4is N, NR4b, or CR4a;

[0250] Gs is N or CR5; and

[0251] Ge is N or CR6;

[0252] wherein R2, R3, R4a, R5, and R6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NRURV;

[0253] or R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring; wherein the heteroaryl ring comprising R4band R6comprises no more than one N and is optionally substituted with alkyl, and the heterocyclic ring comprising R4band R6is optionally substituted with oxo,

[0254] Ruis H or Ci-4alkyl; Rvis H, Ci-4alkyl, monocyclic cycloalkyl, -C(O)Ci-4alkyl, or -C(O)NRwRy; wherein each alkyl present in Rvis unsubstituted or substituted with -OH, -NH2, - NH(Ci-4alkyl), or -N(Ci-4alkyl)2,

[0255] Rwand Ryare each independently H or Ci-4alkyl;

[0256] wherein

[0257]

[0258] not unsubstituted phenyl; and

[0259] R7and R8are each independently hydrogen or Ci-4alkyl,

[0260] or R7and R8are taken together to form -CH2CH2-;

[0261] or a pharmaceutically acceptable salt thereof.

[0262] In certain embodiments, the compound of Formula (I) is a compound selected from those species described or exemplified in the detailed description herein.

[0263] In some embodiments of Formula (I) or any variation thereof, R1is -(CRaRb)m-aryl. In some embodiments, R1is (CRcRd)n-heteroaryl. In some embodiments, R1is (CReRf)o-heterocycloalkyl or (CRgRh)P-cycloalkyl.

[0264] In some embodiments of Formula (I) or any variation thereof, L is -S(O)2-. In some embodiments, L is -C(O)-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or -CRsRt-. In other embodiments, L is -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or -CRsRt. In some embodiments, L is absent.

[0265] Provided in other aspects are compounds of Formula (II):

[0266]

[0267] wherein

[0268] X is O, S, NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), N(SO2cycloalkyl), or CH2;

[0269] Yi, Y2, and Y3 are each independently CH or N;

[0270] G2 is N or CR2; Gs is N or CR3;

[0271] G4is N, NR4b, or CR4a;

[0272] Gs is N or CR5; and

[0273] G6is N or CR6;

[0274] wherein R2, R3, R4a, R5, and R6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NRURV;

[0275] or R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring; wherein the heteroaryl ring comprising R4band R6comprises no more than one N and is optionally substituted with alkyl, and the heterocyclic ring comprising R4band R6is optionally substituted with oxo,

[0276] Ruis H or Ci-4alkyl;

[0277] Rvis H, Ci-4alkyl, monocyclic cycloalkyl, -C(O)Ci-4alkyl, or -C(O)NRwRy;

[0278] wherein each alkyl present in Rvis unsubstituted or substituted with -OH, -NH2, - NH(Ci-4alkyl), or -N(Ci-4alkyl)2, and

[0279] Rwand Ryare each independently H or Ci-4alkyl;

[0280] wherein

[0281]

[0282] not unsubstituted phenyl;

[0283] R7and R8are each independently hydrogen or Ci-4alkyl,

[0284] or R7and R8are taken together to form -CH2CH2-;

[0285] R9aand R9bare each independently hydrogen or halogen;

[0286] R10, R11, R12, R13, and R14are each independently hydrogen, halogen, -OH, -CN, -alkyl, - Oalkyl, -haloalkyl, heterocycloalkyl, -O-haloalkyl, -SO2Ci-4alkyl, or -NRaaRbb;

[0287] Raais hydrogen, Ci-4alkyl, or -Ci-4alkyl-OH;

[0288] Rbbis hydrogen or Ci-4alkyl;

[0289] or R9ais taken together with R10and the interposed atoms to form a heteroaryl or heterocyclic ring;

[0290] or R11is taken together with R12and the atoms to which they are attached to form a heteroaryl or heterocyclic ring;

[0291] or a pharmaceutically acceptable salt thereof.

[0292] Provided in other aspects are compounds of Formula (III):

[0293]

[0294] wherein

[0295] R1is-(CRaRb)m-aryl, -CH=CH-aryl, (CRcRd)n-heteroaryl, (CReRf)0- heterocycloalkyl, or (CRgRh)P-cycloalkyl, wherein when L is SO2, the heteroaryl and the heterocycloalkyl present in R1are each monocyclic;

[0296] m is 0 or 2;

[0297] n, o, and p are each independently 0, 1, or 2;

[0298] Ra, Rb, Rc, Rd, Re, Rf, Rg, and Rhare each independently H, halo, or Ci-4alkyl, or Raand Rbare taken together with the carbon to which they are attached to form a cycloalkyl ring,

[0299] or Raand Rbare taken together to form =CH2 or =0;

[0300] each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl present in R1is unsubstituted or substituted with one or two Rxsubstituents;

[0301] wherein each Rxsubstituent is independently halo, Ci-4alkyl, cycloalkyl, -C1-2- haloalkyl, -OH, -OCi-4alkyl, -O-Ci-2-haloalkyl, cyano, -C(O)Ci-4alkyl, - C(O)NRiRj, -SO2Ci-4alkyl, -SO2NRkRl, -NRqRr, -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -CO2Ci-4alkyl, -CO2aryl, - C(O)CH2-aryl (optionally substituted with methyl or halo), -CH2-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)Ci-4alkyl, or -CO2Ci-4alkyl); wherein Ri, Rj, Rk, and Rlare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, or - Ci-4alkyl-O-Ci-4alkyl,

[0302] wherein Rqand Rrare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, -Ci-4alkyl- O-Ci-4alkyl, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, or -SO2Ci-4alkyl;

[0303]

[0304] wherein

[0305] R10, R11, R12, R13, and R14are each independently hydrogen, halogen, -OH, -CN, -alkyl, -Oalkyl, -haloalkyl, heterocycloalkyl, -O-haloalkyl, -SO2Ci-4alkyl, or - NRaaRbb;

[0306] Raais hydrogen, Ci-4alkyl, or -Ci-4alkyl-OH;

[0307] Rbbis hydrogen or Ci-4alkyl;

[0308] or R10is taken together with R11and the atoms to which they are attached to form a heteroaryl or heterocyclic ring;

[0309] or R11is taken together with R12and the atoms to which they are attached to form a heteroaryl or heterocyclic ring;

[0310] L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or CRsRt-; where Rsand Rtare independently H or alkyl, or Rsand Rtare taken together with the carbon atom to which they are attached to form a cycloalkyl ring;

[0311] X is O, S, NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), N(SO2cyclo-alkyl), or CH2;

[0312] Yi, Y2, and Y3 are each independently CH or N; wherein when L is other than -S(O)2-, Y2 and Y3 are each CH;

[0313] G2 is N or CR2;

[0314] G3 is N or CR3;

[0315] G4is N, NR4b, or CR4a;

[0316] Gs is N or CR5; and

[0317] Ge is N or CR6;

[0318] wherein R2, R3, R4a, R5, and R6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NRURV;

[0319] or R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring; wherein the heteroaryl ring comprising R4band R6comprises no more than one N and is optionally substituted with alkyl, and the heterocylic ring comprising R4band R6is optionally substituted with oxo,

[0320] Ruis H or Ci-4alkyl; Rvis H, Ci-4alkyl, monocyclic cycloalkyl, -C(O)Ci-4alkyl, or -C(O)NRwRy; wherein each alkyl present in Rvis unsubstituted or substituted with -OH, -NH2, -NH(Ci-4alkyl), or -N(Ci-4alkyl)2,

[0321] Rwand Ryare independently H or Ci-4alkyl;

[0322] wherein

[0323]

[0324] not unsubstituted phenyl; and

[0325] R7and R8are each independently hydrogen or Ci-4alkyl,

[0326] or R7and R8are taken together to form -CH2CH2-;

[0327] or a pharmaceutically acceptable salt thereof.

[0328] In some embodiments of any of the compounds of Formula (I), (II), or (III), Yi, Y2, and Y3 are each CH. In some embodiments, Yi is N and Y2 and Y3 are each CH. In some embodiments, Y2 is N and Yi and Y3 are each CH. In some embodiments, Y3 is N and Yi and Y2 are each CH.

[0329] In some embodiments of any of the compounds of Formula (I), (II), or (III), X is O. In some embodiments, X is NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), or N(SO2cyclo-alkyl).

[0330] In some embodiments of any of the compounds of Formula (I), (II), or (III), G2 and G4 are each N, and Ge is CR6. In some embodiments of any of the compounds of Formula (I), (II), or (III), G3 is CR3and Gs is CR5. In some embodiments of any of the compounds of Formula (I), (II), or (III), one of G2 and G4 is N.

[0331] In some embodiments of any of the compounds of Formula (I), (II), or (III), R6is -NRURV. In some embodiments of any of the compounds of Formula (I), (II), or (III), R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring.

[0332] In a further aspect, the present disclosure provides a pharmaceutical composition comprising at least one compound of Formula (I), (II), or (III) or a pharmaceutically acceptable salt thereof. Provided in some embodiments is a pharmaceutical composition that contains (a) at least one compound of Formula (I), (II) or (III), or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable excipient.

[0333] The PI3K inhibitors can be administered in combination with other anti-cancer agents such as autophagy inhibitors, checkpoint inhibitors, CAR T cells, or antibodies. The PI3K inhibitors can be administered by any appropriate formulation, dose, route of administration, and regime. Although the PI3K inhibitors of the present invention are preferably administered locally, they can be administered by any appropriate route of administration.

[0334] In some embodiments, a subject may be diagnosed with, or otherwise known to have, a disease or bodily condition associated with cancer, as described herein.

[0335] In some embodiments the PI3K inhibitor is administered to a subject with an autophagy inhibitor. Cells that receive growth factor signals to divide have high energy demands to fuel the process of cell division. Rapidly dividing cells, including tumor cells, use high levels of glucose. Stresses, including too little nutrient, or glucose, or oxidative stresses, will force the dividing cells to change strategies and use other sources of energy, including fat or protein. Rapidly dividing cancer cells are no exception and use glucose at a very high rate, unless stresses like chemotherapy, force the cells to use an alternate strategy to survive. In all these cells, there is an important pathway that senses the availability of glucose and allows fell division to proceed and, reciprocally, senses nutrient deprivation and forces the cell to use another strategy for survival, called autophagy.

[0336] The phosphatidylinositol 3 -kinase (PI3K) pathway, or more broadly the RTK-RAS-PI3K-AKT-mT0R pathway, is the growth and proliferation cascade most frequently activated in glioblastoma (GBM). VPS34 is a phosphoinositide 3-kinase (PI3K) class III isoform that has a key role in autophagy. The phospholipid phosphoinositide (Ptins) is phosphorylated by VPS34 facilitate docking of Akt and initiation of endosomally mediated autophagy.

[0337] In some embodiments the autophagy inhibitor is a Vps34 inhibitor. Vps34 is a lipid kinase that converts phosphatidylinositol (Ptdins) to phosphatidylinositol 3-phosphate (PtdIns3P). GCT-008 is an exemplary inhibitor of VPS34. Thus in some embodiments GCT-008 is used to inhibit autophagy as a survival strategy for GBM that been treated with PI3K inhibitors. In some embodiments the autophagy inhibitor is GCT-008:

[0338]

[0339] Potential therapeutic compounds such as PI3K inhibitors, while often effective in GBM cultures, are not always effective in vivo. Several problems may contribute to lack of effectiveness in vivo. Many compounds have poor brain penetrability. Enhanced PI3K signaling via feedback loops after initial PI3K blockade and GBM cell signaling redundancy may also cause problems.

[0340] GCT-008 is able to avoid these hurdles and provide effective therapy. It has been established that GCT-008 has good brain penetrance. It also is orally bioavailable, a desirable attribute. GCT-008 inhibits VPS34 selectively, and is involved in intracellular trafficking. In some embodiments the GCT-008 is administered by oral administration. The combined administration of GCT-008 and GCT-007, in some embodiments, particularly when GCT-007 is administered first, followed by GCT-008, produces synergistic responses.

[0341] During cell starvation, i.e. when the amino acids or growth factor concentration in the cell environment drops, inhibition of mTORCl alters two downstream pathways: ULK1 and Vps34 resulting in the nucleation of autophagosomes. In some embodiments other Vps34 inhibitors include but are not limited to nucleic acid inhibitors such as antisense oligonucleotides or siRNA targeting exon 4 of Vps34, Spautin-1 (4-[[3,4-(methylenedioxy)benzyl]amino]-6-chloroquinazoline, MBCQ), pyrimidinone catalytic inhibitors of Vps34 and bisaminopyrimidine catalytic inhibitors of Vps34.

[0342] Spautin-1, 4-[[3,4-(methylenedioxy)benzyl]amino]-6-chloroquinazoline (MBCQ) is a specific and potent autophagy inhibitor. It promotes the degradation of Vps34 complexes without affecting the catalytic activity. Because Spautin-1 is able to promote the death of cancer cells under nutrient deprivation, when autophagy is activated, but not in nutrient-rich conditions it appears to be non cytotoxic to normal cells. Several drugs act as catalytic inhibitors of Vps34, such as pyrimidinones and bisaminopyrimidines. Pyrimidinones include SAR405, a highly potent and selective inhibitor of Vps34. SAR405 has a binding equilibrium constant K D of 1.5 nM. SAR405 appears to prevent the catalytic activity of the two Vps34 complexes, Atgl4L- and UVRAG-containing Vps34 complexes. Bisaminopyrimidines include, for instance, VPS34-IN1 and PIK-III.

[0343] In some embodiments the autophagy inhibitor is a fatty acid oxidation inhibitor, ranolazine, di chloroacetate, chloroquine (CQ), hydroxychloroquine (HCQ), or 3-methyladenine (3-MA). In some embodiments the fatty acid oxidation inhibitor is selected from etomoxir, oxfenicine, perhexiline, mildronate (a carnitine biosynthesis inhibitor), trimetazidine, and pFOX.

[0344] Etomoxir, oxfenicine, and perhexiline function as CPT-I (carnitine palmitoyl transferase) inhibitors. CPT-I converts fatty acyl-CoA to fatty acyl-camitine. Mildronate is a Carnitine biosynthesis inhibitor. 3-KAT (3-ketoacyl-coenzyme A thiolase) inhibitors such as trimetazidine directly inhibit fatty acid beta-oxidation. pFOX directly inhibits fatty acid beta-oxidation.

[0345] The PI3K inhibitor and autophagy inhibitor may be administered to the subject at the same time. Alternatively, the PI3K inhibitor may be administered prior to or after the autophagy inhibitor. The administration of the drugs may be repeated multiple times.

[0346] In some embodiments, the PI3K inhibitors are administered with an immune checkpoint modulator. Immune checkpoint modulators include both stimulatory checkpoint molecules and inhibitory checkpoint molecules (checkpoint inhibitor) e.g., an anti-CTLA4, anti-PDl antibody.

[0347] A checkpoint inhibitor is a compound that inhibits a protein in the checkpoint signaling pathway. Proteins in the checkpoint signaling pathway include for example, PD-1, PD-L1, PD-L2, LAG3, TIM3, and CTLA-4. Checkpoint inhibitors are known in the art. For example, the checkpoint inhibitor can be a small molecule. A “small molecule” as used herein, is meant to refer to a composition that has a molecular weight in the range of less than about 5 kD to 50 daltons, for example less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, less than about 1.5 kD, less than about 1 kD, less than 750 daltons, less than 500 daltons, less than about 450 daltons, less than about 400 daltons, less than about 350 daltons, less than 300 daltons, less than 250 daltons, less than about 200 daltons, less than about 150 daltons, less than about 100 daltons. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. The checkpoint inhibitor may be an antibody or antigen binding fragment thereof.

[0348] The invention involves methods for treating a subject. A subject shall mean a human or vertebrate mammal including but not limited to a dog, cat, horse, goat and primate, e.g., monkey. Thus, the invention can also be used to treat diseases or conditions in non-human subjects. Preferably the subject is a human. In some embodiments, the subject is a female subject (e.g., woman). In some embodiments the subject has a cancer. In some embodiments the cancer is GBM.

[0349] Stimulatory checkpoint inhibitors function by promoting the checkpoint process. Several stimulatory checkpoint molecules are members of the tumor necrosis factor (TNF) receptor superfamily - CD27, CD40, 0X40, GITR and CD 137, while others belong to the B7-CD28 superfamily - CD28 and ICOS. 0X40 (CD 134), is involved in the expansion of effector and memory T cells. Anti-OX40 monoclonal antibodies have been shown to be effective in treating advanced cancer. MEDI0562 is a humanized 0X40 agonist. GITR, Glucocorticoid-Induced TNFR family Related gene, is involved in T cell expansion Several antibodies to GITR have been shown to promote an anti-tumor responses. ICOS, Inducible T-cell costimulator, is important in T cell effector function. CD27 supports antigen-specific expansion of naive T cells and is involved in the generation of T and B cell memory. Several agonistic anti-CD27 antibodies are in development. CD122 is the Interleukin-2 receptor beta sub-unit. NKTR-214 is a CD122-biased immune-stimulatory cytokine.

[0350] Inhibitory checkpoint molecules include but are not limited to PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3. CTLA-4, PD-1 and its ligands are members of the CD28-B7 family of co-signaling molecules that play important roles throughout all stages of T-cell function and other cell functions. CTLA-4, Cytotoxic T-Lymphocyte- Associated protein 4 (CD152) is involved in controlling T cell proliferation.

[0351] The PD-1 receptor is expressed on the surface of activated T cells (and B cells) and, under normal circumstances, binds to its ligands (PD-L1 and PD-L2) that are expressed on the surface of antigen-presenting cells, such as dendritic cells or macrophages. This interaction sends a signal into the T cell and inhibits it. Cancer cells take advantage of this system by driving high levels of expression of PD-L1 on their surface. This allows them to gain control of the PD-1 pathway and switch off T cells expressing PD-1 that may enter the tumor microenvironment, thus suppressing the anticancer immune response. Pembrolizumab (formerly MK-3475 and lambrolizumab, trade name Keytruda) is a human antibody used in cancer immunotherapy. It targets the PD-1 receptor.

[0352] IDO, Indoleamine 2,3 -dioxygenase, is a tryptophan catabolic enzyme, which suppresses T and NK cells, generates and activates Tregs and myeloid-derived suppressor cells, and promotes tumor angiogenesis. TIM-3, T-cell Immunoglobulin domain and Mucin domain 3, acts as a negative regulator of Thl / Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, V-domain Ig suppressor of T cell activation.

[0353] The checkpoint inhibitor is a molecule such as a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein or a combination thereof or a small molecule. For instance, the checkpoint inhibitor inhibits a checkpoint protein which may be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or a combination thereof. Ligands of checkpoint proteins include but are not limited to CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands. In some embodiments the anti-PD-1 antibody is BMS-936558 (nivolumab). In other embodiments the anti-CTLA-4 antibody is ipilimumab (trade name Yervoy, formerly known as MDX-010 and MDX-101).

[0354] In some embodiments the checkpoint inhibitor is a targeted therapy. The targeted therapy may be a BRAF inhibitor such as vemurafenib (PLX4032) or dabrafenib. The BRAF inhibitor may be PLX 4032, PLX 4720, PLX 4734, GDC-0879, PLX 4032, PLX-4720, PLX 4734 and Sorafenib Tosylate. BRAF is a human gene that makes a protein called B-Raf, also referred to as proto-oncogene B-Raf and v-Raf murine sarcoma viral oncogene homolog BL The B-Raf protein is involved in sending signals inside cells, which are involved in directing cell growth. Vemurafenib, a BRAF inhibitor, was approved by FDA for treatment of late-stage melanoma.

[0355] The checkpoint inhibitor in other embodiments is an OX40L. 0X40 is a member of the tumor necrosis factor / nerve growth factor receptor (TNFR / NGFR) family. 0X40 may play a role in T-cell activation as well as regulation of differentiation, proliferation or apoptosis of normal and malignant lymphoid cells.

[0356] In some embodiments the PI3K inhibitor is administered to the subject prior to the autophagy inhibitor and the checkpoint inhibitor. In some embodiments the autophagy inhibitor is administered to the subject prior to the checkpoint inhibitor. In some embodiments the checkpoint inhibitor is administered to the subject prior to the autophagy inhibitor. In some embodiments the autophagy inhibitor is administered to the subject at the same time as the checkpoint inhibitor.

[0357] In some embodiments the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 8 hours of the PI3K inhibitor. In some embodiments the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 24 hours of the PI3K inhibitor. In some embodiments the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 1 week of the PI3K inhibitor. In some embodiments the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 1 month of the PI3K inhibitor. In some embodiments the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 6 months of the PI3K inhibitor. In some embodiments the subject is administered at least 2 doses of PI3K inhibitor.

[0358] As used herein, the term treat, treated, or treating when used with respect to a disorder refers to a prophylactic treatment which increases the resistance of a subject to development of the disease or, in other words, decreases the likelihood that the subject will develop the disease as well as a treatment after the subject has developed the disease in order to fight the disease, prevent the disease from becoming worse, or slow the progression of the disease compared to in the absence of the therapy.

[0359] When used in combination with the therapies of the invention the dosages of known therapies may be reduced in some instances, to avoid side effects.

[0360] The PI3K inhibitor can be administered in combination with the checkpoint inhibitors (or other T cell activators) and such administration may be simultaneous or sequential. When the checkpoint inhibitors are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The administration of the checkpoint inhibitors and the PI3K inhibitor can also be temporally separated, meaning that the checkpoint inhibitors are administered at a different time, either before or after, the administration of the PI3K inhibitor. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.

[0361] In some embodiments, a compound or pharmaceutical composition disclosed herein is administered to the subject in an effective amount for treating a disease or disorder such as cancer (e.g., GBM). An “effective amount”, for instance, is an amount necessary or sufficient to realize a desired biologic effect. An effective amount for treating cancer may be an amount sufficient to reduce proliferation rates or growth of a tumor. According to some aspects of the invention, an effective amount is that amount of a compound of the invention alone or in combination with another medicament, which when combined or co-administered or administered alone, results in a therapeutic response to the disease, either in the prevention or the treatment of the disease. The biological effect may be the amelioration and / or absolute elimination of symptoms resulting from the disease. In another embodiment, the biological effect is the complete abrogation of the disease, as evidenced for example, by the absence of a symptom of the disease.

[0362] The effective amount may vary depending upon the specific compound used, the mode of delivery of the compound or pharmaceutical composition, and whether it is used alone or in combination. The effective amount for any particular application can also vary depending on such factors as the disease being treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular molecule of the invention without necessitating undue experimentation.

[0363] Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject. Toxicity and efficacy of the prophylactic and / or therapeutic protocols of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Prophylactic and / or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and / or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0364] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the prophylactic and / or therapeutic agents for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma or lymph fluid (derived from lymphatic tissues, lymph nodes, or the interstitium), concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma or in lymph fluids may be measured, for example, by high performance liquid chromatography.

[0365] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of a compound (e.g., active compound, GCT-007). In other embodiments, a compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.

[0366] Subject doses of a compound or pharmaceutical composition disclosed herein typically range from about 0.1 pg to 10,000 mg, more typically from about 1 pg / day to 8000 mg, and most typically from about 10 pg to 100 pg. Stated in terms of subject body weight, typical dosages range from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above. The absolute amount will depend upon a variety of factors including the concurrent treatment, the number of doses and the individual patient parameters including age, physical condition, size and weight. These are factors well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

[0367] Multiple doses are also contemplated. In some instances, when a compound or pharmaceutical composition disclosed herein is administered with another therapeutic, a sub-therapeutic dosage of either or both of the compound or pharmaceutical composition may be used. A “sub-therapeutic dose” as used herein refers to a dosage which is less than that dosage which would produce a therapeutic result in the subject if administered in the absence of the other agent.

[0368] In some embodiments, a pharmaceutical composition comprises an effective amount of one or more compounds (e.g., GCT-007), dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. The compounds are generally suitable for administration to humans. This term requires that a compound or composition be nontoxic and sufficiently pure so that no further manipulation of the compound or composition is needed prior to administration to humans.

[0369] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences (1990), incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0370] The agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in creams, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences (1990), incorporated herein by reference).

[0371] In any case, the composition may comprise various antioxidants to retard oxidation of one or more components. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0372] In some embodiments, a compound is formulated into a composition (e.g., pharmaceutical composition) in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g, those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

[0373] In embodiments where the composition (e.g., pharmaceutical composition) is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[0374] A compound or composition (e.g., pharmaceutical composition) may be administered directly to a tissue. Direct tissue administration may be achieved by direct injection. The compound or composition (e.g., pharmaceutical composition) may be administered once, or alternatively they may be administered in a plurality of administrations. If administered multiple times, the compounds may be administered via different routes. For example, the first (or the first few) administrations may be made directly into the affected tissue while later administrations may be systemic.

[0375] In some embodiments, the pharmaceutical composition is in a formulation, administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0376] In some embodiments, the compound or composition (e.g., pharmaceutical composition) may be administered in a pharmaceutical composition. In general, a pharmaceutical composition comprises the compound of the invention and a pharmaceutically-acceptable carrier. Pharmaceutically-acceptable carriers for peptides, monoclonal antibodies, and antibody fragments are well-known to those of ordinary skill in the art. As used herein, a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.

[0377] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials which are well-known in the art. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

[0378] A compound or composition (e.g., pharmaceutical composition) may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections, and usual ways for oral, parenteral or surgical administration. The invention also embraces pharmaceutical compositions which are formulated for local administration, such as by implants.

[0379] A composition (e.g., pharmaceutical composition) for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids, such as a syrup, an elixir or an emulsion.

[0380] For oral administration, the compounds can be formulated readily by combining the compound with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.

[0381] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0382] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0383] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0384] For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, di chlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Techniques for preparing aerosol delivery systems are well known to those of skill in the art.

[0385] Generally, such systems should utilize components which will not significantly impair the biological properties of the active agent (see, for example, Sciarra and Cutie, “Aerosols,” in Remington’s Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing aerosols without resort to undue experimentation.

[0386] The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0387] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.

[0388] In yet other embodiments, the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International Application No. PCT / US / 03307 (Publication No. WO 95 / 24929, entitled “Polymeric Gene Delivery System”, claiming priority to U. S. patent application serial no. 213,668, filed March 15, 1994). PCT / US / 0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing a biological macromolecule. The polymeric matrix may be used to achieve sustained release of the agent in a subject. In accordance with one aspect of the instant invention, the agent described herein may be encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT / US / 03307. The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the agent is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the agent include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted. The size of the polymeric matrix device further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and / or pulmonary areas. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the device is administered to a vascular, pulmonary, or other surface. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.

[0389] Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the agents of the invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers. In general, the agents of the invention may be delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), polyethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.

[0390] Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

[0391] Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.

[0392] Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0393] Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compound, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U. S. Patent 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the platelet reducing agent is contained in a form within a matrix such as those described in U. S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U. S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

[0394] Therapeutic formulations of the compounds may be prepared for storage by mixing a compounds having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0395] The compounds may be administered directly to a cell or a subject, such as a human subject alone or with a suitable carrier.

[0396] The following examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.

[0397] EXAMPLES

[0398] Example 1: Synergy between PI3K inhibitors and immune checkpoint inhibitors in treating mouse GBM

[0399] 1. Experiments were conducted to assess the effect of PI3K inhibitors on GBM tumor growth and properties. It was found that PI3K inhibitors, GCT-007, enhance GL261 surface expression of immune response molecules.

[0400] The ability of GCT-007 to upregulate the surface expression of immune response molecules on GBM cells was examined. GL261 cells were treated with different concentrations of GCT-007 for 24 hours and, using flow-cytometry, the surface expression of MHC-II, CD80, CD74, PD-L1, and CD86 was quantified (Fig. 1 A & IB). GCT-007 treatment at >0.1 pg / ml increased the surface expression of all except CD86 (ANOVA with post-hoc Tukey’s HSD). The results suggest that GCT-007 facilitates antigen presentation by GBM cells, promoting the conversion of immunologically cold GBM tumors into hot, immunotherapy-sensitive tumors at a wide range of doses.

[0401] Additionally, GCT-007 and Paxalisib were compared at submaximal concentrations, examining their effects on the surface expression of MHC-II and PD-L1. The intermediate dose of 0.1 pg / ml was selected based on the MHC-II dose-response plot (Fig. 1 A). The GL261 cells were treated with either drug for 24 or 48 hours before flow-cytometry. Quite surprisingly GCT-007 enhanced MHC-II and PD-L1 surface expression to a significantly larger extent than Paxalisib (Fig. 1C-D), suggesting GCT-007 may be more effective than Paxalisib in synergizing with ICIs and may be used at significantly lower doses to achieve similar results.

[0402] 2. Experiments were conducted to demonstrate that GCT-007 synergizes with anti-PD-1 in the treatment of GL261 syngeneic GBM model. The ability to generate synergy between GCT-007 and anti-PD-1 checkpoint inhibitor in treating GBM was tested. A syngeneic GBM mouse model generated by implanting GL261 GBM cells containing a luciferase reporter into the C57BL / 6 mouse brain was used. Using only female mice, 4 treatment arms were tested: Control (saline), GCT-007 (10 mg / kg daily, oral), anti-PD-1 (10 mg / kg every other day, intraperitoneal), and their combination. The mouse brains after 10 days of treatment showed significant GBM growth in all mice except several in the combination group (GCT-007 + anti-PD-1), which also had significantly smaller tumors on average than the other 3 groups (Fig. 2). The results suggest that combining compounds of Formula 1 with immunotherapy would improve the durable response rate in GBM patients.

[0403] 3. GCT-007 was shown to synergize with anti-PD-1 to improve response in a novel GBM mouse model. Many current syngeneic GBM models have limitations. In order to avoid some of these limitations, GCT-007 was tested in a novel native GBM mouse model generated via in utero electroporation (IDE) of Trp53, Pten, and Nfl CRISPR constructs (schematic in Fig. 3A). Performing IUE at embryonic day E14.5 vs E16.5 results in GBM mice with a median survival of -100 days vs -120 days, respectively, similar across male and female mice, which were used equally in our studies. Using the El 6.5 IUE model, the effects of GCT-007 and Paxalisib vs Control (saline) were tested starting treatment at 8 weeks of age and continuing until death (Fig.

[0404] 3 A). It was found that GCT-007 and Paxalisib both significantly prolonged the median survival by over 30 days vs Control (log-rank p <0.05). The treatment paradigm was altered and the mice were treated only between 4 weeks and 8 weeks of age. The results (Fig. 3B) show survival plots (black lines) very similar to the previous paradigm (gray lines), indicating the shorter treatment period is as effective as the longer treatment if started at an earlier age. Combining the overlapping Fig. 3B plots shows that GCT-007 treatment significantly prolongs (log-rank p=0.0002) the survival of the native mouse model (Fig. 3C). Lastly, the more aggressive E14.5 IUE model was used and 4 different arms were tested, treating between 4 weeks and 8 weeks of age (Fig. 3D). Interestingly, while GCT-007 modestly improved GBM survival compared to Control, the addition of anti-PD-1 made a remarkable difference, resulting in prolonged survival of about 50% of the mice. Ultimately, the anti-PD-1 monotherapy group progressed at -400 days and most of the remaining mice died. However, the combination GCT-007 and anti-PD-1 group continued to demonstrate a durable remission beyond 400 days. While the early (-100 days) and late (-400 days) death dichotomy in the anti-PD-1 group were initially perplexing, further analysis discussed below accounting for the sex of the mice provided an unexpected and important explanation for this.

[0405] Example 2:

[0406] While these results in two different GBM mouse models showed improved durable responses when combining GCT-007 and anti-PD-1 treatment, the durable response rate can be further improved by optimizing the combinations of PI3K inhibitors and ICIs as well as their dosage and treatment regimens. Using our native GBM mouse model, the tumor growth and survival of GBM-bearing mice are treated with placebo, PI3K inhibitors, ICIs, and their combinations. Among PI3K inhibitors, GCT-007 and Paxalisib as well as placebo (3 conditions) are tested. Among ICIs, monoclonal antibodies against PD-1, PD-L1, and CTLA-4 in addition to placebo (4 conditions) are tested. Combinations of these treatments will yield 3 x 4 =12 conditions. GCT-007 or Paxalisib will be administered at 10 mg / kg daily (oral), 4 days on and 3 days off, with monitoring for signs of immune toxicity including weight loss and colonic inflammation in euthanized animals. ICI agents are administered every other day at 10 mg / kg via intraperitoneal injection, as in the studies discussed above. The 12 drug combinations are tested in 32 male and 32 female mice starting at 4 weeks of age and continuing for 4 weeks, based on the preliminary results and power calculations.

[0407] Tumor growth is monitored weekly in select groups using bioluminescence imaging. If tumors do not grow or regress with treatment, the bioluminescence imaging frequency will be reduced. Changes in bioluminescence signal over time are compared across groups using repeated measures ANOVA. The mice in each group are monitored for development of symptoms such as seizure or paralysis or generally appearing unhealthy or unkempt and are euthanized when symptomatic. Inflammation is assessed on H& E stains of fixed colonic tissue from euthanized mice as previously described. Survival data will be collected for each condition and sex and subjected to Kaplan-Meier survival analysis with log-rank statistics. Given the results in Figure 3D, mouse survival will be followed for at least 500 days, at which point the proportions of mice with durable responses will be compared against other groups using Fisher’s exact test.

[0408] GCT-007 and Paxalisib were found to enhance the surface expression of immune response molecules on GL261 cells, facilitating “non-professional” antigen presentation by tumor cells. The net effect of PI3K inhibitors on the tumor immune microenvironment is also in favor of an anti-tumor response. While the immune cell proliferation may be generally reduced, CD4+ helper and CD8+ cytotoxic T cells are expected based on the data disclosed herein to mount a more vigorous response against the tumor, particularly in the presence of ICIs. This combination has not been tested previously in GBM. The GBM tumors generated in the 12 combinatorial treatment conditions above will be tested and the tumors characterized by performing expression profiling via bulk RNA sequencing as well as via histology and pathology evaluation.

[0409] At least 10 tumors (5 male and 5 female) from each treatment condition are harvested fresh with the help of the included GFP signal from the brains of euthanized mice and subjected to bulk RNA isolation and RNA sequencing. Tumors from each condition are harvested at 8 weeks (post-treatment) as well as 12 weeks (before most mice become symptomatic from tumors) for comparison. Early tumors are collected at 4 weeks of age (pre-treatment) to obtain the baseline characterization. Fixed slices from at least 3 male and 3 female brains under each condition are subjected to histology and pathology evaluation and IHC for tumor and immune markers and to assess for full tumor regression or the presence of residual or dormant tumor wherever regression is suspected by bioluminescence analysis. RNA sequencing results are processed using the RSubread package and subjected to geneset enrichment analysis (GSEA) in R.

[0410] Enrichment scores between different conditions in male and female mice are compared against one another to look for statistically significant differences. These measures are compared using Student’s t test or ANOVA where multiple conditions are compared.

[0411] Example 3: Sexual dimorphism

[0412] Focusing on the anti-PD-1 arms of the native mouse model results (Fig. 3D) and stratifying the data based on sex showed that anti-PD-1 treatment results in a significant sexual dimorphism, with a prolonged remission response strongly favoring the female sex (Fig. 4A). Importantly, the data suggest that the combination treatment with GCT-007 and anti-PD-1 improves the durable response rate in both male and female mice, resulting in a long tail of survivors (Fig. 4A). Bulk RNA sequencing was used to compare untreated male and female GBM tumors (Fig. 4B), revealing a baseline sexual dimorphism in the expression of checkpoint molecules (PD-1 and CD96) in these tumors, with a higher expression observed in female tumors consistent with the more favorable response to anti-PD-1 treatment in female mice. After 4 weeks of anti-PD-1 treatment, quantification of the dissected tumor weight and tumor burden (taking GFP signal intensity into account) showed significantly smaller tumors in female mice (Fig.

[0413] 4C). In 6-months-old female mice in remission after GCT-007 + anti-PD-1 treatment, brain slices showed cavitation, presumably where the tumor used to be, with a small residual tumor nodule on the periphery (Fig. 4D-E). IHC analysis of the tumor nodule with anti-CD8 antibody showed sparse CD8+ cells in the periphery (Fig. 4F). The brain of a second female mouse in remission showed a cavity without any visible tumor nodules and only background IHC staining with a high anti-CD8 titer (Fig. 4G), suggesting full regression of the tumor. These findings support a role for CD8+ T cell response only in the presence of a viable tumor.

[0414] To further investigate the immune system’s role in the observed sexual dimorphism, circulating immune cells will be isolated and compared before and during treatment, and at remission in each sex. The immune profiles of peripheral (spleen) and tumor-infiltrating immune cells, as well as GFP+ tumor cells from GBM tissue will be compared using flow cytometry. The T-cell receptor (TCR) repertoire will be determined using spectral flow cytometry and single cell RNA sequencing (scRNAseq) and the populations of T cells that expand in response to the treatment or correlate with tumor cell death in vitro or with tumor regression in vivo will be identified.

[0415] A high dimensional spectral flow cytometry and scRNAseq will be used to analyze the frequency and activation, as well as the TCR repertoire of T cells from tumor bearing mice, comparing these measures in the spleen and tumors of ICI responding and non-responding GBM mice. The frequency and activation state of the T cells are correlated with the treatment groups in male and female mice.

[0416] To provide an unbiased comprehensive view of infiltrating T cell activity before and after our treatment protocols, these cells will be isolated and subjected to scRNAseq in the responding and non-responding tumor bearing mice, analyzed with shotgun metagenomics from cryopreserved tissues from the tumor, the dural meninges, and the cervical lymph nodes. This analysis provide data on both transcriptional function and V(D)J (TCR)-repertoire of tumor infiltrating T cells. Using the native model described above, tumors are harvested from 10 mice (5 of each sex) under select treatment conditions, 21 days after treatment initiation. Immune cells are isolated using the Miltenyi Biotec Dead Cell Depletion Kit. CD45+ cells are selected using microbeads. The cells are then stained for CITE-seq analysis using the Biolegend TotalSeq-A labeled MHC-II tetramers as well as antibodies to TCR-yb, CD4, and CD8 to ensure proper cell identification. Single-cell libraries are prepared using 1 OX-Genomics Chromium System and DNA is sequenced at the BCM core facility. ScRNAseq allows for the characterization of the heterogeneity among different T cell subsets and quantifies the differences between these subsets as well as differences between responding and nonresponding mice at the pre- vs. on-treatment time-points. To decrease batch effects and increase the power of these experiments, cell-hashing antibodies are utilized to tag the cells from each mouse, allowing for the multiplexing of mouse samples in single-cell-isolation wells followed by subsequent computational unmixing.

[0417] Example 4: Assess the transferability and durability of the mouse GBM immune response In order to determine if tumor infiltrating immune cells from mice in remission can mediate an effective immune response experiments are undertaken. The question of both in vitro by killing assays and in vivo by adoptive transfer are studied. For both T cells, and their subpopulations including effector and memory T cells are isolated, and propagated in culture using an in vitro system, followed by adoptive transfer into treatment-naive GBM mice, both male and female. To test for a functional memory response, treated mice that are in remission are challenged by implanting new, treatment-naive GBM cells and monitoring for tumor growth or regression.

[0418] For in vitro cultures, T cells are activated using anti-CD3 and anti-CD28, followed by co-culture with GBM cells (technique example in Fig. 5). Live cell are imaged to measure cell death of GBM tumor cells (targets) mixed with different numbers of each of the different mouse T cell populations (effectors). For all treatment groups, pretreated tumor cells are co-cultured with T cells at Effector: Target ratios of 10:1, 5:1, 1:1, 1:5, and 1:10. GFP-expressing GBM cells are incubated with T cells in the presence of a caspase-sensing dye that fluoresces red when caspase 3 or caspase 7 are cleaved during apoptosis. The IncuCyte live cell imaging system is used to measure cell death in GBM cells. In addition, the cell surface expression of MHCI and MHCII molecules, the costimulatory / coinhibitory molecules PD-L1, CD80, CD86, and CD96 will be measured using flow cytometry. The adoptive transfer of T cells into treatment-naive GBM mice, both male and female will be performed to test whether the transferred T cells could effect an immune response in the treatment-naive mice. Untreated syngeneic GBM cells are transplanted from male and female mice into mice in remission at various times after anti-PD-1 treatment. In both experiments, tumor growth and survival is monitored. These experiments will elucidate the transferability of the immune response to mouse GBM as well as the extent and durability of the immune memory in each sex.

[0419] Cell Isolation, Staining, and Flow Cytometry: Single cell suspensions of red cell depleted splenocytes and single lymphocytes isolated from spleen or the tumor are resuspended in PBS / FBS and stained with fluorochrome-conjugated antibodies. All leukocytes and lymphocytes will be identified in all tissues with antibody to CD45. Subpopulations of T cells will be assessed using anti-CD3 or anti-CD90.2, and counterstaining with antibodies to CD4 and CD8, to distinguish T helper (Th) cells from T cytotoxic cells, respectively. Macrophages are identified using antibodies to CD1 lb, CD14, and CD68. B cells are identified by using anti-CD19 or anti-CD20 and counterstaining with antibody to MHC II. Cells are analyzed using Becton Dickinson FACS Aria Flow Cytometer.

[0420] Adoptive Transfer: Adoptive cell transfer will be used to establish the contribution of immune cells, T cells in particular, in GBM regression. Splenic T cells or tumor infiltrating T cells will be isolated from mice before and during treatment with PI3K inhibitors and anti-PD-1. Cells are cultured with anti-CD3 and anti-CD28 to expand T cells and approximately 1 x 106cells are adoptively transferred intraperitoneally into untreated GBM mice.

[0421] Data and statistical analysis plan: Multivariate regression analysis is performed to longitudinally correlate T cell frequency and activation and also perform an exploratory analysis of other genes in the metagenome that correlate with T cell frequency and activation as well as outcomes using the same techniques, but with less statistical power as we will correct for multiple comparisons with the FDR technique. The frequency and activation of T cells will be compared using one-way ANOVAs, correcting for multiple comparisons using Tukey's multiple comparisons test. The survival based on the levels of T cells or using the log-rank test with high vs. low levels is calculated using receiver operator curve (ROC) analysis. Differences in gene expression are confirmed in cryopreserved samples by flow cytometry with appropriate antibodies.

[0422] Example 5: Evaluate the therapeutic potential of combining PI3K inhibitors and ICIs in human GBM

[0423] Given the results in mouse models disclosed herein, it was next sought to determine if GCT-007 exerts similar functions in human GBM. Using U251 human GBM cells, it was found that GCT-007 inhibits their proliferation and increases cell death (Fig. 6A-B) and increases their surface expression of PD-L1 and MHC-II (Fig. 6C-D). Radiation treatment of cells at 4 Gy amplified this effect (Fig. 6E-F) suggesting synergism with GCT-007 in enhancing GBM sensitivity to immunotherapy in human GBM. PI3K inhibitor and ICI combinations are tested in human GBM tumors, both newly diagnosed and recurrent, to assess whether prior radiation synergizes with our treatments to improve the response. Human clinical samples will be accessed by recruiting GBM patients. The effects of PI3K inhibitors and ICI drugs on established human GBM cell lines and on tumor cell suspensions derived from excised human GBM will be compared. Further, the effects of prior treatment, including radiation, comparing tumors of patients with newly diagnosed GBM vs recurrent GBM will by compared. Analysis of tissue / RNA, PI3K pathway markers, tumor immune infiltrate, markers of tumor regression will be performed. Due to clinical differences between male and female patients with GBM and in view of significant identified significant sex-related differences in response to treatment in the mouse model disclosed herein, GBM from male and female patients will be compared to identify sex-specific mechanistic differences and treatments that adequately target the different biological systems and responsiveness to PI3K inhibitors and ICI drugs in each sex.

[0424] Peripheral blood and fresh tumor tissue will be collected from patients with treatment-naive GBM as well as recurrent, previously treated GBM. T cells are isolated from blood samples taken from patients, expanded in numbers via tissue culturing, and stocks frozen down. The effect of PI3K inhibitors on the surface expression of immune molecules on GBM cells will be tested via flow cytometry. Using stocks of T cells expanded from autologous patient samples, whether PI3K inhibitor treatment of GBM cells enhances their recognition and cytotoxicity by autologous immune cells in cocultures will be determined via flow cytometry. Markers of T cell activation flow cytometrically will be measured and tumor cell death will be quantified using Annexin 5, Caspase assays, and / or TUNEL. Further, studies will test whether pretreatment of autologous tumor cells with PI3K inhibitors enhances their interaction with co-cultured T cells in the presence of different immune checkpoint inhibitors. Using the GBM lines derived from treatment naive patients, dose response assays will be performed to determine the optimal dose of PI3K inhibitors and ICIs in monotherapy and combination regimens. In parallel with these flow cytometry assays, ELISAs, testing the same variables, will measure cytokine release from autologous T cells co-cultured with the GBM cells. These studies will evaluate the use of PI3K inhibitors in enhancing the ICI response in human GBM co-cultures and compare their potential in the treatment-naive versus recurrent setting. Example 6: Determine the mechanism by which pretreatment with PI3K inhibitors in combination with immune checkpoint inhibitor drugs leads to tumor cell death.

[0425] The data included herein has shown that increased expression of MHC-II and PD-L1 resulting from treatment with PI3K inhibitors strongly correlates with improved outcomes when used in combination with checkpoint inhibitors in the native GBM mouse model. How expanding autologous human T cells isolated with anti-CD3 and anti-CD28 can be used to enhance the efficacy of currently available ICI drugs (such as anti-PD-1 or anti-CTLA-4) and next generation ICIs (TIGIT / Lag3 / CD73 / CD47) following treatment with PI3K inhibitors will be further examined. The impact of these novel therapeutic approaches in human GBM cells in vitro, using patient GBM cells and T cells derived from either autologous PBMC or from isolated tumor infiltrating T cells will be tested. While human T cells can kill other types of tumor cells, the ability of autologous T cells to directly kill GBM cells in a subject will be explored.

[0426] Live cell imaging is used to measure cell death of tumor cells (targets) mixed with different numbers of each of the different human T cell populations (effectors). For all treatment groups, PI3K-inhibitor pretreated tumor cells are incubated with T cells at EffectorTarget ratios of 10:1, 5:1, 1:1, 1:5, and 1:10. GFP-expressing tumor cells are incubated with T cells in the presence of a caspase-sensing dye that fluoresces red when caspase 3 or caspase 7 are cleaved during apoptosis MHC Class II protein expression is tightly controlled and cell surface expression is dependent on treatment with the PI3K inhibitor. Cell surface MHC Class II expression can be increased by pretreating cells with GCT-007. The cell surface expression of MHC -I and MHC-II on the GBM cells will be characterized following treatment with GCT-007 or Paxalisib using flow cytometry. Patient-derived GBM cell lines will be incubated with 5 or 10 pg of GCT-007 or Paxalisib prior to seeding into 96-well IncuCyte plates. To block MHC: T cell interactions, performing these assays in the presence of an MHC Class I or MHC Class II blocking-blocking antibody. To determine if blocking PD-1 alters the killing capacity of T cells, performing these experiments in the presence or absence of anti-PD-1. To determine if antigen presenting cells (APCs) are required to prime T cells for killing the GBM cells, GCT-007 pulsed PBMC-derived B cells and monocyte-derived dendritic cells (moDCs) will be incubated with the T cells prior to the killing assays. B cells are isolated from PBMCs using the Miltenyi Biotec B cell isolation kit. Dendritic cells are generated by culturing monocytes isolated from PBMCs using the Miltenyi Biotec monocyte purification kit in vitro for 7 days with 500 U / ml IL-4 and 250 U / ml GM- CSF.94’95T cells (1×106) will be incubated with either GCT-007 or control (PBS) loaded B cells or moDCs (1×105) for 48 hours prior to the killing assays.

[0427] Example 7: Use of PI3K inhibitors in combination with autophagy inhibitors.

[0428] An assay was performed to determine the selectivity profile of several compounds on vsp34, PI3Ka, mTor, pAKT and cell viability. The compounds tested included NPT520-322 (GCT-008), NPT520-319, NPT520-318, PIK-93 and NVP-19. Structures for NPT520-322, PIK-93 and NVP-19 are shown below. The data is shown in Fig. 7.

[0429]

[0430] A titration assay was performed on GCT-007 alone, GCT-008 alone, and various combinations of GCT-007 and GCT-008. The data was measured as the percent change in cellular viability relative to a vehicle control and the results are shown in graph form in Figs. 8A and 8B and in Table A. Table A

[0431]

[0432] Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

[0433] Embodiments

[0434] In some embodiments the invention is: Embodiment 1. A method of treating glioblastoma (GBM) in a subject using a sub-maximum tolerated dose (sub-MTD) of a pharmaceutical composition comprising:

[0435] administering to a subject having GBM a checkpoint inhibitor and a pharmaceutical composition comprising a compound of Formula (I) administered in a sub-MTD to treat the GBM in the subject:

[0436]

[0437] wherein

[0438] R1is -(CRaRb)m-aryl, -CH=CH-aryl, -(CRcRd)n-heteroaryl, -(CReRf)o-heterocycloalkyl, or -(CRgRh)P-cycloalkyl; wherein

[0439] m, n, o, and p are each independently 0, 1, or 2;

[0440] Ra, Rb, Rc, Rd, Re, Rf, Rg, and Rhare each independently H, halo, or Ci-4alkyl, or Raand Rbare taken together with the carbon to which they are attached to form a cycloalkyl ring,

[0441] or Raand Rbare taken together to form =CH2 or =0;

[0442] each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl present in R1is unsubstituted or substituted with one or two Rxsubstituents;

[0443] wherein each Rxsubstituent is independently halo, Ci-4alkyl, cycloalkyl, -C1-2- haloalkyl, -OH, -OCi-4alkyl, -O-Ci-2-haloalkyl, cyano, -C(O)Ci-4alkyl, - C(O)NRiRj, -SO2Ci-4alkyl, -SO2NRkRl, -NRqRr, -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -CO2Ci-4alkyl, -CO2aryl, - C(O)CH2-aryl (optionally substituted with methyl or halo), -CH2-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)Ci-4alkyl, or -CO2Ci-4alkyl); wherein Ri, Rj, Rk, and Rlare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, or -Ci-4alkyl-O-Ci-4alkyl, wherein Rqand Rrare each independently H, Ci-4alkyl, -Ci-4alkyl-0H, -Ci- 4alkyl-O-Ci-4alkyl, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, or -SO2Ci-4alkyl;

[0444] L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or CRsRt-;

[0445] wherein Rsand Rtare independently H or alkyl, or Rsand Rtare taken together with the carbon atom to which they are attached to form a cycloalkyl ring;

[0446] X is O, S, NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), N(SO2cycloalkyl), or CH2;

[0447] Yi, Y2, and Y3 are each independently CH or N; wherein when L is other than -S(O)2-, Y2and Y3 are each CH;

[0448] G2is N or CR2;

[0449] G3 is N or CR3;

[0450] G4is N, NR4b, or CR4a;

[0451] Gs is N or CR5; and

[0452] Ge is N or CR6;

[0453] wherein R2, R3, R4a, R5, and R6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NRURV;

[0454] or R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring; wherein the heteroaryl ring comprising R4band R6comprises no more than one N and is optionally substituted with alkyl, and the heterocyclic ring comprising R4band R6is optionally substituted with oxo,

[0455] Ruis H or Ci-4alkyl;

[0456] Rvis H, Ci-4alkyl, monocyclic cycloalkyl, -C(O)Ci-4alkyl, or -C(O)NRwRy;

[0457] wherein each alkyl present in Rvis unsubstituted or substituted with -OH, -NH2, - NH(Ci-4alkyl), or -N(Ci-4alkyl)2,

[0458] Rwand Ryare each independently H or Ci-4alkyl;

[0459] wherein

[0460]

[0461] not unsubstituted phenyl; and

[0462] R7and R8are each independently hydrogen or Ci-4alkyl,

[0463] or R7and R8are taken together to form -CH2CH2-;

[0464] or a pharmaceutically acceptable salt thereof. Embodiment 2. The method of embodiment 1, wherein the sub-MTD of a compound of Formula (I) is less than 30 mg / day; optionally, the sub-MTD is 0.1 - 30, 1 - 25, 1 - 10, 1 - 5 or 5 - 10 mg / day.

[0465] Embodiment 3. The method of embodiment 1, wherein the sub-MTD of a compound of Formula (I) is 1 - 10 mg / day.

[0466] Embodiment 4. The method of any one of embodiments 1-3, wherein the compound comprises the structure:

[0467]

[0468] ; or salts or combinations thereof.

[0469] Embodiment 5. The method of any one of embodiments 1-3, wherein the compound is GCT-007.

[0470] Embodiment 6. A method of treating glioblastoma (GBM) in a subject using intermittent doses of a pharmaceutical composition comprising:

[0471] administering to a subject having GBM a checkpoint inhibitor, and intermittent doses of a pharmaceutical composition comprising a compound of Formula (I) for at least 2 consecutive days, followed by no administration of the pharmaceutical composition comprising a compound of Formula (I) for at least 2 consecutive days, to treat the GBM in the subject: wherein Formula (I) comprises the structure of Embodiment 1).

[0472] Embodiment 7. The method of embodiment 6, wherein the intermittent doses of a pharmaceutical composition comprising a compound of Formula (I) is for at least 4 consecutive days, followed by no administration of the pharmaceutical composition comprising a compound of Formula (I) for at least 3 consecutive days; optionally, the intermittent doses of a pharmaceutical composition comprising a compound of Formula (I) is for at least 5 consecutive days, followed by no administration of the pharmaceutical composition comprising a compound of Formula (I) for at least 4 consecutive days. Embodiment 8. A method of treating glioblastoma (GBM) in a female subject by rendering the GBM susceptible to a programmed cell death protein 1 (PD1) inhibitor and / or a CD96 inhibitor, comprising:

[0473] (i) measuring a first PD1 level in a first sample from a female subject and determining if the first PD1 level is below a pre-determined PD1 level and / or measuring a first CD96 level in a first sample from a female subject and determining if the first CD96 level is below a pre-determined CD96 level,

[0474] wherein

[0475] (a) if the first PD1 level and / or the first CD96 level is below a predetermined PD1 level and / or a pre-determined CD96 level, administering to the female subject in (i) a phosphoinositide 3 -kinase (PI3K) inhibitor for a sufficient time to increase the first PD1 level to or above the pre-determined PD1 level and / or the first CD96 level to or above the pre-determined CD96 level; or (b) if the first PD1 level and / or the first CD96 level is at or above a predetermined PD1 level and / or a pre-determined CD96 level, administering to the female subject in (i) a first PD1 inhibitor and / or a first CD96 inhibitor to treat the GBM in the female subject.

[0476] Embodiment 9. The method of embodiment 8, further comprising:

[0477] (ii) measuring a second PD1 level in a second sample from the female subject in (a) and determining the second PD1 level and / or measuring a second CD96 level in the second sample from the female subject in (a), and

[0478] (iii) if the second PD1 level in the second sample from the female subject in (ii) is at or above the pre-determined PD1 level and / or the second CD96 level in the second sample from the female subject in (ii) is at or above the pre-determined CD96 level, administering a second PD1 inhibitor and / or a second CD96 inhibitor to treat the GBM in the female subject.

[0479] Embodiment 10. The method of embodiment 9, further comprising repeating (i)(a) - (ii) to produce in the subject a third PD1 level at or above the pre-determined PD1 level and / or a third CD96 level at or above the pre-determined CD96 level.

[0480] Embodiment 11. The method of any one of embodiments 8-10, wherein the first sample, the second sample or both the first sample and the second sample is a population of GBM tumor cells. Embodiment 12. The method of any one of embodiments 1-11, wherein the compound comprises the structure:

[0481]

[0482] ; or salts or combinations thereof.

[0483] Embodiment 13. The method of any one of embodiments 8-12, wherein PI3K inhibitor is GCT-007:

[0484]

[0485] Embodiment 14. The method of any one of embodiments 1-13, wherein the PI3K inhibitor or the pharmaceutical composition is administered to the subject systemically.

[0486] Embodiment 15. The method of any one of embodiments 1-14, wherein the subject is treated with the checkpoint inhibitor within 8 hours of the PI3K inhibitor or the pharmaceutical composition.

[0487] Embodiment 16. The method of any one of embodiments 1-14, wherein the subject is treated with the checkpoint inhibitor within 24 hours of the PI3K inhibitor or the pharmaceutical composition.

[0488] Embodiment 17. The method of any one of embodiments 1-14, wherein the subject is treated with the checkpoint inhibitor within 1 week of the PI3K inhibitor or the pharmaceutical composition.

[0489] Embodiment 18. The method of any one of embodiments 1-14, wherein the subject is treated with the checkpoint inhibitor within 1 month of the PI3K inhibitor or the pharmaceutical composition. Embodiment 19. The method of any one of embodiments 1-14, wherein the subject is treated with the checkpoint inhibitor within 6 months of the PI3K inhibitor or the pharmaceutical composition.

[0490] Embodiment 20. The method of any one of embodiments 1-19, wherein the subject is administered at least 2 doses of PI3K inhibitor or the pharmaceutical composition.

[0491] Embodiment 21. The method of any one of embodiments 1-20, wherein the checkpoint inhibitor is an antibody selected from an anti-PDl antibody or antigenbinding fragment thereof that specifically binds PD1, an anti-PD-Ll antibody or antigenbinding fragment thereof that specifically binds PD-L1, and a combination thereof.

[0492] Embodiment 22. The method of any one of embodiments 1-20, wherein the checkpoint inhibitor or PD1 inhibitor is pembrolizumab, BMS-936558 (nivolumab), REGN-2810 (cemiplimab), orPDROOl (spartalizumab).

[0493] Embodiment 23. The method of any one of embodiments 1-21, wherein the checkpoint inhibitor is an anti-PD-Ll antibody selected from atezolizumab, avelumab, or durvalumab.

[0494] Embodiment 24. The method of any one of embodiments 8-21, wherein the CD96 inhibitor is an antibody.

[0495] Embodiment 25. The method of any one of embodiments 1-24, wherein the GBM is an isocitrate dehydrogenase (IDH) mutant GBM.

[0496] Embodiment 26. The method of any one of embodiments 1-24, wherein the GBM is an IDH-wildtype GBM.

[0497] Embodiment 27. The method of any one of embodiments 1-24, wherein the GBM is characterized by one or more of increased epidermal growth factor receptor (EGFR) amplification relative to a GBM without EGFR amplification, a telomerase reverse transcriptase (TERT)-promoter mutation, or a combined gain of chromosome7 / loss of chromosome 10 copy number changes.

[0498] Embodiment 28. The method of any one of embodiments 1-24, wherein the GBM is characterized by hypermethylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter.

[0499] Embodiment 29. The method of any one of embodiments 1-24, wherein the GBM is characterized by a BRAFv600 mutation, a fibroblast growth factor receptor (FGFR) mutation, a FGFR-TACC gene fusion, a H3K27M mutation, dysregulated CDK4 / 6 and / or dysregulated tumor protein p53 (TP53).

[0500] Embodiment 30. The method of any one of embodiments 1-29, further comprising administering at least one additional treatment; optionally, wherein the at least one additional treatment is chemotherapy, radiation, surgery, administration of temozolomide, or combinations thereof.

[0501] Embodiment 31. The method of any one of embodiments 1-30, wherein the subject is a mammal.

[0502] Embodiment 32. The method of any one of embodiments 1-30, wherein the subject is a human.

[0503] Embodiment 33. A method for treating glioblastoma (GBM) in a subject comprising:

[0504] administering to a subject having GBM an effective amount to treat the GBM in the subject an autophagy inhibitor and a PI3K inhibitor comprising a compound of Formula (I):_wherein Formula (I) comprises the structure of Embodiment 1).

[0505] Embodiment 34. The method of embodiment 33, wherein the PI3K inhibitor comprises the structure of embodiment 12.

[0506] Embodiment 35. The method of any one of embodiments 33-34, wherein the compound is GCT-007 or salts thereof.

[0507] Embodiment 36. The method of any one of embodiments 33-35, wherein the autophagy inhibitor is a Vps34 inhibitor.

[0508] Embodiment 37. The method of any one of embodiments 33-36, wherein the autophagy inhibitor is:

[0509]

[0510] Embodiment 38. The method of embodiment 36, wherein the Vps34 inhibitor is selected from nucleic acid inhibitors such as antisense oligonucleotides or siRNA targeting exon 4 of Vps34, Spautin-1 (4-[[3,4-(methylenedioxy)benzyl]amino]-6- chloroquinazoline, MBCQ), pyrimidinone catalytic inhibitors of Vps34 and bisaminopyrimidine catalytic inhibitors of Vps34.

[0511] Embodiment 39. The method of embodiment 33, wherein the autophagy inhibitor is a fatty acid oxidation inhibitor, ranolazine, dichloroacetate, chloroquine (CQ), hydroxychloroquine (HCQ), or 3 -methyladenine (3 -MA).

[0512] Embodiment 40. The method of embodiment 39, wherein the fatty acid oxidation inhibitor is selected from etomoxir, oxfenicine, perhexiline, mildronate (a carnitine biosynthesis inhibitor), trimetazidine, and pFOX.

[0513] Embodiment 41. The method of any one of embodiments 33-40, wherein the PI3K inhibitor is administered to the subject prior to the autophagy inhibitor.

[0514] Embodiment 42. The method of any one of embodiments 33-41, further comprising administering to the subject a checkpoint inhibitor.

[0515] Embodiment 43. The method of embodiment 42, wherein the PI3K inhibitor is administered to the subject prior to the autophagy inhibitor and the checkpoint inhibitor.

[0516] Embodiment 44. The method of embodiment 43, wherein the autophagy inhibitor is administered to the subject prior to the checkpoint inhibitor.

[0517] Embodiment 45. The method of embodiment 43, wherein the checkpoint inhibitor is administered to the subject prior to the autophagy inhibitor.

[0518] Embodiment 46. The method of embodiment 43, wherein the autophagy inhibitor is administered to the subject at the same time as the checkpoint inhibitor.

[0519] Embodiment 47. The method of any one of embodiments 33-41, wherein 1 - 10 mg / day of the PI3K inhibitor is administered to the subject.

[0520] Embodiment 48. The method of any one of embodiments 33-47, wherein the PI3K inhibitor and / or the autophagy inhibitor is administered to the subject systemically.

[0521] Embodiment 49. The method of any one of embodiments 33-47, wherein the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 8 hours of the PI3K inhibitor.

[0522] Embodiment 50. The method of any one of embodiments 33-47, wherein the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 24 hours of the PI3K inhibitor. Embodiment 51. The method of any one of embodiments 33-47, wherein the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 1 week of the PI3K inhibitor.

[0523] Embodiment 52. The method of any one of embodiments 33-47, wherein the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 1 month of the PI3K inhibitor.

[0524] Embodiment 53. The method of any one of embodiments 33-47, wherein the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 6 months of the PI3K inhibitor.

[0525] Embodiment 54. The method of any one of embodiments 33-47, wherein the subject is administered at least 2 doses of PI3K inhibitor.

[0526] Embodiment 55. The method of any one of embodiments 42-54, wherein the checkpoint inhibitor is an antibody selected from an anti-PDl antibody or antigenbinding fragment thereof that specifically binds PD1, an anti-PD-Ll antibody or antigenbinding fragment thereof that specifically binds PD-L1, and a combination thereof.

[0527] 56. The method of any one of embodiments 42-54, wherein the checkpoint inhibitor orPDl inhibitor is pembrolizumab, BMS-936558 (nivolumab), REGN-2810 (cemiplimab), orPDROOl (spartalizumab).

[0528] Embodiment 57. The method of any one of embodiments 42-54, wherein the checkpoint inhibitor is an anti-PD-Ll antibody selected from atezolizumab, avelumab, or durvalumab.

[0529] Embodiment 58. The method of any one of embodiments 33-57, wherein the GBM is an isocitrate dehydrogenase (IDH) mutant GBM.

[0530] Embodiment 59. The method of any one of embodiments 33-57, wherein the GBM is an IDH-wildtype GBM.

[0531] Embodiment 60. The method of any one of embodiments 33-57, wherein the GBM is characterized by one or more of increased epidermal growth factor receptor (EGFR) amplification relative to a GBM without EGFR amplification, a telomerase reverse transcriptase (TERT)-promoter mutation, or a combined gain of chromosome7 / loss of chromosome 10 copy number changes. Embodiment 61. The method of any one of embodiments 33-57, wherein the GBM is characterized by hypermethylation of the 0-6-methylguanine-DNA methyltransferase (MGMT) promoter.

[0532] Embodiment 62. The method of any one of embodiments 33-57, wherein the GBM is characterized by a BRAFv600 mutation, a fibroblast growth factor receptor (FGFR) mutation, a FGFR-TACC gene fusion, a H3K27M mutation, dysregulated CDK4 / 6 and / or dysregulated tumor protein p53 (TP53).

[0533] Embodiment 63. A pharmaceutical composition comprising a checkpoint inhibitor and an autophagy inhibitor comprising:

[0534]

Claims

1. CLAIMS2.What is claimed is:

1. A method of treating glioblastoma (GBM) in a subject using a sub-maximum tolerated dose (sub-MTD) or intermittent doses of a phosphoinositide 3 -kinase (PI3K) inhibitor comprising:4.administering to a subject having GBM a checkpoint inhibitor and5.a) a PI3K inhibitor comprising a compound of Formula (I) administered in a sub-MTD to treat the GBM in the subject, or6.b) intermittent doses of a PI3K inhibitor comprising the compound of Formula (I) for at least 2 consecutive days, followed by no administration of the PI3K inhibitor comprising the compound of Formula (I) for at least 2 consecutive days, to treat the GBM in the subject7.wherein the compound of Formula (I) comprises:

9. 11.wherein R1is -(CRaRb)m-aryl, -CH=CH-aryl, -(CRcRd)n-heteroaryl, -(CReRf)0-heterocycloalkyl, or -(CRgRh)P-cycloalkyl; wherein m, n, o, and p are each independently 0, 1, or 2;12.Ra, Rb, Rc, Rd, Re, Rf, Rg, and Rhare each independently H, halo, or Ci-4alkyl, or Raand Rbare taken together with the carbon to which they are attached to form a cycloalkyl ring,13.or Raand Rbare taken together to form =CH2 or =0;14.each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl present in R1is unsubstituted or substituted with one or two Rxsubstituents;15.wherein each Rxsubstituent is independently halo, Ci-4alkyl, cycloalkyl, -C1-2- haloalkyl, -OH, -OCi-4alkyl, -O-Ci-2-haloalkyl, cyano, -C(O)Ci-4alkyl, - C(O)NRiRj, -SO2Ci-4alkyl, -SO2NRkRl, -NRqRr, -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -CO2Ci-4alkyl, -CCharyl, - C(O)CH2-aryl (optionally substituted with methyl or halo), -Cbfc-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)Ci-4alkyl, or -CO2Ci-4alkyl); wherein Ri, Rj, Rk, and Rlare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, or -Ci-4alkyl-O-Ci-4alkyl,16.wherein Rqand Rrare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, -Ci- 4alkyl-O-Ci-4alkyl, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, or -SO2Ci-4alkyl;17.L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or-CRsRt-;18.wherein Rsand Rtare independently H or alkyl, or Rsand Rtare taken together with the carbon atom to which they are attached to form a cycloalkyl ring;19.X is O, S, NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), N(SO2cycloalkyl), or CH2;20.Yi, Y2, and Y3 are each independently CH or N; wherein when L is other than -S(O)2-, Y2 and Y3 are each CH;21.G2 is N or CR2;22.G3 is N or CR3;23.G4is N, NR4b, or CR4a;24.Gs is N or CR5; and25.Ge is N or CR6;26.wherein R2, R3, R4a, R5, and R6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NRURV;27.or R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring; wherein the heteroaryl ring comprising R4band R6comprises no more than one N and is optionally substituted with alkyl, and the heterocyclic ring comprising R4band R6is optionally substituted with oxo,28.Ruis H or Ci-4alkyl;29.Rvis H, Ci-4alkyl, monocyclic cycloalkyl, -C(O)Ci-4alkyl, or -C(O)NRwRy;30.wherein each alkyl present in Rvis unsubstituted or substituted with -OH, -NH2, - NH(Ci-4alkyl), or -N(Ci-4alkyl)2,31.Rwand Ryare each independently H or Ci-4alkyl;33.wherein34.

35. not unsubstituted phenyl; and36.R7and R8are each independently hydrogen or Ci-4alkyl,37.or R7and R8are taken together to form -CH2CH2-;38.or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the sub-MTD of the PI3K inhibitor is less than 30 mg / day; optionally, the sub-MTD is 0.1 - 30, 1 - 25, 1 - 10, 1 - 5 or 5 - 10 mg / day, and optionally wherein the sub-MTD of the PI3K inhibitor is 1 - 10 mg / day.

3. The method of claim 1, wherein the intermittent doses of the PI3K inhibitor is for at least 4 consecutive days, followed by no administration of the PI3K inhibitor for at least 3 consecutive days; optionally, the intermittent doses of the PI3K inhibitor is for at least 5 consecutive days, followed by no administration of the PI3K inhibitor for at least 4 consecutive days.

4. A method of treating glioblastoma (GBM) in a female subject by rendering the GBM susceptible to a programmed cell death protein 1 (PD1) inhibitor and / or a CD96 inhibitor, comprising:42.(i) measuring a first PD1 level in a first sample from a female subject and determining if the first PD1 level is below a pre-determined PD1 level and / or measuring a first CD96 level in a first sample from a female subject and determining if the first CD96 level is below a pre-determined CD96 level,43.wherein44.(a) if the first PD1 level and / or the first CD96 level is below a predetermined PD1 level and / or a pre-determined CD96 level, administering to the female subject in (i) a phosphoinositide 3 -kinase (PI3K) inhibitor for a sufficient time to increase the first PD1 level to or above the pre-determined PD1 level and / or the first CD96 level to or above the pre-determined CD96 level; or (b) if the first PD1 level and / or the first CD96 level is at or above a predetermined PD1 level and / or a pre-determined CD96 level, administering to the female subject in (i) a first PD1 inhibitor and / or a first CD96 inhibitor to treat the GBM in the female subject, and optionally, further comprising: (ii) measuring a second PD1 level in a second sample from the female subject in (a) and determining the second PD1 level and / or measuring a second CD96 level in the second sample from the female subject in (a), and45.(iii) if the second PD1 level in the second sample from the female subject in (ii) is at or above the pre-determined PD1 level and / or the second CD96 level in the second sample from the female subject in (ii) is at or above the pre-determined CD96 level, administering a second PD1 inhibitor and / or a second CD96 inhibitor to treat the GBM in the female subject, and optionally further comprising repeating (i)(a) - (ii) to produce in the subject a third PD1 level at or above the pre-determined PD1 level and / or a third CD96 level at or above the pre-determined CD96 level, and optionally wherein the CD96 inhibitor is an antibody.

5. The method of claim 4, wherein the first sample, the second sample or both the first sample and the second sample is a population of GBM tumor cells.

6. The method of any one of claims 1-5, wherein the PI3K inhibitor comprises the structure:

49.

7. The method of any one of claims 1-5, wherein PI3K inhibitor is GCT-007:

53.

54. salts thereof.

8. The method of any one of claims 1-7, wherein the PI3K inhibitor is administered to the subject systemically.

9. The method of any one of claims 1-8, wherein the subject is treated with the checkpoint inhibitor within 8 hours, within 24 hours, within 1 week, within 1 month or within 6 months of the PI3K inhibitor.

10. The method of any one of claims 1-9, wherein the subject is administered at least 2 doses of PI3K inhibitor or the pharmaceutical composition.

11. The method of any one of claims 1-10, wherein the checkpoint inhibitor is an antibody selected from an anti-PDl antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-Ll antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof, optionally wherein the checkpoint inhibitor is an anti-PD-Ll antibody selected from atezolizumab, avelumab, or durvalumab.

12. The method of any one of claims 1-10, wherein the checkpoint inhibitor or PD1 inhibitor is pembrolizumab, BMS-936558 (nivolumab), REGN-2810 (cemiplimab), orPDROOl (spartalizumab).

13. The method of any one of claims 1-12, wherein the GBM is an isocitrate dehydrogenase (IDH) mutant GBM or an IDH-wildtype GBM.

14. The method of any one of claims 1-12, wherein the GBM is characterized by one or more of61.a) increased epidermal growth factor receptor (EGFR) amplification relative to a GBM without EGFR amplification, a telomerase reverse transcriptase (TERT)-promoter mutation, or a combined gain of chromosome7 / loss of chromosome 10 copy number changes,62.b) hypermethylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter, or63.c) a BRAFv600 mutation, a fibroblast growth factor receptor (FGFR) mutation, a FGFR-TACC gene fusion, a H3K27M mutation, dysregulated CDK4 / 6 and / or dysregulated tumor protein p53 (TP53).

15. The method of any one of claims 1-14, further comprising administering at least one additional treatment; optionally, wherein the at least one additional treatment ischemotherapy, radiation, surgery, administration of temozolomide, or combinations thereof.

16. A method for treating glioblastoma (GBM) in a subject comprising: administering to a subject having GBM an effective amount to treat the GBM in the subject an autophagy inhibitor and a PI3K inhibitor comprising a compound of Formula (I):

67. 69.wherein70.R1is -(CRaRb)m-aryl, -CH=CH-aryl, -(CRcRd)n-heteroaryl, -(CReRf)o-heterocycloalkyl, or -(CRgRh)P-cycloalkyl; wherein71.m, n, o, and p are each independently 0, 1, or 2;72.Ra, Rb, Rc, Rd, Re, Rf, Rg, and Rhare each independently H, halo, or Ci-4alkyl, or Raand Rbare taken together with the carbon to which they are attached to form a cycloalkyl ring,73.or Raand Rbare taken together to form =CH2 or =0;74.each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl present in R1is unsubstituted or substituted with one or two Rxsubstituents;75.wherein each Rxsubstituent is independently halo, Ci-4alkyl, cycloalkyl, -C1-2- haloalkyl, -OH, -OCi-4alkyl, -O-Ci-2-haloalkyl, cyano, -C(O)Ci-4alkyl, - C(O)NRiRj, -SO2Ci-4alkyl, -SO2NRkRl, -NRqRr, -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -CO2Ci-4alkyl, -CO2aryl, - C(O)CH2-aryl (optionally substituted with methyl or halo), -CH2-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)Ci-4alkyl, or -CO2Ci-4alkyl); wherein Ri, Rj, Rk, and Rlare each independently H, Ci-4alkyl, -Ci-4alkyl-OH, or -Ci-4alkyl-O-Ci-4alkyl, wherein Rqand Rrare each independently H, Ci-4alkyl, -Ci-4alkyl-0H, -Ci- 4alkyl-O-Ci-4alkyl, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, or -SO2Ci-4alkyl;76.L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or CRsRt-;77.wherein Rsand Rtare independently H or alkyl, or Rsand Rtare taken together with the carbon atom to which they are attached to form a cycloalkyl ring;78.X is O, S, NH, N(CO2Ci-4alkyl), N(SO2Ci-4alkyl), N(SO2cycloalkyl), or CH2;79.Yi, Y2, and Y3 are each independently CH or N; wherein when L is other than -S(O)2-, Y2and Y3 are each CH;80.G2is N or CR2;81.G3 is N or CR3;82.G4is N, NR4b, or CR4a;83.Gs is N or CR5; and84.Ge is N or CR6;85.wherein R2, R3, R4a, R5, and R6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NRURV;86.or R4bis taken together with R6and the atoms to which they are attached to form a heteroaryl or heterocyclic ring; wherein the heteroaryl ring comprising R4band R6comprises no more than one N and is optionally substituted with alkyl, and the heterocyclic ring comprising R4band R6is optionally substituted with oxo,87.Ruis H or Ci-4alkyl;88.Rvis H, Ci-4alkyl, monocyclic cycloalkyl, -C(O)Ci-4alkyl, or -C(O)NRwRy;89.wherein each alkyl present in Rvis unsubstituted or substituted with -OH, -NH2, - NH(Ci-4alkyl), or -N(Ci-4alkyl)2,90.Rwand Ryare each independently H or Ci-4alkyl;93.wherein94.

95. not unsubstituted phenyl; and96.R7and R8are each independently hydrogen or Ci-4alkyl,97.or R7and R8are taken together to form -CH2CH2-;98.or a pharmaceutically acceptable salt thereof.

17. The method of claim 16, wherein the PI3K inhibitor comprises the structure:

100.

18. The method of claim 16, wherein the compound is GCT-007:

104.

105. salts thereof.

19. The method of any one of claims 16-18, wherein the autophagy inhibitor is: a) a Vps34 inhibitor, optionally wherein the Vps34 inhibitor is selected from nucleic acid inhibitors such as antisense oligonucleotides or siRNA targeting exon 4 of Vps34, Spautin-1 (4-[[3,4-(methylenedioxy)benzyl]amino]-6-chloroquinazoline, MBCQ), pyrimidinone catalytic inhibitors of Vps34 and bisaminopyrimidine catalytic inhibitors of Vps34,107.b) a compound comprising:

109. 111.c) a fatty acid oxidation inhibitor, ranolazine, di chloroacetate, chloroquine (CQ), hydroxychloroquine (HCQ), or 3 -methyladenine (3-MA), optionally wherein the fatty acid oxidation inhibitor is selected from etomoxir, oxfenicine, perhexiline, mildronate (a carnitine biosynthesis inhibitor), trimetazidine, and pFOX.

20. The method of any one of claims 16-19, further comprising administering to the subject a checkpoint inhibitor.

21. The method of claim 20, wherein the administration comprises:114.i) the PI3K inhibitor is administered to the subject prior to the autophagy inhibitor,115.ii) the PI3K inhibitor is administered to the subject prior to the checkpoint inhibitor,116.iii) the autophagy inhibitor is administered to the subject prior to the checkpoint inhibitor,117.iv) the checkpoint inhibitor is administered to the subject prior to the autophagy inhibitor, and / or118.v) the autophagy inhibitor is administered to the subject at the same time as the checkpoint inhibitor,119.vi) wherein the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 8 hours of the PI3K inhibitor.120.vii) wherein the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 24 hours of the PI3K inhibitor.121.viii) wherein the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 1 week of the PI3K inhibitor.122.ix) wherein the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 1 month of the PI3K inhibitor.123.x) wherein the subject is treated with the autophagy inhibitor and / or the checkpoint inhibitor within 6 months of the PI3K inhibitor, and / or124.xi) wherein the subject is administered at least 2 doses of PI3K inhibitor.

22. The method of any one of claims 16-21, wherein 1 - 10 mg / day of the PI3K inhibitor is administered to the subject and optionally wherein the PI3K inhibitor and / or the autophagy inhibitor is administered to the subject systemically.

23. The method of any one of claims 20-22, wherein the checkpoint inhibitor is:i) an antibody selected from an anti-PDl antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-Ll antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof,127.ii) pembrolizumab, BMS-936558 (nivolumab), REGN-2810 (cemiplimab), or PDR001 (spartalizumab), or128.iii) an anti-PD-Ll antibody selected from atezolizumab, avelumab, or durvalumab.

24. The method of any one of claims 16-23, wherein the GBM is an isocitrate dehydrogenase (IDH) mutant GBM or an IDH-wildtype GBM.

25. The method of any one of claims 16-23, wherein the wherein the GBM is characterized by one or more of131.a) increased epidermal growth factor receptor (EGFR) amplification relative to a GBM without EGFR amplification, a telomerase reverse transcriptase (TERT)-promoter mutation, or a combined gain of chromosome7 / loss of chromosome 10 copy number changes,132.b) hypermethylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter, or133.c) a BRAFv600 mutation, a fibroblast growth factor receptor (FGFR) mutation, a FGFR-TACC gene fusion, a H3K27M mutation, dysregulated CDK4 / 6 and / or dysregulated tumor protein p53 (TP53).

26. A pharmaceutical composition comprising a checkpoint inhibitor and an136. 138.autophagy inhibitor comprising:

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