WO2023154904A2 - Compositions and methods for treating acute myeloid leukemia - Google Patents

Abstract

Disclosed herein are compositions and methods of treating AML in a subject by administering a TKI in combination with one or more of active vitamin D (such as, 1,25VD₃), an expression construct encoding fructose-bisphosphatase 1 (FBP1) and protein phosphatase 2 catalytic subunit alpha (PPP2CA) or a cell containing thereof, and/or a tumor infiltrating lymphocyte (TIL) expressing a chimeric antigen receptor (CAR) targeting CXCR2 and/or CD33 to the subject. Also provided herein are compositions and methods of reducing viability and/or proliferation of an AML cell.

Description

COMPOSITIONS AND METHODS FOR TREATING ACUTE MYELOID LEUKEMIA

Inventors: Yi (David) Xu and Huynh Cao

RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63/267,891, filed on February 11, 2022, the entire content of which is incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on February 9, 2023, is named L105781_1920WO_SL_XML and is 38.1 kb in size.

TECHNICAL FIELD

[0003] The disclosure relates to compositions and methods of treatment of cancer utilizing a therapeutic agent or a combination of therapeutic agents.

BACKGROUND

[0004] Acute myeloid leukemia (AML) is a heterogeneous bone marrow malignancy with some of the lowest survival rates among the leukemias. AML involves a complex interplay between blasts and their microenvironment in the bone marrow. Despite improvements in understanding of AML and the development of different therapeutic approaches, approximately 50% of patients still will relapse following induction chemotherapy, resulting in a dismal 5-year overall survival rate of 29%.

[0005] Disease relapse is one of the major causes of treatment failure, but the relapse mechanism is not completely understood. Thus, effective therapies of AML based on a better understanding of intra- and extracellular mechanisms underlying AML relapse and developing are urgently needed.

SUMMARY

[0006] Disclosed herein are compositions and methods for treating AML in a subject. Also disclosed herein are methods of reducing viability and/or proliferation of an AML cell or a plurality of AML cells.

[0007] Provided here are pharmaceutical compositions for treating acute myeloid leukemia (AML). Certain embodiments of the pharmaceutical compositions include a therapeutically effective amount of 1,25- dihydroxyvitamin Ds ( 1 .25 VDs) and a tyrosine kinase inhibitor (TKI). In some embodiments, the TKI is Gilteritinib (GILT), Quizartinib (QUIZ), or Midostaurin (MIDO). [0008] In another aspect, provided herein are nucleic acid constructs containing a nucleic acid molecule encoding fructose-bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell. The nucleic acid molecule encoding FBP1 may include a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, wherein the encoded polypeptide retains a FBPl function, or the nucleic acid sequence of SEQ ID NO: 25. Embodiments may include nucleic acid constructs containing a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA) operably linked to a promoter functional in an animal cell. The nucleic acid molecule encoding PPP2CA may include a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 27, wherein the encoded polypeptide retains a PPP2CA function, or the nucleic acid sequence of SEQ ID NO: 27. The nucleic acid construct may include a lentiviral vector.

[0009] In a certain aspect of the present disclosure, a cell containing the nucleic acid construct or the lentiviral vector of the present disclosure is provided. In some embodiments, the cell may be a mesenchymal stem cell (MSC) or a tumor infdtrating lymphocyte (TIL).

[0010] In another aspect of the present disclosure, a tumor infiltrating lymphocyte (TIL) containing a chimeric antigen receptor (CAR) targeting CXCR2 is provided. The TIL can further include a CAR targeting CD33, such that it comprises dual specificity to CXCR2 and CD33.

[0011] In a certain aspect, a method for reducing viability and/or proliferation of an AML cell is provided. The method includes contacting the AML cell with an effective amount of a composition containing a TKI and one or more of: 1 ,25 VDs; a nucleic acid construct containing a nucleic acid molecule encoding fructose- bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell and/or a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell; a cell containing the nucleic acid construct; and a tumor infiltrating lymphocyte (TIL) expressing a chimeric antigen receptor (CAR) targeting CXCR2.

[0012] In some embodiments, the TIL further includes a CAR targeting CD33.

[0013] In some embodiments, the nucleic acid construct may include a nucleic acid molecule encoding FBP1 can include a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, wherein the encoded polypeptide retains a FBP1 function, or the nucleic acid sequence of SEQ ID NO: 25; and/or the nucleic acid molecule encoding PPP2CA comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 27, wherein the encoded polypeptide retains a PPP2CA function, or the nucleic acid sequence of SEQ ID NO: 27. The nucleic acid construct may include a lentiviral vector. In some embodiments, by contacting the AML cell with the nucleic acid construct, the nucleic acid construct is introduced into the AML cell. In some embodiments, the cell containing the nucleic acid construct is a mesenchymal stem cell (MSC) or a TIL.

[0014] In some embodiments, expression and/or function of fructose- bisphosphatase 1 (FBP1) is increased in the AML cell compared to a control AML cell without the treatment. In some embodiments, expression and/or function of cyclin DI and/or cyclin dependent kinase 1 (CDK1) is reduced in the AML cell compared to a control AML cell without treatment. In some embodiments, glycolysis is reduced and/or production of intracellular lactate is reduced in the AML cell compared to a control AML cell without treatment. In some embodiments, expression and/or function of one or more pro-apoptotic or tumorsuppressive genes is reduced in the AML cell compared to a control AML cell. The one or more pro-apoptotic or tumor-suppressive genes can include caspase-3, BAX, or P53. In some embodiments, glycolysis, oxidative phosphorylation, energy production, and/or levels of intracellular pyruvate and/or cytochrome c oxidase subunit 2 (MT-C02) are reduced in the AML cell compared to a control AML cell.

[0015] In some embodiments, the method includes treating a plurality of AML cells with an effective amount of the composition, wherein a number of viable mitotic AML cells are reduced compared to control AML cells.

[0016] In some embodiments, the TKI is GILT, QUIZ, or MIDO. In some embodiments, the AML cell has one or more mutations in FMS- like tyrosine kinase 3 (FLT3) gene. In some embodiments, the AML cell is in a human subject.

[0017] In a certain aspect of the present disclosure, a methods for treating AML in a subject is provided. The method comprises administering a therapeutically effective amount of a composition containing: a TKI; and one or more of: l,25VDs; a nucleic acid construct containing a nucleic acid molecule encoding fructose- bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell, and/or a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell; a cell containing the nucleic acid; and a tumor infdtrating lymphocyte (TIL) expressing a chimeric antigen receptor (CAR) targeting CXCR2 to the subject.

[0018] In some embodiments, the TIL further comprises a CAR targeting CD33.

[0019] The nucleic acid molecule encoding FBP 1 may include a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, wherein the encoded polypeptide retains a FBP1 function, or the nucleic acid sequence of SEQ ID NO: 25; and/or the nucleic acid molecule encoding PPP2CA comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 27, wherein the encoded polypeptide retains a PPP2CA function, or the nucleic acid sequence of SEQ ID NO: 27. The nucleic acid construct may include a lentiviral vector.

[0020] In some embodiments, by administering the nucleic acid construct to the subject, the nucleic acid construct is introduced into an AML cell in the subject. In some embodiments, the cell containing the nucleic acid construct is a mesenchymal stem cell (MSC) or a TIL.

[0021] According to the methods provided herein, the expression and/or function of fructosebisphosphatase 1 (FBP 1) can be increased in the subject compared to the control, expression and/or function of cyclin DI and/or cyclin dependent kinase 1 (CDK1) can be reduced in the subject compared to a control. Production of intracellular lactate and/or glycolysis can be reduced in the subject. In some embodiments, expression and/or function of one or more pro-apoptotic or tumor-suppressive genes is reduced in the subject compared to a control subject. The one or more pro-apoptotic or tumor-suppressive genes can include caspase- 3, BAX, or P53. In some embodiments, glycolysis, oxidative phosphorylation, energy production, and/or levels of intracellular pyruvate and/or cytochrome c oxidase subunit 2 (MT-C02) are reduced in the subject compared to a control subject.

[0022] In some embodiments, the method reduces a number of viable mitotic AML cells in the subject. In some embodiments, the method reduces one or more symptoms associated with AML in the subject. In some embodiments, the TKI is GILT, QUIZ, or MIDO. In some embodiments, the subject has one or more mutations in FLT3 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0024] Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. To facilitate this description, like reference numerals designate like structural elements or procedures in a method. Embodiments are illustrated by way of example and not by way of limitation in the figures of the accompanying drawings.

[0025] FIG. 1 depicts a TKI-treatment-induced increase in gene expression of Vitamin D Receptor (VDR) in AML blasts. Two days after the treatment with GILT or QUIZ in vitro, the MV4- 11 cells were collected for RNA isolation and gene expressions were analyzed by qPCR. Data show mRNA expressions of the genes encoding VDR. Where applicable, data are means ± SEM and were analyzed by student “t” test. ***P<0.005.

[0026] FIGs. 2A-2D depict therapeutic effect of the combination therapy of 80nM 1 .25 VDi and an 80nM TKI inhibitor (GILT or MIDO) on MV4-11. FIGs. 2A-2C depict therapeutic effect of the combination therapy of 80nM 1,25VD3 and 80nM GILT on MV4-11. FIG. 2A shows representative FACS plots of viable CD44⁺ MV4-11 in different treatment groups. FIG. 2B shows cumulative FACS percentage data of viable CD44⁺ MV4-11 cells. FIG. 2C shows mRNA expressions of the genes encoding CYCLIN DI and CDK1. The l,25VDs and/or GILT-treated MV4-11 cells were collected for RNA isolation and gene expressions were analyzed by qPCR. FIG. 2D depicts therapeutic effect of the combination of 80nM MIDO and 80nM l,25VDs on MV4-11. Representative FACS plots of viable Ki67⁺ MV4-11 cells in different treatment groups. Where applicable, data are means ± SEM and were analyzed by student t test. *P<0.05, **P<0.01, ***P<0.005.

[0027] FIGs. 3A-3I depict therapeutic effect of the combination therapy of 80nM 1,25VD3 and 80nM GILT on primary AML-FLT3 BMMNC collected from AML Patient 1 (FIGs. 3A-F) and from AML Patient 2 (FIGs. 3G-I). FIG. 3A shows representative FACS plots of viable CD14⁺ BMMNC cells in different treatment groups. FIGs. 3B shows cumulative FACS percentage data of viable CD14⁺ BMMNC cells. FIG. 3C shows cumulative data of cluster count in different treatment groups. FIG. 3D shows representative FACS plots of viable Ki67⁺CD33⁺CD14" BMMNC cells in different treatment groups. FIG. 3E shows cumulative FACS percentage data of viable Ki67⁺CD33⁺CD14" BMMNC cells. FIG. 3F shows mRNA expressions of the genes encoding CYCLIN DI. The treated BMMNC cells were collected for RNA isolation and gene expressions were analyzed by qPCR. FIG. 3G shows representative FACS plots of viable Ki67⁺CD33⁺CD117⁺ BMMNC cells in different treatment groups. FIG. 3H shows cumulative FACS percentage data of viable CD33⁺CD117⁺ BMMNC cells. FIG. 31 shows cumulative FACS percentage data of viable Ki67⁺CD33⁺CD117⁺ BMMNC cells. Where applicable, data are means ± SEM and were analyzed by student t test. *P<0.05, **P<0.01.

[0028] FIGs. 4A-4F depict RNA-seq analysis of AML cells treated or untreated with l,25VDs. FIG. 4A shows pie distribution of RNA-seq FPKM-based gene expression in MV4-11 cells. FIG. 4B shows data showing a sharp increase in FBP1 expression from the low rank in the 1 ,25VDs- untreated (NO- TX) groups to the high rank in l,25VD3-treated groups in both MV4-11 and MOLM-14 cells. FIG. 4C shows RNA- seq chart of FPKM for the FBP1 expression before or after l,25VDs treatment. FIGs. 4D1-D3 are images from Immunocytochemistry (ICC) to compare FBP1 protein before l,25VDs treatment (FIG. 4D1), after l,25VDs treatment (FIG. 4D2), or an ICC control (the secondary antibody was applied without a primary antibody, FIG. 4D3). FIG. 4E shows representative FACS plots showing the colocalization of FBP1 and VDR in l,25VD3-treated MV4-11 cells. FIG. 4F shows a table of RNA-seq analysis demonstrating that 1,25VD3 modulates enzymes-encoding genes and major metabolic processes in AML blasts.

[0029] FIGs. 5A-5D depict a l,25VD3-induced increase in FBP1 and l,25VD3-induced reduction of the production of lactate in AML blasts. FIG. 5A shows an analysis by RT-qPCR of expression of human FBP 1 (Fold Change). MV4-11 cells were treated with 1,25VD3 or GILT or a combination of 1,25VD3 and GILT for 48 h then harvested and analyzed. FIG. 5B shows a western blotting (WB) analysis of protein expression ofhuman FBPl in treated MV4-11 cells. FIG. 5C shows a lactate assay of treated MV4- 11 cells to measure the concentration of intracellular lactate. FIG. 5D shows cumulative data of the concentration of intracellular lactate. Where applicable, data are means ± SEM and were analyzed by student “t” test. *p < 0.05, ***p < 0.005.

[0030] FIG. 6 depicts a mechanistic model of induction of FBP 1 by vitamin D to block glycolysis and the Warburg effect (increased lactate caused by increased glycolysis in cancer cells) in AML metabolism. The combination therapy of I .25VD1 and TKIs of the present disclosure can effectively treat AML cell lines and primary blasts by inducing CD14⁺ differentiation and inhibiting Cyclin Dl-intiated cell proliferation. The present disclosure also provides a new functional role of 1,25-dihydroxyvitamin Ds in the treatment of AML blasts. I .25VD1 induces -3000-fold increase of FBP1 (qPCR data) in AML blasts, which encodes large amount of Fructose- 1,6-bisphosphatase (extremely big bands in WB) to disrupt the progression of glycolysis and reduce the production of lactate (Warburg Effect), a main energy resource for AML metabolism. Meanwhile, I .25VD1 modulates gene expressions of different enzymes essential for AML metabolic processes.

[0031] FIG. 7 depicts a dual specificity-chimeric antigen receptor (DS-CAR) on the surface of CD33/CXCR2-CAR-TILs (tumor-infiltrating lymphocytes). One of the targeted antigens (Agl) is CXCR2 and the other targeted antigen (Ag2) is CD33. VH refers to the heavy chain of the variable region of the antibody. VL refers to the light chain of the variable region of the antibody.

[0032] FIGs. 8A-8D depict generation of an AML cell line genetically bioengineered to overexpress FBP 1 in vitro (FBP1-MV411 blast). FIG. 8A is a schematic diagram of the lentiviral expression construct containing the human FBP1 open reading frame (ORF) and GFP reporter, with the EFla and IRES promoters, respectively. FIG. 8B is a representative FC histogram showing GFP expression in FBP1-MV4- 11 (FACS sorted) and MV4-11 cells; dying cells have weak GFP expression, indicated by a red circle. FIG. 8C1 is a representative phase-bright image (20x) of FBP1-MV4-11 cells; dying cells are indicated by arrows. FIG. 8C2 is a fluorescent image (20x) of the same FBP1-MV4-11 cells from FIG. 8C1 showing GFP expression in viable cells, while dead cells no longer display fluorescence. FIGs. 8D1-8D2 shows overexpression of the FBP1 gene and protein in the FBP1-MV4-11 cell line, as confirmed by qPCR (fold change, FIG. 8D1) and Western blot assays (FIG. 8D2); the lentiviral empty construct has a GFP reporter without the FBP1 ORF as the control (GFP-MV4-11). Where applicable, data are means ± SEM, and were analyzed by Student’s “f’-test; *** p < 0.005, n = 3.

[0033] FIGs. 9A-9G depict molecular phenotypes of the FBP1-MV4-11 cell line in vitro. FIG. 9A1 is a representative FC histogram showing CD14 expression in different MV4-11 cell lines, including naive MV4-11, MV4-11 with one round of FBPl-lentiviral transduction (FBP1- MV4-11), and MV4-11 with two rounds of FBPl-lentiviral transduction (FBP1-MV4-11 2X transduction). FIG. 9A2 is a histogram ofGFP+ expression in these cell lines. FIG. 9B depicts cumulative FC percentage data of CD 14+ cells in different MV4-11 cell lines from FIGs. 9A1-9A2. FIG. 9C is a representative FC histogram showing the expression of viability dye (more expression representing more cell death) in FBP1- MV4-11 (red line plot), GFP- MV4-11 (green line plot), and MV4-11 cells (fdled gray plot); the fdled orange plot represents the unstained control. FIGs. 9D1-9D2 depict representative phase-bright and fluorescent images (20x) showing some GFP+ FBP1-MV4-11 cells that were abnormally larger (indicated by a large arrow) than their neighboring cells; the small arrow indicates a dying cell with weak GFP+. FIG. 9E depicts gene expression of programmed cell death proteins in FBP1-MV4-11 cells was analyzed by qPCR; data of mRNA expression show the fold change (normalized to [3-actin) of genes encoding caspase-3, BAX, and P53. FIGs. 9F-9G depict P53 promoter assay of blasts and their supernatants; representative live images show luciferase activity in MV4-11 and FBP1-MV4-11 blasts (FIG. 9F) and supernatants from different experimental groups (FIG. 9G). Where applicable, data are means > SEM and were analyzed by Student’s “t”-test; * p < 0.05, *** p < 0.005, n = 3.

[0034] FIGs. 10A-10D depict rescue of the reduced proliferation of FBP1-MV4-11 cells by supplementation of pyruvate and/or glutamine in vitro. FIG. 10A left panel is a representative FC histogram showing the expression of viability dye (more expression representing more cell death) in different FBP1- MV4-11 rescue cell lines in vitro, including naive MV4-11, FBP1-MV4-11 cells, FBP1-MV4-11 cells with supplementation of pyruvate, FBP1-MV4-11 cells with supplementation of glutamine, and FBP1-MV4-11 cells with supplementation of both pyruvate and glutamine. FIG. 10A right panel shows cumulative FC percentage data of viable blasts in the experimental groups from the left panel. FIG. 10B left panel is a representative FC histogram showing Ki67 expression in different FBP1-MV4-11 rescue cell lines in vitro. FIG. 10B right panel shows cumulative FC percentage data of Ki67+ expression in the experimental groups from the left panel. FIG. 10C is a representative film showing WB assay of human P53 protein expression in experimental groups from FIG. 10A. FIG. 10D shows cumulative WB data of human P53 expression in different experimental groups from the left panel. Where applicable, data are means ± SEM and were analyzed by Student’s “t”-test; * p < 0.05, *** p < 0.005, n = 3.

[0035] FIGs. 11A-11D depict mitochondrial adaptation response to impaired metabolic homeostasis in the FBP1-MV411 cell line in vitro. FIG. 11A shows intracellular concentrations of pyruvate in MV4-11 and FBP1-MV4-11 cells were measured by using the Pyruvate Assay Kit. FIG. 11B shows gene expression of biomarkers for mitochondrial dysfunction in FBP 1 -MV4- 11 cells was analyzed by qPCR; data of mRNA expression show the fold change (normalized to [3-actin) of genes encoding FGF21 and GDF15. FIG. 11C shows a representative FC histogram showing the expression of C0X2 (cytochrome c oxidase subunit 2, MT-C02) in FBP1-MV4- 11 (blue line plot) and MV4-11 cells (black line plot); the filled sky-blue plot represents the IgG fluorescent control. FIG. 11D shows a representative film showing WB assay of human PINK1 (PTEN-induced kinase) protein expression in MV4-11 and FBP1-MV4-11 cells; Right panel: Cumulative WB data of human PINK1 expression from FIG. 11C. Where applicable, data are means ± SEM and were analyzed by Student’s “t”-test; * p < 0.05, *** p < 0.005, n = 3.

[0036] FIG. 12 depicts schematic diagram of the anti-leukemic functional roles of FBP1 and FBP- activated P53 in AML blasts in vitro: There are multifaceted anti-leukemic functions of FBP1 in the treatment of AML blasts; FBP1 inhibits the process of glycolysis by reducing key intermediate metabolites such as pyruvate (BOX1) and regulates blast differentiation via changes in cellular signaling. FBP1- disturbed glycolysis can induce stress in the mitochondria (BOX2), leading to the reduction in COX2 in the OXPHOS process. To maintain metabolic homeostasis and survive, FBP1-MV4-11 blasts can activate the quality control system of mitochondria via upregulation of PINK 1 (BOX3). The activated mitophagy and autophagy can replenish key metabolites such as fatty acids and glutamine, to either promote the regeneration of dysfunctional mitochondria (BOX3) or prevent cell death (BOX4). In addition to its glycolytic regulatory function, FBP1 can increase pro-apoptotic proteins and tumor suppressors — such as caspase-3, BAX, and P53 — while decreasing hypoxic oncogenes such as HIF1A (not shown), leading to programmed leukemic cell death (BOX4).

[0037] FIG. 13 depicts an example of double-phosphatase lentiviral constructs for dual phosphatase-based targeted therapy for relapsed or refractory AML. FBP1 encodes fructose-1,6 bisphosphatase. PPP2CA encodes protein phosphatase 2 catalytic subunit alpha.

DETAILED DESCRIPTION

I. Overview

[0038] Acute myeloid leukemia (AML) is a heterogeneous blood cancer with no available cure now, partly due to the common relapse of the disease. Thus, finding an effective treatment for AML patients with a protective benefit from COVID- 19 is an urgent need. Vitamin D gene therapy was reported to effectively treat AML mouse models in vivo. Vitamin D might also play advantageous roles in the prevention and treatment of COVID-19. Provided herein is a novel therapeutic approach combining active vitamin D (l,25VDs or 1,25(OH)2D3) and TKI to treat AML cancer cells (blasts) in vitro and ex vivo. The pharmaceutical compositions containing a combination of 1,25VD3 and GILT can effectively suppress the proliferation of blasts and induced their maturation. Importantly, 1,25VD3 can block glycolysis-based metabolism in AML blasts, known as the “Warburg Effect” that was idealized a century ago. Mechanistically, the compositions and methods of the present disclosure increases expression of fructose-

I,6-bisphosphatase, encoded by fructose- bisphosphatase 1 (FBP1), in response to 1 ,25VDs treatment, such as approximately by 3000-fold, in multiple different AML-FLT3 cell lines. Fructose- 1,6-bisphosphatase can co-localize with Vitamin D Receptor in AML cells. The compositions and methods of the present disclosure containing l .25VDs can modulate the expression of different genes encoding key enzymes for AML metabolic progresses including gluconeogenesis, glycolysis, TCA, de novo nucleotide synthesis. The compositions and methods of the present disclosure reduce lactate concentration in AML blasts after treatment with either l .25VDs or its combination with GILT. Further, the present disclosure provides constructs ( such as lentiviral vector) encoding FBP1 and protein phosphatase 2 catalytic subunit alpha (PPP2CA), and TILs expressing a chimeric antigen receptor (CAR) targeting CXCR2 and/or CD33. The present disclosure provides methods of treating AML cells and/or AML using the dual phosphatase (FBP1 and PPP2CA) constructs and/or CAR-CXCRs, along with a TKI. Combination therapies of the present disclosure can treat AML more effectively compared to a TKI therapy alone.

II. Definitions

[0039] A “therapeutically effective amount” is an amount sufficient to effect desired clinical results (that is, achieve therapeutic efficacy). A therapeutically effective dose can be administered in one or more administrations. “Administering” refers to the physical introduction of a therapeutic agent to a subject in need thereof. Exemplary routes of administration for agents to inhibit proliferation of mesenchymal cancer stem cells, include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. A therapeutic agent may be administered via a non-parenteral route, or orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. Therapeutic agents can be constituted in a composition, s u c h a s a pharmaceutical composition containing an antibody and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. [0040] The present disclosure describes various embodiments related to compositions and methods for management or treatment of cancers, such as AML, gastric cancer, or breast cancer. In the following description, numerous details are set forth in order to provide a thorough understanding of the various embodiments. Before the present methods and compositions are described, it is to be understood that these embodiments are not limited to particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present embodiments will be limited only by the appended claims. The description may use the phrases “in certain embodiments,” “in various embodiments,” “in an embodiment,” or “in embodiments,” which may each refer to one or more of the same or different embodiments. Furthermore, the terms “comprising,” “including,” “having,” and the like, as used with respect to embodiments of the present disclosure, are synonymous.

[0041] A “subject” refers an animal, such as a mammal, including a primate (such as a human, a nonhuman primate, such as a monkey) and a non-primate (such as a mouse). In some aspects of the disclosure, the subject is a human. In some aspects, the subject is a pediatric subject, such as a neonate, an infant, or a child. In other aspects, the subject is an adult subject.

[0042] A “patient” refers to a subject who shows symptoms and/or signs of a disease, is under treatment for disease, has been diagnosed with a disease, and/or is at risk of developing a disease. A “patient” can be human and veterinary subjects. Any reference to subjects in the present disclosure, should be understood to include the possibility that the subject is a “patient” unless clearly dictated otherwise by context. More specifically, the subject in certain aspects is a patient who has a liquid cancer, such as a leukemia.

[0043] As used herein, the terms “treating”, “treatment” and the like, shall include the management and care of a subject or patient for the purpose of combating a disease, condition, or disorder and includes the administration of a composition to prevent the onset of the symptoms or complications, alleviate the symptoms or complications, reduce at least one associated sign, symptom, or condition, or eliminate the disease, condition, or disorder. Treatment also refers to a prophylactic treatment, such as prevention of a disease ( such as AML) or prevention of at least one sign, symptom, or condition associated with the disease ( such as relapse of AML). Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0044] As used herein with respect to a parameter, the term “decreased” or “decreasing” or “decrease” or “reduced” or “reducing” or “reduce” or “lower” refers to a detectable ( such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) negative change in the parameter from a comparison control, such as an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “decreased”, “reduced”, and the like encompass both a partial reduction and a complete reduction compared to a control.

[0045] As used herein with respect to a parameter, the term “increased” or “increasing” or “increase” or “enhanced” or “enhancing” or “enhance” refers to a detectable ( such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%) positive change in the parameter from a comparison control, such as an established normal or reference level of the parameter, or an established standard control.

III. Pharmaceutical compositions for treating AML

[0046] In one aspect, the present disclosure provides pharmaceutical compositions for treating AML, containing a therapeutically effective amount of 1,25- dihydroxyvitamin Ds ( l .25VDs) and a TKI. In some embodiments, the TKI is GILT, QUIZ, or MIDO. In some embodiments, pharmaceutical compositions comprise 80 nM l,25VDs and 80 nM of a TKI, such as GILT, QUIZ, or MIDO.

[0047] Pharmaceutical compositions according to the present disclosure may comprise additional pharmaceutically acceptable components. As used herein, “pharmaceutically acceptable” refers to the compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. For example, pharmaceutical compositions of the present disclosure can include a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, such as sugars, starches, cellulose, malt, gelatin, agar, buffering agents, saline, pH buffered solutions, serum component, and non-toxic compatible substances employed in pharmaceutical formulations.

IV. Constructs containing nucleic acid molecules encoding fructose-bisphosphatase 1 (FBP1) and/or protein phosphatase 2 catalytic subunit alpha (PPP2CA)

[0048] FBP1 and PPP2CA are phosphatases that also possess a tumor suppressor function. The present disclosure provides a double phosphatase expression vector 1) to utilize double-effect of FBP1 and PPP2CA as tumor suppressors, 2) to de-phosphorylate pBAD, which does not have the function of BAD due to phosphorylation and cannot bind to BCL-2 to initiate the cell death of AML blasts, and 3) to prevent self-phosphorylation of FBP1, which leads to loss of its function as a tumor suppressor, by adding the phosphatase PPP2CA. The present disclosure provides the therapeutic effect of FBP1 and PPP2CA on AML blasts by overexpression of these 2 phosphatases at the same time.

[0049] FBP 1 is also referred to as fructose- 1 ,6-bisphosphatase 1 , EC 3.1.3.11, FBPase 1 , growth-inhibiting protein 17, and EC 3.1.3. Additional information on FBP1 can be found, for example, on the world wide web at https://www.ncbi.nlm.nih.gov/gene/2203. The sequence of human FBP1 mRNA transcript can be found in, for example, GenBank Reference Sequence: NM_00507.4 (transcript variant 1) (SEQ ID NO: 25) and NM_001127628.2 (transcript variant 2) (SEQ ID NO: 26).

[0050] PPP2CA is also referred to as PP2Calpha, serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform, replication protein C, EC 3.1.3.16, PP2A-Alpha, PP2AC, RP- C, and NEDLBA. Additional information on PPP2CA can be found, for example, on the world wide web at https://www.ncbi.nlm.nih.gov/gene/5515. The sequence of a human PPP2CA mRNA transcript can be found in, for example, GenBank Reference Sequence: NM_002715.2 (SEQ ID NO: 27).

[0051] In one aspect, the present disclosure provides a nucleic acid construct containing: (a) a nucleic acid molecule encoding fructose-bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell; and/or (b) a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell. In some embodiments, the nucleic acid molecule encoding FBP1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, wherein the encoded polypeptide retains a FBP1 function, or the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the nucleic acid molecule encoding PPP2CA comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 27, wherein the encoded polypeptide retains a PPP2CA function, or the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the promoter operably linked to the FBP 1 and/or the PPP2CA coding sequences are an EF la promoter. For example, the construct may include the FBP1 coding sequence and an operably-linked EFla promoter. In some embodiments, the construct comprises double open reading frames (ORFs) and comprises, in operable linkage, an EFla promoter, an FBP1 coding sequence, an internal ribosome entry site (IRES), and a PPP2CA entry site. In some embodiments, the nucleic acid construct includes a viral vector, such as a lentiviral vector. In some embodiments, the double ORF construct (such as the double ORF lentiviral vector) can be used for dual phosphatase-based targeted therapy for relapsed or refractory AML.

[0052] The construct can additionally include a reporter gene operably linked to a promoter, to facilitate identification and/or selection of cells expressing the construct. Any reporter gene can be used in the construct provided herein containing the FBP1 and/or the PPP2CA coding sequences, including GFP and mCherry, and can be operably linked to any suitable promoter. For example, the construct may include, in an operable linkage, the EFla promoter, the FBP1 coding sequence, the IRES, and the GFP coding sequence, as shown in FIGs. 8 A and 13. The construct may include, in an operable linkage, the EFla promoter, the mCherry coding sequence, the IRES, and the PPP2CA coding sequence, as shown in FIG. V. Reducing viability and/or proliferation of an AML cell or a plurality of AML cells

[0053] Provided herein are methods for reducing viability and/or proliferation of AML cells (such as AML blasts). In one aspect, the methods include increasing the amount of concentration of FBP1 and/or PPP2CA in AML cells or in the environment of AML cells, and/or treating the AML cell with an effective amount of 1,25-dihydroxyvitamin Ds ( l .25VDs) and a TKI. In a certain aspect, a method for reducing viability and/or proliferation of an AML cell is provided. The method comprises contacting the AML cell with an effective amount of a composition containing: a TKI; and one or more of: 1,25-dihydroxyvitamin Ds (l,25VDs); a nucleic acid construct containing a nucleic acid molecule encoding fructose- bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell and/or a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell; a cell containing the nucleic acid construct; and a tumor infiltrating lymphocyte (TIL) expressing a chimeric antigen receptor (CAR) targeting CXCR2. The TKI can be GILT, QUIZ, or MIDO.

[0054] The method may include contacting the AML cell with an effective amount of a composition containing a TKI, and a TIL expressing a chimeric antigen receptor (CAR) targeting CXCR2. The TIL can further comprises a CAR targeting CD33.

[0055] The method may include contacting the AML cell with an effective amount of a composition containing a TKI, and a nucleic acid construct containing a nucleic acid molecule encoding fructose- bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell and/or a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell. The nucleic acid molecule encoding FBP1 may include a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, wherein the encoded polypeptide retains a FBP1 function, or the nucleic acid sequence of SEQ ID NO: 25; and/or the nucleic acid molecule encoding PPP2CA comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 27, wherein the encoded polypeptide retains a PPP2CA function, or the nucleic acid sequence of SEQ ID NO: 27. The nucleic acid construct may include a lentiviral vector. By contacting the AML cell with the nucleic acid construct, the nucleic acid construct can be introduced into the AML cell, such that FBP1 and/or PPP2CA can be overexpressed by the AML cell.

[0056] The method may include contacting the AML cell with an effective amount of a composition containing a TKI, and a cell containing the nucleic acid construct with a nucleic acid molecule encoding fructose- bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell and/or a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell, the cell containing the nucleic acid construct is a mesenchymal stem cell (MSC) or a TIL. For example, the methods may include introducing the nucleic acid construct, such as a lentiviral vector encoding FBP1 and/or PPP2CA, into mesenchymal stem cells (MSCs) and contacting the AML cell with the MSCs expressing FBP1 and/or PPP2CA.

[0057] The AML cell can be cultured in vitro. Alternatively, the AML cell can be in a subject, such as a human, such as a human AML patient. The cells can be human AML cell lines, such as MV4-11 (ATCC CRL- 9591) and MOLM-14 (DSMZ ACC-777). The cells can be primary AML blasts of AML patients. The AML cell can have one or more mutations in the FLT gene, and can be a AML-FLT3 cell.

[0058] In the methods provided herein, contacting the AML cell with an effective amount of the composition increases expression and/or function of FBP1 in the AML cell compared to a control AML cell. For example, expression and/or function of FBP1 can be increased in the AML cell contacted with an effective amount of the composition by about 10-100 fold, 200-1000 fold, 300-1000 fold, 400-1000 fold, 500-1000 fold, 600-2000 fold, 700-2000 fold, 800-2000 fold, 200-2000 fold, 30-2000 fold, 40-2000 fold, 50- 2000 fold, 60-2000 fold, 70-2000 fold, 1000-2000 fold, 200-3000 fold, 300-3000 fold, 400- 3000 fold, 500-3000 fold, 600-3000 fold, 700-3000 fold, 800-3000 fold, 200-3000 fold, 300- 3000 fold, 400- 3000 fold, 500-3000 fold, 600-3000 fold, 700-3000 fold, or more than 3000 fold ( such as by about 10- 20 fold, 20-30 fold, 30-40 fold, 40-50 fold, 50-60 fold, 60-70 fold, 70- 80 fold, 80-90 fold, 90-100 fold, 100-200 fold, 200-300 fold, 300-400 fold, 400-500 fold, 500- 600 fold, 600-700 fold, 700-800 fold, 800- 900 fold, 900-1000 fold, 1000-3000 fold, or more than 3000 fold), such as by about 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80 fold, 85 fold, 90 fold, 95 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold or more as compared to a control AML cell without being contacted with the composition provided herein.

[0059] In the methods provided herein, contacting the AML cell with an effective amount of the composition reduces expression and/or function of cyclin DI and/or cyclin dependent kinase 1 (CDK1) in the AML cell compared to a control AML cell. For example, expression and/or function of cyclin D 1 and/or cyclin dependent kinase 1 (CDK1) can be reduced in the AML cell contacted with an effective amount of the composition by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80- 100%, 20-90%, 30-90%, 40-90%, 50- 90%, 60-90%, or 70-90% ( such as by about 10-20%, 20-30%, 30- 40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), such as by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to a control AML cell without being contacted with the composition provided herein. [0060] In the methods provided herein, contacting the AML cell with an effective amount of the composition can increase expression or function of one or more pro-apoptotic or tumor-suppressive genes (such as caspase-3, BAX, and P53). For example, expression or function of one or more pro-apoptotic or tumor-suppressive genes can be increased in the AML cell contacted with an effective amount of the composition by about 10-100%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-2000%, 700- 2000%, 800-2000%, 200-2000%, 30-2000%, 40-2000%, 50- 2000%, 60-2000%, 70-2000%, 1000-2000%,

200-3000%, 300-3000%, 400- 3000%, 500-3000%, 600-3000%, 700-3000%, 800-3000%, 200-3000%,

300- 3000%, 400-3000%, 500-3000%, 600-3000%, 700-3000%, or more than 3000% ( such as by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70- 80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500- 600%, 600-700%, 700-800%, 800-900%, 900-1000%, 1000-3000%, or more than 3000%), such as by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000% or more as compared to a control AML cell without being contacted with the composition provided herein.

[0061] In the methods provided herein, contacting the AML cell with an effective amount of the composition can reduce glycolysis, oxidative phosphorylation, energy production, and/or intracellular pyruvate and/or cytochrome c oxidase subunit 2 (MT-CO2) levels in the AML cell compared to a control AML cell. For example, glycolysis, oxidative phosphorylation, energy production, and/or intracellular pyruvate and/or cytochrome c oxidase subunit 2 (MT-CO2) levels can be reduced in the AML cell contacted with an effective amount of the composition by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50- 90%, 60-90%, or 70-90% ( such as by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), such as by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to a control AML cell without being contacted with the composition provided herein.

[0062] In the methods provided herein, viability and/or proliferation of the AML cell contacted by the composition provided herein can be reduced by about 10-100%, 20-100%, 30-100%, 40- 100%, 50-100%,

60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, or 70-90% (such as by about

10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80- 90%, or 90-100%), such as by about

10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or

100%, or by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%_;

85%, 90%, 95%, or 100%, as compared to a control cell not contacted with the composition. [0063] The method may include contacting a plurality of AML cells with an effective amount of the composition, wherein a number of viable mitotic AML cells are reduced compared to control AML cells. For example, the number of viable mitotic AML cells can be reduced by about 10-100%, 20-100%, 30- 100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, or 70-90% ( such as by about 10-20%, 20-30%, 30- 40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90- 100%), such as by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the AML cells (blasts) contacted with the composition provided herein, as compared to control AML cells not contacted with the composition.

[0064] The remodeling of cellular metabolism is a fundamental progress to meet higher demands of energy in cancer. An “Warburg Effect,” defined as an increase in the rate of glucose uptake and preferential production of lactate, even in the presence of oxygen, has been identified in cancer cells. An increased glycolysis was confirmed in leukemic blasts and correlated with clinical prognosis for AML. Increased production of lactate was attributed to chemoresistance in AML patients who had increased levels of lactate dehydrogenase (LDH). AML metabolism also involves diverse processes of nucleotides, amino acid, lipids and their end metabolites to perform signaling functions and produce energy to support tumorigenesis.

[0065] FIG. 6 depicts a mechanistic model of induction of FBP1 by vitamin D to block glycolysis and the Warburg effect (increased lactate caused by increased glycolysis in cancer cells) in AML metabolism. Vitamin D ( l .25VDs) has ability to induce differentiation and inhibit proliferation. In some aspects, l,25VDs increases expression of FBP1. FBP1, as used herein, refers to a gene known to encode Fructose- 1,6-bisphosphatase, a key rate-limiting gluconeogenic enzyme. In addition to being a metabolic enzyme, FBP1 is also a tumor suppressor in solid tumors by inhibiting multiple oncogenic transcription factors and their downstream signaling pathways. l .25VDs up-regulates FBP1, which suppresses glycolysis. FBP1 colocalizes with VDR.

[0066] Further, as summarized in FIG. 12, FBP1 can simultaneously stimulate the apoptosis of blasts while also preventing leukemic growth through the regulation of pro-apoptotic and tumor-suppressive genes, such as caspase-3, BAX, and P53. FBP1 can stimulate the expression of the tumor-suppressor P53 gene, whose downregulation or mutation characterizes a distinct feature of AML. FBP1 can also activate mitochondrial reprogramming by reducing pyruvate and COX2 (MT-C02). The FBP1 -subverted glycolysis (BOX1, FIG. 12). can be linked to an impaired OXPHOS process, leading to mitochondrial stress or dysfunction (BOX2, FIG. 12).

VI. Combination Therapy for AML [0067] Methods for treating AML in a subject are provided. The method includes administering a therapeutically effective amount of a composition to a subject, where the composition comprises a TKI; and one or more of: 1,25 -dihydroxyvitamin Ds (l,25VDs); a nucleic acid construct containing a nucleic acid molecule encoding fructose- bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell and/or a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell; a cell containing the nucleic acid construct; and a tumor infdtrating lymphocyte (TIL) expressing a chimeric antigen receptor (CAR) targeting CXCR2. The TKI can be Gilteritinib (GILT), Quizartinib (QUIZ), or Midostaurin (MIDO).

[0068] The method may include administering to the subject an effective amount of a composition containing a TKI, and a TIL expressing a chimeric antigen receptor (CAR) targeting CXCR2. The TIL can further comprises a CAR targeting CD33, such that the CAR-TIL targets both CXCR2 and CD33 (“CXCR2/CD33-CAR-TILs”) .

[0069] The method may include administering to the subject an effective amount of a composition containing a TKI, and a nucleic acid construct containing a nucleic acid molecule encoding fructose- bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell and/or a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell. The nucleic acid molecule encoding FBP1 may include a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, wherein the encoded polypeptide retains a FBP1 function, or the nucleic acid sequence of SEQ ID NO: 25; and/or the nucleic acid molecule encoding PPP2CA comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 27, wherein the encoded polypeptide retains a PPP2CA function, or the nucleic acid sequence of SEQ ID NO: 27. The nucleic acid construct may include a lentiviral vector. By contacting the AML cell with the nucleic acid construct, the nucleic acid construct can be introduced into the AML cell, such that FBP1 and/or PPP2CA can be overexpressed by the AML cell. The construct can be the FBP1-PPP2CA double phosphatase construct (such as the double-ORF lentiviral construct described herein).

[0070] The method may include administering to the subject an effective amount of a composition containing a TKI, and a cell containing the nucleic acid construct containing a nucleic acid molecule encoding fructose-bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell and/or a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell, the cell containing the nucleic acid construct is a mesenchymal stem cell (MSC) or a TIL. For example, the methods may include introducing the nucleic acid construct, such as a lentiviral vector encoding FBP 1 and/or PPP2CA, into mesenchymal stem cells (MSCs) and contacting the AML cell with the MSCs expressing FBP1 and/or PPP2CA. [0071] In the methods provided herein, administering to the subject an effective amount of the composition can increase expression and/or function of FBP1 in the subject compared to a control subject. For example, expression and/or function of FBP1 can be increased in the subject contacted with an effective amount of the composition by about 10-100 fold, 200-1000 fold, 300-1000 fold, 400-1000 fold, 500-1000 fold, 600- 2000 fold, 700-2000 fold, 800-2000 fold, 200-2000 fold, 30-2000 fold, 40-2000 fold, 50- 2000 fold, 60- 2000 fold, 70-2000 fold, 1000-2000 fold, 200-3000 fold, 300-3000 fold, 400- 3000 fold, 500-3000 fold, 600-3000 fold, 700-3000 fold, 800-3000 fold, 200-3000 fold, 300- 3000 fold, 400-3000 fold, 500-3000 fold, 600-3000 fold, 700-3000 fold, or more than 3000 fold (such as by about 10-20 fold, 20-30 fold, 30- 40 fold, 40-50 fold, 50-60 fold, 60-70 fold, 70- 80 fold, 80-90 fold, 90-100 fold, 100-200 fold, 200-300 fold, 300-400 fold, 400-500 fold, 500- 600 fold, 600-700 fold, 700-800 fold, 800-900 fold, 900-1000 fold, 1000-3000 fold, or more than 3000 fold), such as by about 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80 fold, 85 fold, 90 fold, 95 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold or more as compared to a control subject without being administered the composition provided herein.

[0072] In the methods provided herein, administering to the subj ect an effective amount of the composition reduces expression and/or function of cyclin DI and/or cyclin dependent kinase 1 (CDK1) in the subject compared to a control subject. For example, expression and/or function of cyclin DI and/or cyclin dependent kinase 1 (CDK1) can be reduced in the subject contacted with an effective amount of the composition by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50- 90%, 60-90%, or 70-90% ( such as by about 10-20%, 20-30%, 30-40%, 40- 50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), such as by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to a control subject without being administered the composition provided herein.

[0073] In the methods provided herein, administering to the subj ect an effective amount of the composition can increase expression or function of one or more pro-apoptotic or tumor-suppressive genes (such as caspase-3, BAX, and P53). For example, expression or function of one or more pro-apoptotic or tumorsuppressive genes can be increased in the subject contacted with an effective amount of the composition by about 10-100%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-2000%, 700-2000%, 800-2000%,

200-2000%, 30-2000%, 40-2000%, 50- 2000%, 60-2000%, 70-2000%, 1000-2000%, 200-3000%, 300- 3000%, 400- 3000%, 500-3000%, 600-3000%, 700-3000%, 800-3000%, 200-3000%, 300- 3000%, 400- 3000%, 500-3000%, 600-3000%, 700-3000%, or more than 3000% ( such as by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70- 80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400- 500%, 500- 600%, 600-700%, 700-800%, 800-900%, 900-1000%, 1000-3000%, or more than 3000%), such as by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000% or more as compared to a control subject without being administered the composition provided herein.

[0074] In the methods provided herein, administering to the subj ect an effective amount of the composition can reduce glycolysis, oxidative phosphorylation, energy production, and/or intracellular pyruvate and/or cytochrome c oxidase subunit 2 (MT-C02) levels in the subject compared to a control subject. For example, glycolysis, oxidative phosphorylation, energy production, and/or intracellular pyruvate and/or cytochrome c oxidase subunit 2 (MT-C02) levels can be reduced in the subject contacted with an effective amount of the composition by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-

100%, 20-90%, 30-90%, 40-90%, 50- 90%, 60-90%, or 70-90% ( such as by about 10-20%, 20-30%, 30-

40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), such as by about 10%, 15%, 20%, 25%,

30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or by at least

10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or

100% as compared to a control subject without being administered the composition provided herein.

[0075] The method can reduce a number of viable mitotic AML cells in the subject. In some embodiments, the treating reduces a number of viable mitotic AML cells by about 10-100%, 20-100%, 30-100%, 40- 100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40- 90%, 50-90%, 60-90%, or 70-90%

( such as by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), such as by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,

85%, 90%, 95%, or 100%, or by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,

65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the subject. In some embodiments, the treating reduces one or more symptoms associated with AML in the subject. The method can reduce one or more symptoms associated with AML by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%,

80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, or 70-90% ( such as by about 10-20%, 20-30%,

30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90- 100%), such as by about 10%, 15%,

20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the subject. In some embodiments, the subject has one or more mutations in the FLT3 gene.

[0076] The composition provided herein can be administered to the subject at any dose and intervals that are suited for treating AML. A skilled artisan can identify and practice an appropriate dosing regimen suited for the clinical purpose . For example, For example, the composition may be administered once per week, twice per week, three times per week, four times per week, or five times per week. In some embodiments, the schedule involves regularly spaced administrations, e.g., hourly, every four hours, every six hours, every eight hours, every twelve hours, daily, every 2 days, every 3 days, every 4 days, every 5 days, weekly, biweekly, or monthly. In some embodiments, the composition is administered at the frequency required to achieve a desired effect.

[0077] The schedule can involve closely spaced administrations followed by a longer period of time during which the agent is not administered. For example, the schedule may involve an initial set of doses that are administered in a relatively short period of time (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours) followed by a longer time period (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) during which the composition is not administered. For example, the composition can be initially administered daily and is later administered at a longer interval (e.g., weekly, biweekly, or monthly). In certain embodiments, the longer interval increases over time or is determined based on the achievement of a desired effect.

[0078] The composition can be administered to the subject via any desired route. For example, the composition can be administered to the subject orally, intravenously, subcutaneously, intramuscularly, or intrathecally. Further, any standard methods for treating AML, including chemotherapy, radiation, bone marrow transplant, and stem cell transplant, can be used with (such as sequentially or concurrently) the methods provided herein.

EXAMPLES

[0079] The following examples are offered by way of illustration and not by way of limitation.

Example 1: TKI-treatment induces vitamin D receptor (VDR) in AML blasts

[0080] To understand the survival mechanisms of AML blasts after the treatment of tyrosine kinase inhibitors (TKIs), in vitro studies of MV4-11 and MOLM-14, monoblast cell lines harboring a FLT3 internal tandem duplication mutation (FLT3-ITD), were performed. MV4- 11 cells or MOLM-14 cells were treated with one dose of 80nM GILT, a second generation TKI drug, for 48 h in vitro. The mRNA was isolated from experimental cells and their change was determined by real-time polymerase chain reaction (qPCR). Notably, data revealed that the GILT treatment significantly increased the gene expression of VDR by 59- fold when compared to no treatment (NO-TX) (FIG. 1). A similar result of a 40-fold upregulation was obtained with one dose of 80nM quizartinib, another second generation TKI drug. This phenomenon of treatment-activated VDR expression is consistent with the previous report that one dose of 5-Azacytidine (AZA), a FDA -approved hypomethylating agent for AML, can increase VDR expression in MOLM-14 cells, and that supplementing active vitamin D ( I .25VD1. also abbreviated as 1,25(OH)2D3) to AZA suppressed FLT3 and displayed superior therapeutic effects compared to single agents ex vivo and in vivo. The data suggest that adding 1,25VD3 to the regimen may overcome the resistance to TKI treatment in AML blasts.

Example 2: The combination of 1,25VD3 and TKI reduces proliferating MV4-11 cells and AML- FLT3 bone marrow mononuclear cells (BMMNC)

[0081] Whether the combination of 1 .25VD1 and TKIs could effectively eliminate AML blasts was tested . MV4-11 cells were treated with one dose of 80nM 1,25VD3, 80nM GILT, or their combination for 48 h in vitro. The flow cytometry (FC) data demonstrated that the combination of GILT plus I .25VD1 significantly reduced the percentage of viable CD44⁺ MV4-11 blasts (27.3% versus 71.6% in GILT only, 38.2% in l,25VDs only and 91.8% in untreated control, FIG. 2A, B). Similar results of anti-leukemia effects were observed in the experimental studies combining 1 .25VD1 with midostaurin (MIDO), a 1^st generation TKI, approved by FDA for AML. The combination of MIDO plus I .25VD1 significantly reduced the percentage of viable Ki67⁺ proliferating blasts (3.79% versus 25.6% in GILT only, 17.2 % in 1,25VD3 only, and 51.6% in untreated control, FIG. 2D). To examine the cellular mechanism underlying the inhibition of blast proliferation after the combination therapy, gene expression of all Cyclin and Cyclin Dependent Kinase (CDK) family members (primer information in Table 2) was studied. The qPCR data showed the combination of 1,25VD3 and GILT significantly reduced CYCLIN DI mRNA (91% downregulation) and CDK1 mRNA (89% downregulation) when compared to the untreated control (NO- TX) (FIG. 2C). A significantly more decrease in expression was observed in the combination therapy than 1,25VD3 only treatment (81% and 55% downregulation in CYCLIN DI and CDK1) or GILT only treatment (38% and 69% downregulation in CYCLIN DI and CDK1) (FIG. 2C).

[0082] The anti -leukemic effect of combining GILT with 1,25VD3 was examined on primary BMMNC from AML patients with FLT3 mutations ex vivo. BMMNC was treated with one dose of 80nM 1,25VD3, 80nM GILT, or their combination for 48 h ex vivo. The flow cytometry data showed that GILT + 1,25VD3 not only significantly increased the percentage of viable CD14⁺ primary cells (19.5% versus 0.76% in GILT only, 15.9% in 1,25VD3 only, and 0.71% in untreated control, Patient #1 FIG. 3A, B), but also significantly inhibited the cluster formations in primary BMMNC (FIG. 3C). According to the clinical profile, Patient #1, an elderly AML patient, has a large population of CD33⁺ blasts. The flow cytometry data confirmed that the combination therapy significantly reduced Ki67⁺CD33⁺ proliferating blasts which are CD14 negative (29.7% versus 60% in GILT only, 48.5% in 1,25VD3 only, and 68.4% in untreated control (FIG. 3D, E). Mechanistically, the qPCR data showed the combination of GILT and 1,25VD3 significantly reduced the gene expression of CY CLIN D 1, a molecule responsible for Gl/S transition of the cell cycle (FIG. 3F). To confirm the therapeutic efficacy, treatment experiments were performed on BMMNC samples from a different AML-FLT3 patients. In Patient #2, the flow cytometry' data showed that the combination therapy also significantly reduced both viable CD33⁺CD117⁺ BMMNC and Ki67⁺CD33⁺CD117⁺ proliferating blasts (FIG. 3G-I). Collectively, the flow cytometry and qPCR data demonstrated that the combination of GILT and 1 .25 VDs significantly reduced the viable AML blasts (cell lines as well as primary BMMNC) by inhibiting their proliferation (Ki67⁺) and inducing blast differentiation (CD14⁺).

Example 3: Vitamin D (1,25VD3) activates FBP1 gene and modulated the AML metabolism

[0083] The functional role of l .25VDs on AML metabolism was studied. AML blasts from MV4-11, MOLM-14, MV4-11-TKI-resistant, and MOLM-14-TKI-resistent cell lines were pretreated for 48 hours with 80nM l,25VDs, 80nM MIDO, or a combination of 80nM l,25VDs and 80nM MIDO. The MV4-11- TKI-resistant and MOLM-14-TKI-resistent cell lines were generated as reported in Xu et al., 2020 Transl Oncol 13(12): 100869, which is incorporated by reference in its entirety.

[0084] The RNA-Seq results from MV4-11 and MOLM-14 were analyzed. According to the RNA-seq library for MV4-11, there are 17,757 genes with FPKM numbers after the treatment of l,25VDs: 10,947 genes with FPKM numbers greater than 1; 8052 genes with FPKM numbers greater than 5; 5931 genes with FPKM numbers greater than 10; 1473 genes with FPKM numbers greater than 50; 686 genes with FPKM numbers greater than 100; and 318 genes with FPKM numbers greater than 200 (distribution pie, FIG. 4A). The data revealed that l,25VDs induced an increase of~254-fold in the expression of the FBP1 gene, which was ranked at the 8413th among 17,757 genes in the untreated group and was found to rise to the 94th rank among 17,757 genes after l,25VDs treatment (FIG. 4B, C). Similar changes in FPKM and ranks were observed in RNA-seq results of MOLM-14 blasts (~263-fold increase in FBP1, FIG. 4B, C) and both TKI-resistant cell lines. The increased FBP1 expression was confirmed by immunocytochemistry, which showed low FBP1 expression in non-treated MV4- 11 (FIG. 4D1) and a much higher staining in l,25VDs-treated MV4-11 (FIG. 4D2). The flow cytometry data similarly confirmed the increased FBP1, and 95.9% of the FBP1+ cells colocalized with VDR+ cells (FIG. 4E). Table 1 below summarizes the FBP1 expression and rank in MV4-11 and MOLM-14 cells that received different treatments. As shown in Table 1, the FBP1 expression (FPKM) increased sharply from the low rank in non-treated (NO-TX) group to the high rank in 80 nM 1, 25 VD3 -treated groups in MV4-11, MOLM-14, midostaurin-resistant MV4-11 cells, and midostaurin-resistant MOLM-14 cells.

Table 1. FBP1 Expression and Rank in Different Blasts

MIDO-R: resistant to midostaurin (80 nM).

[0085] Tumor metabolism supports oncogenic pathways and is presumed to play essential roles in relapse and metastasis. To understand the vitamin D’s role in AML metabolism, modulation by l,25VDs of the genes essential for different metabolite processes including gluconeogenesis, glycolysis, TCA cycle, oxidative phosphorylation, glycogenesis, glycogenolysis, and nucleotide synthesis was analyzed (FIG. 4E). Notably, l,25VDs increased gene expressions of enzymes related to gluconeogenesis, TCA cycles, oxidative phosphorylation, glycogenesis, and reduced gene expressions of enzymes related to glycolysis, glycogenolysis and nucleotide synthesis (FIG. 4E). In summary, the data suggests that 1 .25 VDs activates FBP1 and modulates the AML metabolism.

Example 4: Vitamin D (1,25VD3) suppresses glycolysis-based production of lactate in AML blasts

[0086] The changes of FBP1 expression after the combination therapies were studied. MV4- 11 cells were treated with l .25VDs or GILT or a combination of l .25VDs and GILT for 48 hours, then harvested and analyzed by qPCR for expression of human FBP1 (Fold Change). The data showed that l .25VDs or the combination of l .25VDs and GILT significantly up- regulated gene expression of human FBP1 by 2678-fold and 3358-fold when compared to non- treatment (FIG. 5A). Furthermore, western blotting analyses of experimental groups confirmed the substantial increase of FBP1 protein by visualizing significantly larger bands after 1 .25VD1 treatment orthe combination (FIG. 5B). Finally, a functional assay was performed to examine the production of lactate in experimental groups. Each experimental group supplemented with I .25VD1 has a lighter color, indicating less activity, than untreated or GILT-treated groups (FIG. 5C). Quantitative measurement of OD of experimental groups confirmed that the combination therapy of GILT and I .25VD1 significantly reduced the production of intracellular lactate (Concentration of lactate: 0.04 nmol/pl versus 0.05 nmol/pl in GILT only, 0.05 nmol/pl in l,25VDs only, and 0.12 nmol/pl in untreated control (FIG. 5D). These findings are consistent with reports through both gain of function and loss of function experimental approaches showing that FBP1 can function as a negative regulator of the Warburg Effect in patients with hepatocellular carcinoma. Collectively, the findings suggest that l,25VD3-induced dramatic increases of FBP1 may block glycolysis and reduce the production of intracellular lactate in AML blasts.

[0087] Anti-tumor effects of the combination therapy of 1,25VD3 and a TKI is tested in vivo. Vitamin D’s effects on metabolisms of blasts and neighboring cells in the AML microenvironment is also tested.

Example 5: Effect of fructose-bisphosphatase 1 (FBP1) on AML blasts Generation of an AML Cell Line Overexpressing FBP1 (FBP1-MV411 Blast)

[0088] To investigate novel anti -leukemic functional roles of FBP1 in leukemic cells, the FBP1 gene was overexpressed in MV4-11 blasts via lentiviral transduction in vitro. FBP1 overexpression was assessed by GFP expression from the same lentiviral construct (FIG. 8A) and confirmed with PCR and Western blots. The FC data demonstrated that about 92.8% of the MV4-11 blasts were successfully transduced, confirming the establishment of a viable FBP1-MV4-11 blast line (FIG. 8B). Additionally, both phase-bright and fluorescent microscope imaging confirmed that viable leukemic cell clusters were GFP+ high, dying cells were GFP+ low (indicated by red circles, Figure IB), and dead cells were not fluorescent (indicated by arrows, FIG. 8C1). Finally, both qPCR and Western blotting assays confirmed the successful generation of the FBP1-MV4-11 cell line by showing significantly increased FBP1 transgene and protein expressions when compared to the native MV4-11 or GFP-MV4-11 control cell lines (FIG. 8D1, 8D2).

Molecular Phenotypes of the FBP1-MV4-11 Cell Line In Vitro

[0089] The functional properties of the FBP1-MV4-11 cell line were analyzed in vitro. To examine FBPlOs function in blast differentiation, three cell lines were analyzed by flow cytometry, including MV4- 11, an FBP1-MV4-11 cell line with one round of FBP1 lentiviral transduction, and an FBP1-MV4-11 cell line with two rounds of FBP1 lentiviral transduction (FIG. 9A1). The cell line with two rounds of lentiviral transduction (higher expression of FBP1, FIG. 9A2) demonstrated a significantly higher percentage (CD14+: 27.05%) of viable CD 14+ blasts (a biomarker of monocytic differentiation) when compared to both the naive control (CD14+: 1.287%) and the cell line (CD14+: 5.14%) with only one round of FBP1 lentiviral transduction, indicating that increased FBP1 overexpression could lead to enhanced differentiation of leukemia cells (FIG. 9B).

[0090] Flow cytometry (FC) analysis was also conducted to observe the effects of FBP1 gene expression on cell viability. The FC histogram demonstrated that increased FBP1 expression was directly correlated with increased death of FBP1-MV4-11 cells when compared to MV4-11 or GFP-MV4-11 blasts (FIG. 9C), consistent with a role of FBP1 in suppressing glycolysis and promoting apoptosis, leading to inhibition of tumorigenic proliferation. Interestingly, the phase-bright analysis showed certain FBP1-MV4-11 cells with membrane blebbing that were abnormally larger than their neighboring blasts, which displayed cellular shrinkage and fragmentation (indicated by arrows, FIG. 9D) — a finding associated with caspase-3 -induced apoptosis. To elucidate the mechanism of blast death, additional qPCR analysis was conducted on each cell line. The FBP1-MV4-11 cell line was found to have significantly increased gene expression of key pro- apoptotic mediators, including a 4.7-fold increase in caspase-3 and a 3.4-fold increase in Bcl-2 -associated X protein (BAX, FIG. 9E). In addition, FBP1 expression also significantly increased the gene expression of P53 — a tumor-suppressor gene (2.5-fold increase, FIG. 9E) known to initiate programmed cell death in leukemic cells. Finally, to confirm whether FBP1 activates the promoter of the P53 gene, a P53 promoter reporter lentiviral construct was transduced into FBP1-MV4-11 and MV4- 11 cells. Luciferase activity was measured in both blasts and secreted supernatants of each cell line via the dual-luciferase assay kit (GeneCopoeia) (FIGs. 9F, 9G). Increased luciferase radiance in the FBP1-MV4-11 cell line suggests a strong correlation between FBP1 overexpression and P53 induction. More specifically, luciferase analysis demonstrated FBPl’s role in activating the P53 gene promoter, supporting our hypothesis that increased FBP1 gene expression leads to increased anti-tumorigenesis properties.

Pyruvate and/or Glutamine Reversed Cell Death and Reduced the Proliferation ofFBPl-MV4-ll Cells by Inhibiting P 53 Expression In Vitro

[0091] To investigate the possibility of reversing the inhibitory effect of FBP1 on blast proliferation, we rescued FBP1-MV4-11 cells by supplementing them with pyruvate and/or glutamine in vitro. We hypothesized that the supplementation of key metabolites would allow blasts to bypass the glycolytic pathway that was inhibited by FBP1 through alternate energy-producing pathways to facilitate their continued survival and proliferation. FBP1- MV4-11 blasts were found to have significantly increased cell death (viable cells: 88.3%) and decreased expression of Ki67 (34.7%) — a biomarker for the proliferation of AML blasts — when compared to MV4-11 (viable cells: 95.6% and Ki67+: 59.1%) in vitro (FIGs. 10A, 10B). Interestingly, the addition of pyruvate (viable cells: 94.1% and Ki67+: 53.3%), glutamine (viable cells: 95.2% and Ki67+: 58.4%), and pyruvate + glutamine (viable cells: 97.4% and Ki67+: 61.3%) increased the percentage of viable blasts and accelerated the proliferation of FBP1-MV4-11, as shown by the increased percentage of Ki67+ expression (FIGs. 10A, 10B). Finally, to investigate a mechanistic link between FBP1 -activated P53 and phenotypes of leukemic progression, we compared the protein expression of P53 in each cell line. Both pyruvate and glutamine individually reduced P53 in FBP1-MV4-11 cells in vitro. However, the combination of pyruvate and glutamine had a much stronger inhibitory effect on P53 expression than each single agent (P53 protein density normalized to [3-actin: 0.39 in MV4- 11 versus 0.98 in FBP1-MV4-11, and 0.63 in FBP1-MV4-11 (+P/G); FIGs. 10C, 10D), showing an additive effect between glutamine and pyruvate in leukemic metabolism. These results also suggest that increased shunting of metabolism past the bottleneck point of our induced FBP1 overexpression led to decreased cellular apoptosis and continued leukemic growth.

Mitochondrial Metabolic Reprogramming Responds to Disturbed Metabolic Homeostasis in the FBP1-MV411 Cell Line

[0092] The correlation between FBP1 -altered carbohydrate metabolism and mitochondrial adaptive pathways was investigated. The mitochondria, the central hub of energy production to support leukemic survival and proliferation, largely depend on metabolites generated from carbohydrate pathways, including major products of glucose such as pyruvate and NADH, whose deprivation induces leukemic apoptosis. Pyruvate molecules produced by glycolysis are actively transported into the mitochondrial citric acid cycle (TCA or Krebs cycle) to generate ATP. Therefore, the intracellular concentration of pyruvate in FBP1- MV4-11 blasts was measured. FBP1-MV4-11 blasts were found to have significantly decreased concentrations of pyruvate (0.99 pM) in comparison to those of MV4-11 blasts (6.83 pM) in vitro (FIG. 11A). Although the TCA cycle utilizing oxidative phosphorylation (OXPHOS, aerobic respiration) can generate far more ATP (36 ATPs) than glycolysis (anaerobic respiration, 2 ATPs), the process depends on the availability of oxygen and metabolites in the leukemic microenvironment, emphasizing the vulnerability of biochemical processes in the mitochondria. Therefore, these results suggest an inverse relationship between FBP 1 expression and glycolytic activity.

[0093] To elucidate the mechanism by which FBP1 overexpression affects the mitochondria, additional qPCR studies were conducted. The FBP1-MV4-11 cell line was found to have significantly increased gene expression of fibroblast growth factor 21 (FGF21, 2.95-fold increase) and growth differentiation factor 15 (GDF15, 1.66-fold increase) (FIG. 11B), which are biomarkers of mitochondrial dysfunction and OXPHOS deficiency. Additionally, targeting electron transport chain (ETC) complex proteins has been found to shut down energy machinery and facilitate the leakage of pro-apoptotic proteins in the mitochondria. Cytochrome c oxidase subunit 2 (COX2, or MT-C02) — the functional core of cytochrome c oxidase — plays an essential role in the transfer of electrons in the OXPHOS process and, unfortunately, serves as an adverse prognostic marker for adult AML. Interestingly, the results showed FBP1-MV4-11 blasts had significantly reduced MT-C02, suggesting that FBP1 may also play a disruptive role in mitochondrial aerobic respiratory chains (FIG. HD).

[0094] In addition to building an abnormal energy production pathway to support uncontrolled blast proliferation, leukemic cells also utilize metabolic adaptation to create resistance to treatment. The protein analysis showed increased expression of PTEN-induced kinase (PINK1) - a biomarker of mitophagy - indicating the activation of the mitochondrial quality control system in FBP1-MV4-11 blasts (PINK1 protein density normalized to [3-actin: 0.4 in MV4-11 versus 0.56 in FBP1-MV4-11, FIG. HD). In summary, the results suggest that FBP 1 -altered leukemic metabolism leads to the activation of mitochondrial adaptive pathways to maintain metabolic homeostasis, supporting the survival and continued proliferation of AML blasts.

Anti-Leukemic Functional Roles of FBP 1 and FBP -Activated P53 in AML Blasts In Vitro

[0095] The results show that FBP1 has a multifaceted impact on the regulation of leukemic survival and growth in vitro. As summarized in FIG. 12, FBP1 simultaneously stimulated the apoptosis of blasts while also preventing leukemic growth through the regulation of pro-apoptotic and tumor-suppressive genes, such as caspase-3, BAX, and P53. Furthermore, the P53 promoter assay results suggest that one of the most significant non-catalytic regulatory functions of FBP 1 is to stimulate the expression of the tumor-suppressor P53 gene, whose downregulation or mutation characterizes a distinct feature of AML. Interestingly, increased P53 production in FBP1-MV4-11 blasts could be reversed by adding post-glycolytic metabolites, such as pyruvate and glutamine. Mechanistically, it was found that pyruvate-deprivation-induced cell-cycle arrest is associated with augmented P53 pathways via the upregulation of the P21 protein — a cyclin- dependent kinase inhibitor and target of P53. Moreover, P53 has been found to be regulated by aerobic glycolysis in cancer cells. A similar relationship was also seen between glutamine and P53. The data suggest that pyruvate and glutamine might provide the blasts with alternative energy-producing pathways to facilitate continued leukemic survival and proliferation through the decrease in P53 gene expression.

[0096] Another significant phenotype in FBP1-MV4-11 blasts is the activation of mitochondrial reprogramming. The FBPl-mediated reduction in pyruvate and COX2 (MT-C02) provided herein demonstrates the tightly coupled relationship between glycolysis and oxidative phosphorylation (OXPHOS), because pyruvate — the end product of glycolysis — is fuel for OXPHOS. Therefore, FBP1- subverted glycolysis (BOX1, FIG. 12). can be linked to an impaired OXPHOS process, leading to mitochondrial stress or dysfunction (BOX2, FIG. 12). To maintain metabolic homeostasis and survival, FBP1-MV4-11 blasts can activate the mitochondrial repair process ofmitophagy via upregulation of PINK1 (BOX3, FIG. 12). Activated mitophagy and autophagy replenish key metabolites such as fatty acids and glutamine, to either promote the regeneration of dysfunctional mitochondria or prevent cell death (BOX4, FIG. 12)

Example 6: Effect of fructose-bisphosphatase 1 (FBP1) and protein phosphatase 2 catalytic subunit alpha (PPP2CA) on AML blasts

[0097] As disclosed herein, vitamin D induces FBP1 expression ( such as an approximately 3000- fold increase according to the Examples disclosed herein) and vitamin D and FBP1 reduce energy production in AML metabolism. FBP1 and PPP2CA are phosphatases that also possess a tumor suppressor function. To test effects of FBP1 and PPP2CA on AML blast proliferation and metabolism, a double-ORF lentiviral plasmid encoding both FBP1 and PPP2CA was generated and validated. The diagram of the double ORF lentiviral vector as well as the control lentiviral vectors are depicted in FIG. 13.

[0098] Mesenchymal stem cells (MSCs) are transfected with the FBP1-PPP2CA double phosphatase construct (such as the double-ORF lentiviral construct described herein, such as a dual- phosphatase lentiviral vector) or a control construct. The effect of FBP1 and/or PPP2CA on cell viability, proliferation, and metabolism in vitro and ex vivo is tested, including their effects on lactic acid production and expression of cytokines and various genes, using qPCR, western blotting, functional assays, and any other methods known in the art.

Example 7: Combination Therapy of TKIs, CXCR2-CAR-TILs, and MSCs- FBP1/PPP2CA to treat AML

[0099] CAR-TILs targeting CXCR2 (“CXCR2-CAR-TILs”), or dual specificity CAR-TILs targeting CXCR2 and CD33 (“CXCR2/CD33 -CAR-TILs”) are generated as described for instance in Cao et al. 2021 Neoplasia 23(12): 1252-1260, which is incorporated by reference in its entirety. An exemplary diagram of a dual specific CAR is depicted in FIG. 7.

[0100] AML-FLT3 blasts are harvested from a subject and are contacted ex vivo with TKIs, CXCR2-TILs (or CXCR2/CD33-CAR-TILs), and MSCs transfected with the FBP1-PPP2CA double phosphatase construct (such as the double-ORF lentiviral construct described herein) (“MSCs-FBPl/PPP2CA”). The effect of the combination therapy on tumor viability, proliferation, and metabolism ex vivo is tested, including their effects on lactic acid production, AML blast cell numbers, and expression of cytokines and various genes, using qPCR, western blotting, functional assays, and any other methods known in the art.

[0101] Further, AML-FLT3 blasts are xenografted in mice and are contacted in vivo with TKIs, CXCR2- TILs (or CXCR2/CD33 -CAR-TILs), and MSCs transfected with the FBP1-PPP2CA double phosphatase construct (such as the double-ORF lentiviral construct described herein) (“MSCs-FBPl/PPP2CA”). The effect of the combination therapy on tumor viability, proliferation, and metabolism in vivo is tested, including their effects on lactic acid production, AML blast cell numbers, and expression of cytokines and various genes, using qPCR, western blotting, functional assays, and any other methods known in the art.

Materials and Methods

Human samples

[0102] AML bone marrow mononuclear cells (BMMNC) were obtained from the City of Hope National Medical Center (COHNMC). All donor patients signed an informed consent form. Sample acquisition was approved by the Institutional Review Boards at the LLUMC and the COHNMC in accordance with an assurance filed with and approved by the Department of Health and Human Services, and it met all requirements of the Declaration of Helsinki.

Cell culture of AML cells and treatment of blasts by active vitamin D (1,25VD3 or 1,25(OH)2D3) and TKIs

[0103] MV4-11 (ATCC CRL-9591) and MOLM-14 (DSMZ ACC-777) are human-derived AML blast cell lines with FLT3-ITD. The AML cells (either cell lines or primary AML BMMNC) were cultured in RPMI- 1640 medium (HyClone) supplemented with 10% heat- inactivated fetal bovine serum (HyClone) and 1% penicillin/streptomycin. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2. As single agents to treat blasts in vitro, one dose of 80nM of 1,25VD3, GILT, QUIZ, or a combination of 80nM 1,25VD3 and GILT was added to 1 ml of lx 106 MV4-11 cells for each experimental group in 24 well plates. Two days after the one dose treatment, cells were then collected for further analyses of gene or protein expressions.

RNA-sequencing and data processing

[0104] AML cells from MV4-11, MOLM-14, MV4-11-TKI-resistant and M0LM-14-TKI- resistant cell lines were treated for 48 hours with or without 80nM 1,25VD3. Cell samples were collected and sent to BGI for RNA sequencing (RNA-seq). The RNA-seq libraries were prepared and fragments per kilobase of exon per million fragments mapped (FPKM) were applied to compare gene expression of samples between different treatment groups (FIG. 4).

Preparation ofFBPl Lentivirus and Generation ofFBP-MV4-ll Cell Lines In Vitro

[0105] The lentiviral transfer plasmid contains a full-length open reading frame (ORF) of human FBP1 (GeneCopoeia catalog: EX-Z5880-Lv225, Rockville, MD, USA). This plasmid expresses FBP1 and GFP, controlled by the EFl and IRES promoters, respectively (FIG. 8A). A GFP empty vector (GeneCopoeia catalog: EX-NEG-Lv225) was used as the vector control, which as an EFla promoter and IRES-GFP.

[0106] Lentiviruses were prepared as previously described (Xu et al. 2021 Int. J. Mol. Sci. 22, 9516). Briefly, HEK-293T cells were cultured in complete Dulbecco’s modified Eagle medium (DMEM, Gibco, Waltham, MA, USA) containing 10% FBS and 100 U/mL penicillin/streptomycin. When the cells were 70-80% confluent, the culture medium was replenished, and a transfection solution containing an envelope, packaging, and transfer plasmid (FBP1) was added dropwise to the cells. After the cells were cultured at 37 °C and in 5% CO2 for 48 h, the supernatants were collected, filtered through a 0.45 pm filter, and centrifuged at 4800 g at 4 °C for 24 h. The virus pellet was reconstituted in PBS containing 5% glycerol and titrated by a GFP-based flow cytometry method. The typical titer of virus was 108-109 transducing units/mL. The MV4-11 cells were transduced with FBP1 lentivirus at a multiplicity of infection (MOI) of 5. Twenty-four hours later, the virus was removed, and the culture medium was replenished. The cells were cultured for 24 h and examined for transduction efficiency via fluorescence microscopy and flow cytometry. When necessary, the above transduction procedure was repeated one more time to create a cell line with two rounds of transduction. FBP1-MV4-11 cells were purified by fluorescence-activated cell sorting (FACS; Research Core Facility, School of Medicine, University of California, Riverside). To generate FBP1-MV4-11 rescue cell lines, extra doses of 1 mM sodium pyruvate (Gibco 11360070) and/or 2 mM L- glutamine (Gibco 25030081) were added to the culture medium to treat blasts in vitro.

P53 Promoter Assay In Vitro [0107] A lentiviral plasmid containing the promoter reporter clone with Gaussia luciferase reporter for human TP53 (NM_001126115; GeneCopoeia catalog: HPRM34629-EvPG04- 50) was acquired. The preparation of the lentivirus of the P53 promoter reporter was performed similarly to that of the FBP1 lentivirus previously mentioned. The P53 promoter reporter was transduced into MV4-11 and FBP-MV4- 11 cell lines to generate P53-MV4-11 and P53-FBP1-MV4-11 blasts in vitro. The detection of P53 activity in blasts and their secreted supernatants was performed using the Secrete-Pair™ Dual Luminescence Assay Kit (GeneCopoeia, Rockville, MD, USA). Images were acquired through a high-resolution CCD camera (Perkin Elmer IVIS Lumina III, Waltham, MA, USA).

Flow cytometry (FC)

[0108] Cells were harvested and examined for the expression of cell surface biomarkers and intracellular proteins by multichromatic FC as previously described [14], About 1 x 10⁴ - 1 x 10⁶ cells in 100 pl FC buffer (PBS containing 1% FBS and 0.05% sodium azide) were stained with various fluorescence- conjugated antibodies specific for the desired cell surface proteins at 4oC for 30min. The surface-stained cells were then fixed and permeabilized using the appropriate reagents (e.g. the BD Pharmingen Cytofix/Cytoperm buffer) and stained with different fluorescence-conjugated antibodies specific for the desired intracellular proteins at 4°C for 2 hours in the permeabilizing buffer (e.g. the BD Perm/Wash buffer). Concentrations of the Abs were used per the manufacturers’ recommendations. Finally, the cells were washed twice in the permeabilizing buffer and twice in the FC buffer before being analyzed on the BD FACSAria II. Data was analyzed using the FlowJo software (Tree Star Inc., Ashland, OR).

RNA isolation and real-time polymerase chain reaction (qPCR) analysis

[0109] MV4-11 or primary BMMNC was cultured with the presence of appropriate treatments for 48 hours. Cells were isolated for RNA isolation and qPCR analysis as previously described [12], Total RNA was isolated using the RNeasy Micro Kit (Qiagen) according to the manufacturer’s instruction. First-strand cDNA was synthesized using the SuperScript III Reverse Transcriptase (Invitrogen). With an Applied Biosystems 7900HT Real-Time PCR machine, qPCR was performed and analyzed. Primers used in this study are available in Table 2. The PCR conditions were 10 minutes at 95°C followed by 40 cycles of 10 seconds at 95°C and 15 seconds at 60°C. The relative expression level of a gene was determined using the AACt method and normalized to GAPDH or [3-actin.

Western blotting (WB) analysis

[0110] AMU cells were collected for WB as previously described (Xu et al. 2020 Transl. Oncol. 13, 100869). Briefly, cells were homogenized in ice-cold lysis buffer composed of 20mM Hepes, pH 7.5, lOmM KC1, 1.5mM MgC12, ImM ethylenediaminetetraacetic acid, ImM dithiothreitol, ImM phenylmethylsulfonyl fluoride, 2ug/ml of aprotinin, and lOug/ml of leupeptin, followed by incubation on ice for 30 min. The homogenates were ultrasonicated and centrifuged at 20000 X g for 30 min at 4°C. Samples with equal quantities of protein were loaded onto 10% SDS- polyacrylamide gel and separated by electrophoresis at 100 V for 1 h. Proteins were then transferred onto Immobilon-P membranes (Millipore Corporation, Billerica, MA) and were probed with the primary antibody of FBP1 (Invitrogen). After incubation with horseradish peroxidase-conjugated secondary antibody (Amersham, Arlington Heights, IL), proteins were visualized with a chemiluminescence reagent, and the blots were exposed to Hyperfilm (Amersham). Results were quantified using the Kodak electrophoresis documentation and analysis system and Kodak ID image analysis software (Eastman Kodak, Rochester, NY).

Immunocytochemistry (ICC) and imaging acquisition

[oni] ICC staining of treated MV4-11 cells was performed according to the established protocol in the previous report. Fluorescent images were taken using an Olympus 1X71 microscope and were processed using an Olympus cellSens Dimension 1.15 Imaging Software (Tokyo, Japan).

Lactate assay

[0112] MV4-11 cells were treated with or without l,25VDs for 48 hours. The cell number of each experimental group was counted to have equal cell numbers per sample. Then, the cell samples were processed according to the manufacturer’s protocol (Catalog Number: MAK064, Sigma- Aldrich). Briefly, the lactate concentration of each sample was determined by an enzymatic assay, resulting in a colorimetric product, proportional to the lactate present. The experimental plate (FIG. 5) was read with a spectrophotometric microplate reader at 570 nm. The concentration of lactate within samples was calculated by comparing the sample OD to the standard curve.

Pyruvate assay

[0113] The cell numbers of each experimental group were counted to ensure equal cell numbers per sample. The cell samples were then processed according to the manufacturer’s protocol (Sigma- Aldrich catalog: MAK332, St. Louis, MO, USA). Briefly, the lactate concentration of each sample was determined by an enzymatic assay, resulting in a colorimetric product proportional to the concentration of pyruvate present. The experimental plates were read on a spectrophotometric microplate reader at 570 nm. The pyruvate concentration within the samples was calculated by comparing the sample OD to the standard curve.

Statistical analysis

[0114] Statistical analyses were performed with GraphPad software (Prism 5.02, San Diego, CA, USA). The quantitative analyses were analyzed with 1- or 2-way ANOVA, followed by Dunnett’s multiple comparisons test, Bonferroni’s post hoc analysis, or an unpaired t-test, as appropriate. All values were presented as mean ± SEM. Results were considered significant when the P value was <0.05. Table 2. List of Exemplary Reagents

Table 3. List of Exemplary Primers

Table 4. FBP1 and PPP2CA mRNA Sequences

Claims

What is claimed is: A pharmaceutical composition comprising a therapeutically effective amount of 1,25- dihydroxyvitamin D3 (1,25VD3) and a tyrosine kinase inhibitor (TKI). The pharmaceutical composition of claim 1, wherein the TKI is gilteritinib, quizartinib, or midostaurin. A nucleic acid construct comprising:

(a) a nucleic acid molecule encoding fructose-bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell; and/or

(b) a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell. The nucleic acid construct of claim 3, wherein:

(a) the nucleic acid molecule encoding FBP1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, wherein a corresponding encoded polypeptide retains a FBP1 function, or a nucleic acid sequence of SEQ ID NO: 25; and/or

(b) the nucleic acid molecule encoding PPP2CA comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 27, wherein the corresponding encoded polypeptide retains a PPP2CA function, or a nucleic acid sequence of SEQ ID NO: 27. The nucleic acid construct of claim 3 or 4, comprising a lentiviral vector. A cell comprising the nucleic acid construct of claim 3 or 4, or the lentiviral vector of claim 5. The cell of claim 6, wherein the cell is a mesenchymal stem cell or a tumor infdtrating lymphocyte. A pharmaceutical composition comprising a tumor infiltrating lymphocyte (TIL) comprising a chimeric antigen receptor (CAR) targeting CXCR2. The pharmaceutical composition of claim 8, wherein the TIL further comprises a CAR targeting CD33. A method for reducing viability and/or proliferation of an acute myeloid leukemia (AML) cell, comprising: contacting the AML cell with an effective amount of a composition comprising: a tyrosine kinase inhibitor (TKI); and one or more of:

(i) 1,25-dihydroxyvitamin D3 (1,25VD3);

(ii) a nucleic acid construct comprising: (a) a nucleic acid molecule encoding fructose- bisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell; and/or (b) a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell;

(iii) a cell comprising the nucleic acid construct of (ii); and

(iv) a tumor infiltrating lymphocyte (TIL) expressing a chimeric antigen receptor (CAR) targeting CXCR2. The method of claim 10, wherein the TIL further comprises a CAR targeting CD33. The method of claim 10 or 11, wherein:

(a) the nucleic acid molecule encoding FBP1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, wherein a corresponding encoded polypeptide retains a FBP1 function, or the nucleic acid sequence of SEQ ID NO: 25; and/or

(b) the nucleic acid molecule encoding PPP2CA comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 27, wherein the corresponding encoded polypeptide retains a PPP2CA function, or the nucleic acid sequence of SEQ ID NO: 27. The method of any one of claims 10 to 12, wherein the nucleic acid construct comprises a lentiviral vector. The method of any one of claims 10 to 13, wherein by contacting the AML cell with the nucleic acid construct, the nucleic acid construct is introduced into the AML cell. The method of any one of claims 10 to 14, wherein the cell comprising the nucleic acid construct is a mesenchymal stem cell (MSC) or a TIL. The method of any one of claims 10 to 15, wherein expression and/or function of fructose- bisphosphatase 1 (FBP1) is increased in the AML cell compared to a control AML cell. The method of any one of claims 10 to 16, wherein expression and/or function of cyclin DI and/or cyclin dependent kinase 1 (CDK1) is reduced in the AML cell compared to a control AML cell. The method of any one of claims 10 to 17, wherein expression and/or function of one or more pro-apoptotic or tumor-suppressive genes is reduced in the AML cell compared to a control AML cell. The method of claim 18, wherein the one or more pro-apoptotic or tumor-suppressive genes comprise caspase-3, BAX, or P53. The method of any one of 10 to 19, wherein glycolysis, oxidative phosphorylation, energy production, and/or levels of intracellular pyruvate and/or cytochrome c oxidase subunit 2 (MT- CO2) are reduced in the AML cell compared to a control AML cell. The method of any one of claims 10 to 20, comprising contacting a plurality of AML cells with an effective amount of the composition, wherein a number of viable mitotic AML cells are reduced compared to control AML cells.

22. The method of any one of claims 10 to 21, wherein the TKI is gilteritinib, quizartinib, or midostaurin.

23. The method of any one of claims 10 to 22, wherein the AML cell has one or more mutations in FMS-like tyrosine kinase 3 (FLT3) gene.

24. The method of any one of claims 10 to 23, wherein the AML cell is in a subject.

25. A method for treating acute myeloid leukemia (AML) in a subject, the method comprising: administering to the subject an effective amount of a composition comprising: a tyrosine kinase inhibitor (TKI); and one or more of:

(i) 1,25-dihydroxyvitamin D3 (1,25VD3);

(ii) a nucleic acid construct comprising: (a) a nucleic acid molecule encoding fructosebisphosphatase 1 (FBP1), operably linked to a promoter functional in an animal cell; and/or (b) a nucleic acid molecule encoding protein phosphatase 2 catalytic subunit alpha (PPP2CA), operably linked to a promoter functional in an animal cell;

(iii) a cell comprising the nucleic acid construct of (ii); and

(iv) a tumor infiltrating lymphocyte (TIL) expressing a chimeric antigen receptor (CAR) targeting CXCR2.

26. The method of claim 25, wherein the TIL further comprises a CAR targeting CD33.

27. The method of claim 25 or 26, wherein:

(a) the nucleic acid molecule encoding FBP1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 25, wherein the encoded polypeptide retains a FBP1 function, or the nucleic acid sequence of SEQ ID NO: 25; and/or

(b) the nucleic acid molecule encoding PPP2CA comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 27, wherein the encoded polypeptide retains a PPP2CA function, or the nucleic acid sequence of SEQ ID NO: 27.

28. The method of any one of claims 25 to 27, wherein the nucleic acid construct comprises a lentiviral vector.

29. The method of any one of claims 25 to 28, wherein by administering the nucleic acid construct to the subject, the nucleic acid construct is introduced into an AML cell in the subject.

30. The method of any one of claims 25 to 29, wherein the cell comprising the nucleic acid construct is a mesenchymal stem cell (MSC) or a TIL.

31. The method of any one of claims 25 to 30, wherein expression and/or function of fructose- bisphosphatase 1 (FBP1) is increased in the subject compared to a control. The method of any one of claims 25 to 31, wherein expression and/or function of cyclin DI and/or cyclin dependent kinase 1 (CDK1) is reduced in the subject compared to a control. The method of any one of claims 25 to 32, wherein expression and/or function of one or more pro-apoptotic or tumor-suppressive genes is reduced in the subject compared to a control. The method of claim 33, wherein the one or more pro-apoptotic or tumor-suppressive genes comprise caspase-3, BAX, or P53. The method of any one of claims 25 to 34, wherein glycolysis, oxidative phosphorylation, energy production, and/or levels of intracellular pyruvate and/or cytochrome c oxidase subunit 2 (MT- CO2) are reduced in the subject compared to a control. The method of any one of claims 25 to 35, wherein a number of viable mitotic AML cells are reduced in the subject compared to a control. The method of any one of claims 25 to 36, wherein one or more symptoms associated with AML is reduced in the subject. The method of any one of claims 25 to 37, wherein the TKI is gilteritinib, quizartinib, or midostaurin. The method of any one of claims 25 to 38, wherein the subject has one or more mutations in FMS-like tyrosine kinase 3 gene.