Compositions and methods for nutrient-based immunotherapy to treat acute myeloid leukemia

Abstract

A nucleic acid construct encoding glucose transporter 1 or mitochondrion transcription factor A, and a vector or a cell containing such a nucleic acid construct are provided. An engineered T cell overexpressing GLUT1 and / or TFAM is also provided. Also provided are methods for reducing viability and / or proliferation of an AML cell by contacting the AML cell with the engineered T cell composition, as well as methods for treating AML in a subject by administering the subject the engineered T cell composition.

Description

COMPOSITIONS AND METHODS FOR NUTRIENT-BASED IMMUNOTHERAPY TO TREAT ACUTE MYELOID LEUKEMIAInventors: Yi (David) Xu and Huynh CaoCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 512,401, filed July 7, 2023, which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] The disclosure relates to compositions and methods of treatment of cancer utilizing a therapeutic composition, more specifically for tire treatment of leukemia.BACKGROUND

[0003] 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. Comorbidity burden, such as with hyperglycemia / diabetes mellitus, has been associated with an increased risk of up to 20% for leukemia and a significant increase in tire odds of AML mortality. 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 30.5%.

[0004] 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

[0005] 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.

[0006] In one aspect, the present disclosure provides a first nucleic acid construct comprising a nucleic acid molecule encoding glucose transporter 1 (GLUT1), operably linked to a promoter functional in an animal cell, or a second nucleic acid molecule encoding mitochondrion transcription factor A (TFAM), operably linked to a promoter functional in an animal cell, or both nucleic acid molecules.

[0007] In some embodiments, the nucleic acid molecule encoding GLUT1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT1 function, or a nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the nucleicacid molecule encoding TFAM comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 16, wherein the corresponding encoded polypeptide retains a TFAM function, or a nucleic acid sequence of SEQ ID NO: 16. In some embodiments, tire nucleic acid construct includes a reporter gene operatively linked to the promoter.

[0008] Certain embodiments include a vector with the nucleic acid construct provided herein containing a first nucleic acid molecule encoding GLUT 1 , operably linked to a promoter functional in an animal cell, and / or a second nucleic acid molecule encoding TFAM, operably linked to a promoter functional in an animal cell. In some embodiments, the vector is a lentiviral vector.

[0009] Certain embodiments include a cell comprising the nucleic acid construct or the vector provided herein. In some embodiments, the cell is a T cell. Certain embodiments include an engineered T cell overexpressing GLUT1 and / or TFAM relative to a control T cell. In some embodiments, treatments with the engineered T cell leads to attenuated cell exhaustion, increased tumor immunosuppression, and / or increased cell proliferation relative to treatment with a control T cell.

[0010] In some embodiments, the cells exhibit increased level or activity of nuclear protein Kiel 67 (Ki67), IL-2, and / or cyclin-dependent kinase 1 (CDK1), and / or decreased level or activity of nuclear receptor 4A1 (Nur77) relative to a control cell. Certain embodiments include a composition containing the cells comprising the nucleic acid construct or the vector provided herein, and the engineered T cell overexpresses GLUT1 and / or TFAM relative to a control T cell.

[0011] Certain embodiments include 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 containing an engineered T cell overexpressing GLUT1 and / or TEAM relative to a control T cell. In some embodiments, the engineered T cell contains a nucleic acid construct comprising (a) a nucleic acid molecule encoding GLUT1. operably linked to a promoter functional in an animal cell, or (b) a nucleic acid molecule encoding TFAM, operably linked to a promoter functional in an animal cell, or both (a) and (b). In some embodiments, the nucleic acid molecule encoding GLUT1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT1 function, or the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the nucleic acid molecule encoding TFAM comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 16, wherein the corresponding encoded polypeptide retains a TFAM function, or the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the engineered T cell is generated by transfecting a T cell with a viral vector containing a nucleic acid construct described herein. In some embodiments, the viral vector is a lentiviral vector.

[0012] In some embodiments, treatments with the engineered T cell lead to attenuated cell exhaustion, increased tumor immunosuppression, and increased cell proliferation relative to treatment with a control T cell. In some embodiments, the engineered T cell exhibits increased level or activity of Ki67, IL-2, and / orcyclin-dependent kinase 1 (CDK1), and / or decreased level or activity of nuclear receptor 4A1 (Nur77) relative to a control T cell.

[0013] Certain embodiments include a method for decreasing proliferation of AML cells that includes contacting a plurality of AML cells with an effective amount of the composition, wherein the number of viable mitotic AML cells are reduced compared to control AML cells. In some embodiments, the AML cell is in a subject. In some embodiments, mitosis and / or glucose uptake is decreased in the AML cell compared to a control AML cell. In some embodiments, level or activity of cyclin Bl is reduced in the AML cell compared to a control AML cell.

[0014] Certain embodiments include a method for treating AML in a subject by administering to the subject an effective amount of a composition containing an engineered T cell overexpressing GLUT1 and / or TFAM relative to a control T cell. Certain embodiments include the engineered T cell contains a nucleic acid construct with (a) a nucleic acid molecule encoding GLUT1, operably linked to a promoter functional in an animal cell and / or (b) a nucleic acid molecule encoding TFAM, operably linked to a promoter functional in an animal cell. In some embodiments, the nucleic acid molecule encoding GLUT1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT1 function, or the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the nucleic acid molecule encoding TFAM comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 16, wherein the corresponding encoded polypeptide retains a TFAM function, or the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the engineered T cell is generated by transfecting a T cell with a viral vector comprising the nucleic acid construct. In some embodiments, tire viral vector is a lentiviral vector.

[0015] In some embodiments, treatments with the engineered T cell lead to attenuated cell exhaustion, increased tumor immunosuppression, and increased cell proliferation relative to a control T cell. In some embodiments, the engineered T cell exhibits increased level or activity of Ki67, IL-2, and / or cyclin-dependent kinase 1 (CDK1), and / or decreased level or activity of nuclear receptor 4A1 (Nur77) relative to a control T cell. In some embodiments, administering the composition decreases mitosis and / or glucose uptake in AML cells in the subject. In some embodiments, administering the composition decreases level or activity of cyclin Bl in AML cells in the subject. In some embodiments, administering the composition decreases a number of viable mitotic AML cells in the subject. In some embodiments, administering the composition decreases one or more symptoms associated with AML in the subject. Embodiments include methods of selecting a T cell for a T-cell based immunotherapy by evaluating glucose sensitivity / tolerance of the T cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. Embodiments are illustrated by way of example and not by way of limitation in the accompanying drawings.

[0018] FIGs. 1A-1D depict effects of high doses of glucose on the size of T cell clusters in AML patient samples ex vivo. FIG. 1A depicts representative phase-bright images (4x) of T cells isolated from peripheral blood (PB) of a healthy control subject (#1) and AML patient (#1). FIG. IB is a set of representative phase- bright images in higher magnification (20x) of the T cell clusters. FIG. 1C is a graphical presentation of the cumulative data of cluster size of T cells isolated from healthy control PB and AML patient PB. FIG. ID is a set of representative phase-bright images (4x) of T cells isolated from peripheral blood (PB) of another healthy control (#2) and two AML patient (#2 and 3). Where applicable, data are means ± SEM and were analyzed by student “t” test. *P < 0.05, **P < 0.01, n = 3.

[0019] FIG. IE presents an RNA-sequencing heatmap of two AML patients-derived T cells (AML-T1 or T2), which were treated with 50mM glucose or left untreated. FIG. IF presents a partial list of key ATP / metabolic processes-related genes encoded by mitochondrial genome and nuclear genome of AML T1 / T2. Cumulative TPM data of MT-ATP8 gene expression in untreated (NO-TX) and 50mM glucose-treated AML-T cells; n=2.

[0020] FIGs. 1G-1I depict results of transcriptomic analyses (RNA-sequencing or RNA-seq) of AML-T cells (n=2 AML patients). FIG. 1G depicts cumulative TMP data of MT-ATP8 (mitochondrially encoded ATP synthase membrane subunit 8) gene expression in untreated (NO-TX) and 50 mM glucose-treated AML-T cells; N = 2. FIG. 1H (upper panel) is a heatmap showing three mitochondria-related RNA expression changes in NO-TX and 50mM glucose-treated AML-T cells. FIG. 1H (lower panel) presents the cumulative TPM data of FIS 1 gene expression in NO-TX versus 50mM glucose-treated AML-T cells. FIG. II presents expression levels measured as FPKM (Fragments Per Kilobase per Million mapped fragments) of PD-1 (programmed cell death protein 1) in AML-T cells with or without 50mM glucose treatment. n=2 AML patient T cells. Where applicable, data are means ± SEM and were analyzed by student “t” test. *P<0.05, **P<0.01. FIG. 1J is an illustration of how the mitochondrion (Mito)-nucleus crosstalk would be affected by high-dose glucose.

[0021] FIGs. 2A-2C depict molecular phenotypes of glucose-treated Primary T cells ex vivo. FIG. 2A presents representative flow cytometry (FC) plots showing CD62L and CCR7 expression in healthy control- derived T cells and AML PB -derived T cells which were pretreated with different doses of glucose (left panel), and graphs showing cumulative percentage data of CCR7+cells in control and AML glucose-treated T cellsfrom the FC plots (top and bottom, right panel, respectively). FIG. 2B presents representative flow cytometry (FC) plots (left panel) showing CD3 and CD8 expression in healthy control and AML PB -derived T cells, which were pretreated with different doses of glucose, and graphs showing cumulative percentage data of CD3+CD8+cells in control and AML glucose-treated T cells from the FC plots (top and bottom, right panel, respectively). FIG. 2C depicts representative flow cytometry (FC) plots (left panel) showing CD3 and Ki67 expression in healthy control-derived T cells and AML PB-derived T cells, which were pretreated with different doses of glucose, and graphs showing cumulative percentage data of Ki67+CD3+cells in control and AML glucose-treated T cells from tire FC plots (top and bottom, right panel, respectively). Where applicable, data are means ± SEM and were analyzed by student “t” test. *P < 0.05, n = 3.

[0022] FIGs. 3A-3C depict effects of high doses of glucose on GLUT1 and TFAM expression in primary T cells and Jurkat cells. FIG. 3A presents graphical representation of the gene expression of TFAM analyzed by qPCR. Data of mRNA expressions show the fold change (normalized to (Lactin) of genes encoding TFAM in T cells from healthy control and AML patient samples, which were pretreated with different doses of glucose. FIG. 3B depicts representative FC histograms showing the expression of GLUT1 and Ki67 in Jurkat cells (left and right panels, respectively), which were pretreated with 25mM glucose for 48h (red line plot) or persistent supplementation of 25mM glucose for 4 weeks in vitro (blue line plot). The filled grey line and pink line plots represent Jurkat cells without treatment and IgG-fluore scent control. FIG. 3C (left panel) depicts representative FC histograms showing the expression of TFAM in Jurkat cells which were pretreated with 25mM glucose for 48h (red line plot) or persistent supplementation of 25mM glucose for 4 weeks in vitro (blue line plot); the filled grey line and pink line plots represent Jurkat w ithout treatment and IgG-fluore scent control. FIG. 3C (right panel) is a graphical representation of the results from qPCR data of mRNA expressions showing tire fold change (normalized to [3-actin) of the gene encoding TFAM in Jurkat cells, which were pretreated with or without different time-point of 25mM glucose. Where applicable, data are means ± SEM and were analyzed by student ‘‘t’’ test. *p < 0.05, **P < 0.01, n = 3.

[0023] FIGs. 4A-4D depict generation of Jurkat cell lines genetically bioengineered to overexpress SLC2A1 (GLUT1) and / or TFAM in vitro. FIG. 4A is an illustration of the lentiviral expression constructs containing human GLUT1 or TFAM open-reading frames and GFP or Cherry reporters, operably linked to promoters EFla and IRES, respectively. FIG. 4B is a representative FC histogram showing GFP and / or Cherry expression in GLUTl-Jurkat. TFAM-Jurkat and GLUT 1 / TFAM- Jurkat (FACS-sorted cells). FIG. 4C presents graphical representations of the expression of GLUT1 and TFAM analyzed by qPCR. Expressed by the fold change (normalized to [Lactin) of genes encoding GLUT 1 and TFAM in new Jurkat cell lines (left and right panels, respectively). FIG. 4D depicts representative FC histograms showing GLUT1 and TFAM expression (left and right panels, respectively) in GLUTl-Jurkat (red line plots) versus vector-control Jurkat (GFP-Jurkat, the filled grey line) and IgG-fluorescent control (pink line plots). Where applicable, data are means ± SEM and were analyzed by student "4” test. *p < 0.05, **P < 0.01, ***P < 0.005, n = 3.

[0024] FIGs. 5A-5C depict molecular and genetic phenotypes of the engineered Jurkat cell lines of the present disclosure in vitro. FIG. 5A (left panel) is a representative FC histogram showing the expression of Ki67 in the Jurkat cell lines, including GLUT 1 -expressing Jurkat (green line plot), TFAM-expressing Jurkat (orange line plot),GLUTl / TFAM-expressing Jurkat (blue line plot), as compared to the control, GFP- expressing Jurkat (the filled grey line) and IgG-fluore scent control (pink line plot). FIG. 5A (right panel) is a graphical representation of the cumulative data of the mean fluorescence intensity (MFI) levels of Ki67 in the four Jurkat cell lines. FIG. 5B is a set of graphical representations of qPCR analysis of the gene expression of different cytokines and biomarkers for cell divisions, inhibitory signals, and exhaustion. Data of mRNA expressions show the fold change (normalized to [3-actin) of genes encoding IL-2, CDK1 and Nur77 in the engineered Jurkat cell lines. FIG. 5C depicts images of blot films developed for proteomic analyses of cell- free supernatants from the engineered Jurkat cell lines, including GLUT 1 -Jurkat, TFAM-Jurkat, versus GFP- Jurkat. Where applicable, data are means ± SEM and were analyzed by student “t” test. *P < 0.05. **P < 0.01. n = 3.

[0025] FIGs. 6A - 6E depict the ability of GLUT1- or / TFAM-expressing Jurkats to compete for glucose and reduce viable blasts in vitro. FIG. 6A (left panel) depicts representative FC plots showing the expression of CD15. a biomarker for MV4-11 blasts, and CD3 in different cocultures, including GLUTl-Jurkat coculturing with MV4-11, GLUTl / TFAM-Jurkat coculturing with MV4-11 versus GFP-Jurkat coculturing with MV4-11, MV4-11 only and GLUTl-Jurkat only in vitro. FIG. 6A (right panel) is a graphical representation of the cumulative FC percentage of viable CD15+cells in different experimental groups. FIG. 6B depicts representative FC plots showing the expression of an Annexin V / PI apoptosis and necrosis assay in transwell co-cultures, including GLUTl-Jurkat coculturing with MV4-11, GLUTl / TFAM-Jurkat coculturing with MV4-11 versus GFP-Jurkat coculturing with MV4-11, and MV4-11 only in vitro. FIG. 6C depicts representative FC histograms showing the uptake of a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa- l,3-diazol-4-yl) amino] -2 -deoxy-D-glucose (2-NBDG) in viable MV4-11 cocultured with GLUTl-Jurkat (red line plots) and GLUTl / TFAM-Jurkat (blue line plots) versus vector-control Jurkat (GFP-Jurkat, green line) and viable MV4-11 alone (the filled grey line plots). FIG. 6C inset is an illustration of the transwell culture with MV4-11 cells (black) and Jurkat cells (blue). FIG. 6D is a graphical representation of the mean fluorescence intensity (MFI) levels of 2-NBDG uptake in the four MV4-11 cell lines. FIG. 6E is a graphical representation of tire gene expression of CY CLIN B 1 (CCNB 1 gene), a biomarker for mitosis in the cell cycle, analyzed by qPCR. Data of mRNA expressions show the fold change (normalized to [Lactin) of CYCLIN Bl gene in MV4-11 alone orMV4-l 1 co-cultured with different Jurkat cell lines. Where applicable, data are means ± SEM and were analyzed by student “f’ test. *p < 0.05, ** p < 0.01, n=3.

[0026] FIG. 7 is an illustration of tire mechanism of a competitive glucose-based T cell immunotherapy to treat AML.DETAILED DESCRIPTION

[0027] AML is an aggressive hematologic malignancy with common relapse. As one of the most successful immunotherapies developed for cancer treatment, T-cell-based adoptive cell therapies can be essential to track and eliminate residue leukemic cells (blasts) to prevent AML relapse. However, the applicability of CAR-T to AML remains to be fully proven because multiple recurring problems hinder their clinical effectiveness, including the lack of persistence of transplanted T cells due to cellular exhaustion / or senescence, and the ensuing failed restoration of anti-leukemic immunity in vivo.

[0028] In addition to enhancement of the specificity of CAR or TCR for the cancer cells, a successful T cell therapy can also depend on bioengineering T cells with competitive capabilities against cancer cells to utilize local nutrients to recover / rejuvenate themselves and survive in the AML microenvironment so that the T cells could maintain their long-term anti-leukemic surveillance in vivo. One decisive factor responsible for T-cell fate and exhaustion is the functionality of mitochondria. Decreased calcium buffering, impaired ATP synthesis, and increased mitochondrial reactive oxygen species are typical mitochondrion pathologies, leading to T-cell exhaustion / senescence and dysfunction. Recently, deficiency of TFAM, a multifunctional transcription factor essential for organizing mitochondrial chromatin and activating mtDNA transcription was linked to T cell dysfunction with phenotypes of earlier senescence. Adequate nutrients are essential for the survival of functional T cells and recovery from critical illness for cancer patients. T-cell activation / proliferation and effector functions required an abundance of glucose, which is often inadequate in the hostile AML microenvironment where disease activity is high. Notably, the depletion and direct competition for nutrients like glucose by rapidly proliferating blasts dampened the T-cell activation, induces T-cell exhaustion / senescence, and enables leukemic progression. In contrast to the increase of GLUT1 in cancer cells, suppressed T cells exhibited a downregulation in GLUT1 expression, indicating an alternative inhibitory mechanism of cancer cells to elude the T cell therapies by bottle-necking T-cell access to necessary metabolites.

[0029] In investigating how glucose affects primary T lymphocyte cells and Jurkat (a human T cell line) ex vivo, the inventors developed the compositions and methods disclosed herein. High doses of glucose were found to affect (such as reduce) the proliferation and differentiation of AML patient-derived T cells more greatly than heathy control-derived T cells, including causing a decrease in expression levels of mitochondrially encoded ATP synthase membrane subunit 8 (MT-ATP8), a crucial subunit for ATP synthase, and an increase in expression levels of programmed cell death protein 1 (PD-1). an inhibitory' receptor associated with T cell exhaustion, leading to cell cycle arrest and T cell dysfunction. Furthermore, high glucose was found to significantly reduce gene and protein expressions of SLC2A1 (GLUT1), the main glucose transporter, and TFAM, the mitochondrion transcription factor. Glucose tolerance was recognized to be a crucial criterion for the selection of T cell sources from either autologous AML patients or allogenic healthy controls for T-cell based immunotherapies.

[0030] Provided herein are compositions containing GLUT1 and / or TF AM-bioengineered T cells and immunotherapies for AML using the same. GLUT1 and / or TF AM-bioengineered T cell immunotherapy is a novel strategy of bioengineering T cells to be competitive against blasts for glucose, meanwhile utilizing enhanced GLUT1 and / or TFAM to activate mitochondrial biogenesis, reverse T cell exhaustion, and / or build a durable nutrient-based T cell immunotherapy for AML. Compositions provided herein can include engineered cells genetically overexpressing GLUT1 and / or TFAM. GLUT1 and / or TFAM-overexpressing cells have significantly enhanced production and release of IL-2 and reduced expression of Nur77, an exhaustion biomarker to promote T cell proliferation. They can significantly outgrow cocultured blasts in vitro. Bioengineering of GLUT1 and / or TFAM offers one or more of tire following advantages: (i) enhance the competitive capability of up-taking glucose, (ii) enhance the mitochondrial biogenesis, (iii) facilitate a nutrientbased supply chain of metabolites and ATP energy in T cells, (iv) enhance the production and release of cytokines, (v) accelerate tire T cell proliferation (CDK1). and (vi) reverse the exhaustion or senescence in T cells. Bioengineering of GLUT1 and / or TFAM can provide a novel, efficient, and durable T cell immunotherapy for AML.

[0031] Tire present disclosure describes various embodiments related to compositions and methods for management or treatment of cancers, such as hematologic malignancy, such as AML. 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 tire 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.

[0032] A “subject” refers an animal, such as a mammal, including a primate (such as a human), a non-human 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.

[0033] 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 a human or a veterinary subject. 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.

[0034] As used herein, the terms “treating”, “treatment” and the like, shall include the management and careof 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.

[0035] 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 temrs “decreased”, “reduced”, and the like encompass both a partial reduction and a complete reduction compared to a control.

[0036] 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.Compositions containing GLUT1 and / or TFAM

[0037] Provided herein are nucleic acid constructs, vectors, cells, and compositions containing GLUT1, TFAM, or both. The nucleic acid constructs, vectors, cells, and compositions provided herein can be used for reducing proliferation and / or viability of an AML cell, or treating a subject having AML.

[0038] GLUT1 is also referred to as solute carrier family 2 member 1 (SLC2A1), CSE. PED. DYT9, GLUT. DYT17. DYT18, EIG12. GLUT1, HTLVR, GLUT-1. SDCHCN. and GLUT1DS. GLUT1 is an integral membrane hydrophobic protein. GLUT1 helps in the transport of glucose, galactose, mannose, glucosamine and ascorbic acid across the cell membrane. Additional information on GLUT1 can be found, for example, on the world wide web at https: / / www.ncbi.nlm.nih.gov / gene / 6513. The sequence of human GLUT1 mRNA transcript can be found in, for example, GenBank Reference Sequence: NM 006516.4 (SEQ ID NO: 15).

[0039] TFAM is also referred to as TCF6. MTTF1, MTTFA, TCF6L1, TCF6L2, TCF6L3, and MTDPS15. TFAM is a mitochondrial DNA (mtDNA) binding protein essential for genome maintenance. TFAM functions in determining the abundance of the mitochondrial genome by regulating packaging, stability, and replication. TFAM also plays a central role in the mtDNA stress-mediated inflammatory response. Additional information on TFAM can be found, for example, on the world wide web at https: / / www.ncbi.nlm.nih.gov / gcnc / 7019. Tire sequence of a human TFAM mRNA transcript can be found in, for example, GenBank Reference Sequences: NM 003201.3 (SEQ ID NO: 16, homo sapiens TFAM transcript variant 1). NM 001270782.2 (SEQ ID NO:17, homo sapiens TFAM transcript variant 2), XM 054366663.1 (SEQ ID NO: 18, predicted homo sapiens TFAM transcript variant XI), and XM 054366664.1 (SEQ ID NO: 19, predicted homo sapiens TFAM transcript variant X2).

[0040] Tire inventors demonstrated that exposure to high doses of glucose reduces expression of both GLUT1 and TFAM, and thereby reducing glucose uptake and mitochondrial activities in human T cells.

[0041] Nucleic acid constructs encoding GLUT1 and / or TFAM are provided. These nucleic acid constructs can contain a nucleic acid molecule encoding GLUT1, operably linked to a promoter functional in an animal cell, or a nucleic acid molecule encoding TFAM, operably linked to a promoter functional in an animal cell, or both.

[0042] In some embodiments, the nucleic acid molecule encoding GLUT1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT1 function. For example, the nucleic acid molecule encoding GLUT1 can comprise a nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the nucleic acid molecule encoding TFAM comprises a nucleic acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 16-19, wherein the corresponding encoded polypeptide retains a TFAM function. For example, the nucleic acid molecule encoding TFAM comprises a nucleic acid sequence of any one of SEQ ID NO: 16-19. In specific embodiments, the nucleic acid molecule encoding TFAM comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 16. wherein the corresponding encoded polypeptide retains a TFAM function. For example, the nucleic acid molecule encoding TFAM comprises a nucleic acid sequence of SEQ ID NO: 16. In further specific embodiments, the nucleic acid construct contains a nucleic acid molecule encoding GLUT1 having the sequence of SEQ ID NO: 15, operably linked to a promoter functional in an animal cell, and / or a nucleic acid molecule encoding TFAM, having the sequence of SEQ ID NO: 16, operably linked to a promoter functional in an animal cell.

[0043] The construct can additionally include a reporter gene operably linked to the 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 GLUT1 and / or the TFAM coding sequences, including GFP and Cherry, and can be operably linked to any suitable promoter. For example, the construct may include, in an operable linkage, the EFla promoter, the GLUT1 coding sequence, tire internal ribosome entry site (IRES) sequence, and the GFP coding sequence. The construct may include, in an operable linkage, the EFla promoter, the TFAM coding sequence, the IRES sequence, and the Cherry coding sequence. FIG. 4A is an illustration of the lentiviral expression constructs containing human GLUT1 or TFAM open-reading frames and GFP or Cherry reporters, operably linked to promoters EF la and IRES, respectively. An IRES sequence can be used to express two proteins from a single promoter in an expression construct or a transgenic construct. By using an IRES, single RNA encoding the two protein can be produced with a second translational start located on the same RNA.

[0044] Embodiments include a vector with the nucleic acid construct provided herein, comprising a nucleic acid molecule encoding GLUT1, operably linked to a promoter functional in an animal cell, and / or a nucleic acid molecule encoding TFAM, operably linked to a promoter functional in an animal cell. For example, the nucleic acid molecule contained in the vector and encoding GLUT1 can comprise a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT 1 function, or a nucleic acid sequence of SEQ ID NO: 15. The nucleic acid molecule contained in the vector and encoding TFAM can comprise a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 16, wherein the corresponding encoded polypeptide retains a TFAM function, or a nucleic acid sequence of SEQ ID NO: 16.

[0045] Any vector can be used to contain one or more of the nucleic acid constructs provided herein and. for example, introduce the construct into a cell. In some embodiments, the vector is a viral vector. In specific embodiments, the vector is a lentiviral vector. In some embodiments, the promoter operably linked to the GLUT1 and / or the TFAM coding sequences are an EFla promoter. For example, the construct may include the GLUT1 coding sequence and an operably-linked EFla promoter.

[0046] Embodiments include cells containing the nucleic acid construct or the vector (such as viral vector, lentiviral vector) provided herein. In some embodiments, the cell is a T cell. Tire nucleic acid construct or the vector can comprise a nucleic acid molecule encoding GLUT1, operably linked to a promoter functional in an animal cell, and / or a nucleic acid molecule encoding TFAM. operably linked to a promoter functional in an animal cell. For example, as disclosed elsewhere herein, the nucleic acid molecule encoding GLUT1 can comprise a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT1 function, or a nucleic acid sequence of SEQ ID NO: 15. The nucleic acid molecule encoding TFAM can comprise a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 16, wherein the corresponding encoded polypeptide retains a TFAM function, or a nucleic acid sequence of SEQ ID NO: 16.

[0047] Embodiments include an engineered cell, such as an engineered T cell, overexpressing GLUT1 and / or TFAM relative to a control cell, such as a control T cell. A “control” cell as used herein is a native cell or a wild-type cell. For example, an exogenous polynucleotides has not been introduced or the genetic or plasmid sequence have not been modified in a control cell. One skilled in the art is able to select an appropriate control. The engineered cell (such as the engineered T cell) can contain a nucleic acid construct comprising a nucleic acid molecule encoding GLUT1, operably linked to a promoter functional in an animal cell, and / or a nucleic acid molecule encoding TFAM, operably linked to a promoter functional in an animal cell, such that the engineered cell overexpress GLUT1 and / or TFAM. For example, the nucleic acid molecule encoding GLUT1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT1 function, or a nucleic acid sequence of SEQ ID NO: 15. The nucleic acid molecule encoding TFAM comprises a nucleic acid sequence having at least 90% sequenceidentity with SEQ ID NO: 16, wherein the corresponding encoded polypeptide retains a TFAM function, or a nucleic acid sequence of SEQ ID NO: 16.

[0048] Without wishing to be bound by theory, the engineered cell (such as an engineered T cell) overexpressing GLUT1 can have an increased ability to uptake glucose across cell membrane and / or increased glucose metabolism. The engineered cell (such as an engineered T cell) overexpressing TFAM can have an increased mitochondrial activity. Overexpression of GLUT1 and TFAM can have additive or synergistic effect on level or activity of each of GLUT1 and TFAM. For example, overexpressing both GLUT1 and TFAM in a cell (such as by introducing into the cell a nucleic acid construct encoding GLUT1 operably linked to a functional promoter and a nucleic acid molecule encoding TFAM operably linked to a functional promoter) can increase the level or activity of GLUT1 as compared to overexpressing only GLUT1 in a cell (such as by introducing into the cell a nucleic acid construct encoding only GLUT1 operably linked to a functional promoter), and can increase the level or activity of TFAM as compared to overexpressing only TFAM in a cell (such as by introducing into the cell a nucleic acid construct encoding only TFAM operably linked to a functional promoter).

[0049] For example, level or activity of GLUT1 and / or TFAM can be each increased in the engineered cell (such as the engineered T cell) 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 cell. In specific embodiments, the expression of GLUT1 is increased to about 1 .5-3 fold and / or the expression of TFAM is increased to about 1.3-5 fold in the engineered cell (such as the engineered T cell) as compared to a control cell (such as a control T cell). Level of GLUT1 and / or TFAM gene expression can be measured by any standard method for measuring mRNA levels, such as PCR and quantitative PCR. Level of GLUT 1 and / or TFAM protein can be measured by any standard method for measuring protein levels, such as Western blot. ELISA, and dot blot. Activity of GLUT1 and / or TFAM can be measured by standard functional assays, such as measuring level of downstream target molecules, measuring cellular glucose uptake, and measuring mitochondrial activity.

[0050] The cell containing the nucleic acid construct or tire vector provided herein, or the engineered cell ovcrcxprcssing GLUT1 and / or TFAM is a T cell, and can lead to attenuated cell exhaustion, increased tumor immunosuppression, and / or increased cell proliferation relative to a control T cell. In some embodiments, the cell (e.g., cell comprising the construct, engineered cell) has an increased level or activity of Ki67, IL-2, and / orcyclin-dependent kinase 1 (CDK1), and / or decreased level or activity of nuclear receptor 4A1 (Nur77) relative to a control cell. Ki67 can be a cell proliferation marker. IL-2 is one of the cytokines secreted by T cells and can promote T cell proliferation, and thus, can be a marker for T cell activity and potential for proliferation. CDK1 is an essential kinase and central regulator of cell proliferation, and can be a marker for cell proliferation. Nur77, also referred to as nuclear receptor 4A1, suppresses T cell immunometabolism by controlling oxidative phosphorylation and glycolysis during T cell activation and proliferation, and can be a marker of cell exhaustion.

[0051] For example, level or activity of Ki67, IL-2, or CDK 1 can be each increased in the cell provided herein (such as the engineered T cell) 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 cell. Level or activity of Nur77 can be decreased in the cell provided herein 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. Level of Ki67 can be measured by any standard methods, including labeling and flow cytometry (e.g.. fluorescence-activated cell sorting) or immunocytochemistry. Level of IL-2. CDK1. or Nur 77 gene expression can be measured by any standard method for measuring mRNA levels, such as PCR and quantitative PCR. Level of IL-2, CDK1, or Nur 77 protein can be measured by any standard method for measuring protein levels, such as Western blot, ELISA, and dot blot. Activity of Ki67, IL-2, CDK1, or Nur77 can be measured by standard functional assays known in the art.

[0052] Embodiments include a composition containing the cell provided herein, such as the cell comprising the nucleic acid construct or the vector provided herein the engineered T cell overexpressing GLUT1 and / or TFAM relative to a control T cell. The composition provided herein can be used to reduce viability and / or proliferation of an AML cell or a plurality of AML cells, or to treat a subject having AML.

[0053] Tire composition provided herein can be a pharmaceutical composition. 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 formswhich are, within the scope of sound medical judgment, suitable for use in contact with tire 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.Reducing viability and / or proliferation of AML cells

[0054] Provided herein are methods for reducing viability and / or proliferation of one or more AML cells (such as AML blasts). One such method includes contacting the AML patient with an effective amount of a composition comprising an engineered cell, such as an engineered T cell overexpressing GLUT1 and / or TFAM relative to a control T cell. These methods can be performed in vitro, in vivo, or ex vivo. When performed in vivo, the method includes administering to a subject an effective amount of a composition provided herein or an active variant thereof.

[0055] An “effective amount” or “therapeutically effective amount” as used herein is an amount sufficient to effect desired pharmacological or clinical results (for example to achieve therapeutic efficacy). For instance, an effective amount of the composition comprising the engineered cells (such as the engineered T cells) provided herein can reduce mitosis and / or glucose uptake, reduce level or activity of cyclin Bl, and / or reduce viability and / or proliferation of an AML cell or a population of AML cells. An effective amount of the composition comprising the engineered cells (such as the engineered T cells) provided herein can reduce the number of viable mitotic AML in the subject, and / or reduce one or more symptoms and signs associated with AML in the subject. When the method provided herein is performed in vivo, an effective amount of the composition (e.g., pharmaceutical composition) is determined based on the intended goal.

[0056] The engineered cell (such as an engineered T cell) of the composition can comprise a nucleic acid construct comprising (a) a nucleic acid molecule encoding GLUT1, operably linked to a promoter functional in an animal cell, and / or (b) a nucleic acid molecule encoding TFAM, operably linked to a promoter functional in an animal cell. For example, the nucleic acid molecule encoding GLUT1 can comprise a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT1 function, or the nucleic acid sequence of SEQ ID NO: 15. The nucleic acid molecule encoding TFAM can comprise a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 16, wherein the corresponding encoded polypeptide retains a TFAM function, or the nucleic acid sequence of SEQ ID NO: 16.

[0057] In some embodiments, the engineered cell (such as an engineered T cell) is generated by transfecting a T cell with a viral vector comprising the nucleic acid construct containing a nucleic acid molecule encoding GLUT1. operably linked to a promoter functional in an animal cell, and / or a nucleic acid molecule encodingTFAM, operably linked to a promoter functional in an animal cell. The viral vector can be a lentiviral vector.

[0058] Without wishing to be bound by theory, AML-T cells exposed to high doses of glucose can have reduced T cell proliferative potential, as evidenced by the abnormal mitochondrial gene expressions essential for ATP synthesis and reduced size of growing T cell clusters. As illustrated by FIG. 1 J, significant reduction of mitochondria-encoded genes and potentially impaired oxidative phosphorylation process (OXPHOS) under the condition of high-dose glucose will activate the crosstalk between mitochondrial genome and nuclear genome (mitochondrial retrograde signaling) to up-regulate or down-regulate T cells’ nuclear transcriptional machinery to compensate the genetic loss and recover the ATP / metabolic processes of stressed mitochondria. As shown in FIG. 7, overexpression of the GLUT1 transgene can enhance competitive capability or T cells against leukemic blasts to uptake tire glucose, leading to tire metabolic / energy dysfunction in blasts and their ultimate death. Meanwhile, GLUT1 overexpression can enhance the production and release of IL-2 from the T cells to promote the T cell proliferation, which can be further facilitated by increased expression of CDK1 and reduced expression of exhaustion genes, such as Nur77.

[0059] There is a collaborative balance between glycolysis and mitochondrial biogenesis in T cells. Tire inventors have demonstrated that high glucose can reduce TFAM, a key regulator for mitochondrial genome transcription. GLUT1 overexpression can enhance both gene and protein expression of TFAM. Overexpression of GLUT1 and TFAM in T cells can confer the T cells superior capability to perform anti-leukemic function. In certain embodiments, methods provided herein using engineered T cells that overexpress GLUT1 and / or TFAM aim to utilize glycolysis and mitochondrial biogenesis to rejuvenate T cells and create durable and effective T cell therapies to treat AML in vivo. These engineered T cells can lead to attenuated cell exhaustion, increased tumor immunosuppression, and increased cell proliferation relative to a control T cell. In some embodiments, the engineered T cell has an increased level or activity of Ki67, IL-2, and / or cyclin-dependent kinase 1 (CDK1), and / or decreased level or activity of nuclear receptor 4A1 (Nur77) relative to a control T cell.

[0060] Upon being contacted with the composition provided herein, mitosis and / or glucose uptake of the AML cell can be decreased compared to a control AML cell. A control AML cell can be an AML cell that has not been contacted with the composition provided herein, or that has been contacted with a control composition not containing the engineered cell. One skilled in the art can select an appropriate control. For example, mitosis and / or glucose uptake 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 (such as an AML cell without being contacted with the composition provided herein). Mitosis of AMLcells can be measured by standard methods, including measuring level of biomarker for mitosis (such as cyclin Bl, level of which can be detected by for instance qPCR or Western blot; or phosphorylation of histone H3 at serines 10 and 28, which can be detected by fluorescent, colorimetric, or chemiluminescent assays). Glucose uptake by AML cells can be measured by any standard glucose uptake assays, including using 2-NBDG fluorescent tracer for glucose uptake.

[0061] Additionally or alternatively, in the methods provided herein, contacting the AML cell with an effective amount of the composition can reduce level or activity of cyclin Bl in the AML cell compared to a control AML cell. For example, level or activity of cyclin Bl 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%, or100% as compared to a control AML cell without being contacted with the composition provided herein. Level or activity of cyclin Bl can be measured by standard methods, such as qPCR, western blot, ELISA, and a cyclin-dependent kinase 1 (CDK1) enzymatic assay.

[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. In some embodiments, the AML cell is in a subject. 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. Viability and / or proliferation of the AML cells can be measured by any standard method for measuring cell viability and / or proliferation, including an MTT assay, a dye exclusion assay (e g., using trypan blue), a live / dead assay (e.g., using calcein-AM, propidium iodide, and / or a Hoechst dye), optionally incombination with flow cytometry and / or staining for a biomarker (for example, CD 15 can be used as a biomarker for MV4-11 AML cell).Methods of treating AML

[0064] Embodiments include methods for treating AML in a subject. One such method for treating AML in a subject includes administering to the subject an effective amount of a composition containing an engineered cell (such as an engineered T cell) overexpressing GLUT1 and / or TFAM relative to a control T cell.

[0065] 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 of the composition to the subject include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. Tire 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. Therapeutic agents can be constituted in a composition, such as a pharmaceutical composition containing an engineered cell (e.g.. an engineered T cell) 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.

[0066] The engineered cell (such as an engineered T cell) of the composition can contain a nucleic acid construct comprising (a) a nucleic acid molecule encoding GLUT1. operably linked to a promoter functional in an animal cell, and / or (b) a nucleic acid molecule encoding TFAM, operably linked to a promoter functional in an animal cell. For example, the nucleic acid molecule encoding GLUT1 can comprise a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT1 function, or the nucleic acid sequence of SEQ ID NO: 15. The nucleic acid molecule encoding TFAM can comprise a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 16. wherein the corresponding encoded polypeptide retains a TFAM function, or the nucleic acid sequence of SEQ ID NO: 16.

[0067] The engineered cell (such as an engineered T cell) can be generated by transfecting a T cell with a viral vector comprising the nucleic acid construct containing a nucleic acid molecule encoding GLUT1, operably linked to a promoter functional in an animal cell, and / or a nucleic acid molecule encoding TFAM, operably linked to a promoter functional in an animal cell. The viral vector can be a lentiviral vector. The engineered T cell provided herein can lead to attenuated cell exhaustion, increased tumor immunosuppression.or increased cell proliferation relative to a control T cell. In some embodiments, the engineered T cell has an increased level or activity of Ki67, IL-2, and / or cyclin-dependent kinase 1 (CDK1), and / or decreased level or activity of nuclear receptor 4A1 (Nur77) relative to a control T cell.

[0068] In some embodiments, administering an effective amount of tire composition decreases mitosis and / or glucose uptake in AML cells in the subject. For example, mitosis and / or glucose uptake can be reduced in the AML cells in the subject 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 (such as an AML cell in a subject without being administered the composition provided herein). Mitosis of AML cells can be measured by standard methods, including measuring level of biomarker for mitosis (such as cyclin Bl, level of which can be detected by for instance qPCR or Western blot: or phosphorylation of histone H3 at serines 10 and 28, which can be detected by fluorescent, colorimetric, or chemiluminescent assays). Glucose uptake by AML cells can be measured by any standard glucose uptake assays, including using 2-NBDG fluorescent tracer for glucose uptake.

[0069] Additionally or alternatively, administering an effective amount of the composition can reduce level or activity of cyclin B 1 in the AML cells in the subject compared to a control AML cell. For example, level or activity of cyclin Bl can be reduced in the AML cells in the subject 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. Level or activity of cyclin Bl can be measured by standard methods, such as qPCR, western blot, ELISA, and a cyclin-dependent kinase 1 (CDK1) enzymatic assay.

[0070] Administering an effective amount of the composition can decrease viability and / or proliferation of AML cells in the subject. For example, viability and / or proliferation of the 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%, as compared to a control cell.

[0071] Administering an effective amount of the composition can decrease the number of viable mitotic AML cells in the subject. 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 subject as compared to a control (e.g., before the administration of the composition, or in a subject who has not been administered the composition). Viability and / or proliferation of the AML cells can be measured by any standard method for measuring cell viability and / or proliferation, including an MTT assay, a dye exclusion assay (e.g., using trypan blue), a live / dead assay (e.g., using calcein-AM, propidium iodide, and / or a Hoechst dye), optionally in combination with flow cytometry and / or staining for a biomarker for AML cells(such as CD 15).

[0072] Administering an effective amount of the composition can decrease one or more signs or symptoms associated with AML in the subject. Signs and symptoms of AML can include tiredness, shortness of breath, weight loss, failure to thrive, frequent infections, fever, night sweats, and unusual and frequent bleeding.

[0073] The composition can be administered in one dose or in two or multiple doses. In some embodiments, the number, frequency, or amount of subsequent doses is dependent on the achievement of a desired therapeutic effect. In some embodiments, the composition is administered to a subject at the frequency and amount required to achieve a therapeutic effect. The subject can be monitored for desired therapeutic effects and unwanted side effects associated with administration of the composition.

[0074] 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, the composition may be administered once, a plurality of times, and / or over one or more extended periods. In some embodiments, the administration is repeated, for example, hourly, twice per day, daily, twice per week, weekly, biweekly, or monthly. In some embodiments, the administration is for a prescribed time period, e.g., for one month, two months, three months, four months, five months, six months, one year, longer, or indefinitely. After an initial treatment, the subsequent treatments can be administered less frequently relative to the initial treatment. In some embodiments, the composition is administered at the frequency required to achieve a desired effect.

[0075] 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, tire 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.

[0076] 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

[0077] The following examples are offered by way of illustration and not by way of limitation.Example 1: Effect of high doses of glucose on primary T cells ex vivo

[0078] T cells were isolated from peripheral blood (PB) samples of both AML patients (AML-T, n=3) and normal healthy controls (control T, n=2), and expanded T cells ex vivo according to the protocol previously reported (Cao et al. 2021 Neoplasia 23(12): 1252-1260). T cells were then treated with different doses of glucose for 48 hours. FIG. 1A depicts representative phase-bright images (4x) of T cells isolated from peripheral blood (PB) of a healthy control subject (# 1 ) and AML patient (# 1 ). FIG. 1 B is a set of representative phase-bright images in higher magnification (20x) of the T cell clusters. FIG. 1C is a graphical presentation of the cumulative data of cluster size of T cells isolated from healthy control PB and AML patient PB. FIG. ID is a set of representative phase-bright images (4x) of T cells isolated from peripheral blood (PB) of another healthy control (#2) and two AML patient (#2 and 3). Where applicable, data are means ± SEM and were analyzed by student ’1” test. *P < 0.05, **P < 0.01, n = 3. No significant difference in the size of T cell clusters were found between different experimental glucose-treated groups of control T cells of healthy control # 1 (FIG. 1A); however, the size of T cell clusters significantly decreased by 1.6- and 3-fold in AML-T cells of patient #1 when treated with higher glucose doses (50 mM and 100 mM), respectively (FIGs. 1A-1C). A similar phenomenon was found in AML-T cells of tw o other patients and control T cells of another healthy control (FIG. ID).

[0079] To investigate the mechanism underlying the reduced T cell growth, transcriptome analyses (RNA- sequencing or RNA-seq) was performed involving two AML patient primary T cell samples (AML T1 / T2) that were treated with 50 mM glucose or were not treated (NO-TX). Tire RNA-seq analysis was specifically- requested to include the bioinfomiatics of mitochondrion-encoded genes. The RNA-sequencing heatmap of AML-T cells treated or not treated with glucose revealed expression level differences of several genes (FIG. IE). FIG. IE presents an RNA-sequencing heatmap of two AML patients-derived T cells (AML-T1 or T2), which were treated with 50mM glucose or left untreated. FIG. IF presents a partial list of key ATP / metabolic processes-related genes encoded by mitochondrial genome and nuclear genome of AML T1 / T2. Cumulative TPM data of MT-ATP8 gene expression in untreated (NO-TX) and 50mM glucose-treated AML-T cells; n=2.

[0080] Among the 26,421 genes (both nuclear and mitochondrial genomes) responding to 50mM glucose treatment in the transcriptome database, mitochondrially encoded ATP synthase membrane subunit 8 (MT- ATP8), a mitochondrial gene that encodes an important protein subunit for ATP synthase, was identified ashaving the highest expression in both AML patients.

[0081] FIG. 1G depicts cumulative TMP data of MT-ATP8 (mitochondrially encoded ATP synthase membrane subunit 8) gene expression in untreated (NO-TX) and 50 mM glucose-treated AML-T cells; N = 2. Notably, a significant decrease in MT-ATP8 transcripts per million (TPM) was observed for 50 mM glucose treated AML-T cells, from 63203 TPM in the non-treated AML-T cells to 34986 TPM in AML-T cells treated with 50 mM glucose (FIG. 1G). The effect of 50 mM glucose on key genes of the mitochondrial quality control system compared to NO-TX was investigated in the two AML-T cell samples. FIG. 1H (upper panel) is a heatmap showing three mitochondria-related RNA expression changes in NO-TX and 50mM glucose-treated AML-T cells. FIG. 1H (lower panel) presents the cumulative TPM data of FIS 1 gene expression in NO-TX versus 50mM glucose-treated AML-T cells. Mitochondrial fission 1 protein (FIS 1) (essential for mitochondrial fission) increased by 1.3-fold TPM (FIG. 1H, lower bar panel) upon glucose treatment. Both AML patients also showed increased PTEN-induced kinase 1 (PINK1) (a key activator for mitophagy) expression but decreased mitochondrial dynamin like GTPase encoding OPA1 (mitochondrial fusion) expression (FIG. 1H, upper heatmap panel).

[0082] PD-1 (programmed cell death protein 1) is a well-known inhibitory receptor associated with T cell exhaustion, leading to cell cycle arrest and T cell dysfunction. FIG. II presents expression levels measured as FPKM (Fragments Per Kilobase per Million mapped fragments) of PD-1 (programmed cell death protein 1) in AML-T cells with or without 50mM glucose treatment. n=2 AML patient T cells. Notably, the RNA-seq results revealed that PD-1 was significantly increased from 34 FPKM to 90 FPKM after 50 mM glucose treatment in AML-T cells (FIG. II). The results provide a novel finding that high glucose-induced PD-1 may play a crucial role in reducing Ki67+proliferation (small AML-T cell clusters) and CD8+differentiation (cytotoxic T cells) in AML patients derived T cells.

[0083] In summary, the ex vivo experiments demonstrated that AML-T cells exposed to high doses of glucose have reduced T cell proliferative potential, as evidenced by the abnormal mitochondrial gene expressions essential for ATP synthesis and reduced size of growing T cell clusters, while healthy patient T cells were unaffected.

[0084] FIG. 1J is an illustration of how the mitochondrion (Mito)-nucleus crosstalk would be affected by high-dose glucose. Briefly, significant reduction of mitochondria-encoded genes and potentially impaired oxidative phosphory lation process (OXPHOS) under the condition of high-dose glucose will activate the crosstalk between mitochondrial genome and nuclear genome (mitochondrial retrograde signaling) to up- regulate or down-regulate T cells’ nuclear transcriptional machinery to compensate the genetic loss and recover the ATP / mctabolic processes of stressed mitochondria.

[0085] Next, flow cytometry (FC) was perfonned to investigate the molecular phenotypes of glucose-treated primary T cells. Both CCR7 and CD62L are biomarkers for naive or central memory T cells.

[0086] FIG. 2A presents representative flow cytometry’ (FC) plots showing CD62L and CCR7 expression inhealthy control-derived T cells and AML PB -derived T cells which were pretreated with different doses of glucose (left panel), and graphs showing cumulative percentage data of CCR7+cells in control and AML glucose-treated T cells from the FC plots (top and bottom, right panel, respectively). There is no significant difference in CCR7+T cell populations in experimental groups of control T cells pretreated with different doses of glucose when compared to the no-treatment (NO TX) control (FIG. 2A, left panels). Interestingly. CCR7+T cell populations were increased in AML-T cell experimental groups pretreated with increasing doses of glucose when compared to NO TX control by up to 5.2% in the highest glucose dose of 100 mM (FIG. 2A, right panels).

[0087] Next, effect of glucose on T cell differentiation was examined. FIG. 2B presents representative flow cytometry (FC) plots (left panel) showing CD3 and CD8 expression in healthy control and AML PB -derived T cells, which were pretreated with different doses of glucose, and graphs showing cumulative percentage data of CD3+CD8+cells in control and AML glucose-treated T cells from the FC plots (top and bottom, right panel, respectively). There was no significant difference in CD8+cytotoxic T cells in experimental groups of control T cells pretreated with different doses of glucose when compared to NO TX control (FIG. 2B, left panels). However, there are significantly decreased percentages of CD8+cytotoxic T cells in experimental groups of AML-T cells pretreated with different doses of glucose when compared to NO TX control by up to a 4.7% decrease in the 100 mM glucose group (FIG. 2B, right panels).

[0088] Finally, effect of glucose on T cell proliferation was investigated. FIG. 2C depicts representative flow cytometry (FC) plots (left panel) showing CD3 and Ki67 expression in healthy control -derived T cells and AML PB-derived T cells, which were pretreated with different doses of glucose, and graphs showing cumulative percentage data of Ki67+CD3+cells in control and AML glucose-treated T cells from tire FC plots (top and bottom, right panel, respectively). A significant increase in Ki67+T cells in experimental groups of control T cells pretreated with 25mM or 50mM glucose when compared to NO TX control by 3.6% and 2.7%. respectively (FIG. 2C, left panels). However, there is a significantly decreased percentage of Ki67+T cells in experimental groups of AML-T cells pretreated with 50mM or lOOmM glucose when compared to NO TX control by 3.8% and 6.7%, respectively (FIG. 2C, right panels). Hris decrease might be caused by the increased naive CCR7+T cells in FIG. 2A as naive T cells are kept in a quiescent state to maintain their survival. In summary, the ex vivo experiments demonstrated that AML-T cells exposed to high doses of glucose have reduced proliferative potential, as evidenced by the reduced expression of Ki67 and size of T cell clusters, while healthy patient T cells were unaffected.Example 2: High doses of glucose reduce GLUT1 and TFAM expression in T cells

[0089] To dissect the mechanism underlying the T cell responses to high doses of glucose, qPCR was performed to screen genes responsible for metabolic adaptation. Among many candidates, TFAM gene expression (mitochondrion transcription factor A) was found to be consistently decreased in gene expression by about 1.2-fold in different experimental groups of both AML-T cells and control T cells. FIG. 3A presentsgraphical representation of the gene expression of TFAM analyzed by qPCR. Data of mRNA expressions show the fold change (normalized to P-actin) of genes encoding TFAM in T cells from healthy control and AML patient samples, which were pretreated with different doses of glucose.

[0090] To confinn this phenomenon, Jurkat, a human T lymphoblast cell line, was treated with glucose (25 mM) over a short tenn (48 h) and long term (4 weeks) time span. FIG. 3B depicts representative FC histograms showing the expression of GLUT1 and Ki67 in Jurkat cells (left and right panels, respectively), which were pretreated with 25mM glucose for 48h (red line plot) or persistent supplementation of 25mM glucose for 4 weeks in vitro (blue line plot). The filled grey line and pink line plots represent Jurkat cells without treatment and IgG-fluorescent control. FIG. 3C (left panel) depicts representative FC histograms showing the expression of TFAM in Jurkat cells which were pretreated with 25mM glucose for 48h (red line plot) or persistent supplementation of 25mM glucose for 4 weeks in vitro (blue line plot); the filled grey line and pink line plots represent Jurkat without treatment and IgG-fluorescent control. In the 48h experimental groups, 25 mM glucose treatment increased GLUT1 (glucose transporter 1) expression when compared to the NO TX Jurkat (FIG. 3B, left panel), which was consistent with increased Ki67+proliferation (FIG. 3B, right panel). However, in the 4-week experimental groups, 25 mM glucose treatment reduced both GLUT1 expression and Ki67+proliferation when compared to the NO TX Jurkat (FIG. 3B). In contrast to minor changes in gene and protein expressions of TFAM in 48-hour glucose-treated Jurkat, there are significantly reduced gene (by 1.2- fold) and protein expressions of TFAM in 4-week glucose-treated Jurkat. FIG. 3C (right panel) is a graphical representation of the results from qPCR data of mRNA expressions showing the fold change (normalized to [3- actin) of the gene encoding TFAM in Jurkat cells, which were pretreated with or without different time-point of 25mM glucose. In sum, the results demonstrated that long-term exposure to high doses of glucose results in reduced GLUT1 expression and decreased mitochondrial activities in T cells.Example 3: Generation of Jurkat cell lines overexpressing GLUT1 (SLC2A1) and / or TFAM in vitro

[0091] To reverse the glucose-induced deficiency of GLUT1 and TFAM and to investigate their functional roles in enhancing T cell activation and proliferation, the GLUT1 and / or TFAM transgenes were overexpressed in Jurkat cells by lentiviral transduction in vitro (FIG. 4A). GLUT1 and TFAM overexpression in new Jurkat cell lines were assessed by fluorescent reporter-dye GFP and Cherry (FIG. 4B) and confirmed by qPCR analyses of transgene expressions (FIG. 4C). The new Jurkat cell lines were purified by FACS sorting (FIG. 4B). GLUT1 overexpression increased TFAM gene expression by 1.3-fold (FIG. 4C, right panel) while TFAM overexpression did not exhibit a reciprocal increase in GLUT1 gene expression (FIG. 4C, left panel). Furthermore, there was greater expressions of GLUT1 and TFAM genes in the combined GLUT1 / TFAM- Jurkat group (2.9-fold GLUT1 increase, 4.6-fold TFAM increase relative to GFP-Jurkat) when compared to the GLUTl-Jurkat group (1.5-fold GLUT1 increase, 1.3-fold TFAM increase) or thc TFAM-Jurkat group (0- fold GLUT1 increase, 3.6-fold TFAM increase) (FIG. 4C). In addition to increased GLUT1 protein expression (FIG. 4D, left plot). GLUT1 overexpression increased the protein expression of TFAM (FIG. 4D, right plot).which is consistent with increased TFAM gene expression in GLUTl-Jurkat (FIG. 4C, right panel).

[0092] Flow cytometry analysis was conducted to observe the effects of GLUT1 and TFAM expression on T cell proliferation. Tire FC histogram (FIG. 5A, left panel) and mean fluorescence intensity (MFI) levels of Ki67 (FIG. 5A, right panel) demonstrated that increased GLUT1 and TFAM expression was directly correlated to increased cell proliferation of GLUTl-Jurkat, TFAM-Jurkat and GLUTl / TFAM-Jurkat when compared to GFP-Jurkat.

[0093] To elucidate the mechanism of enhanced T cell proliferation, additional qPCR analysis was conducted on the new Jurkat cell lines. All of GLUTl-Jurkat, TFAM-Jurkat and GLUTl / TFAM-Jurkat had significantly increased gene expression of key cytokine IL-2 by 4.1-fold, 2.2-fold, and 6.5-fold, respectively (FIG. 5B). As a result, there was an additive effect in IL-2 expression in GLUTl / TFAM-Jurkat when compared to GLUTl- Jurkat or TFAM-Jurkat (FIG. 5B). Furthennore, all of GLUTl-Jurkat, TFAM-Jurkat and GLUTl / TFAM- Jurkat had significantly increased gene expression (by 1.5-, 1.6-, and 1.5-fold, respectively) of cyclin- dependent kinase 1 (CDK1), an essential kinase and central regulator that drives cell proliferation (Enserink et al. 2010 Cell Div 5: 11) (FIG. 5B). To elucidate the mechanism by which GLUT1 and / or TFAM overexpression reverse the T cell exhaustion / senescence, additional qPCR screens was conducted on exhaustion genes and inhibitory signaling pathways. Jurkats with GLUT1, TFAM, and GLUT1 / TFAM overexpression had significantly decreased gene expression (by 1.6-, 1.5-, and 1.3-fold, respectively) of nuclear receptor 4A1 (Nur77, FIG. 5B), a key exhaustion biomarker, which has been found to suppress T cell immunometabolism by? controlling oxidative phosphorylation and glycolysis during T cell activation and proliferation (Liebmann et al. 2018 Proc Natl Acad Sci USA 115(34): E8017-E8026). To investigate cytokine release in Jurkats with GLUT1 or TFAM overexpression, proteomic screenings of human cytokines secreted in the cell-free supernatants from the new Jurkat cell lines were perforated. Both GFP-Jurkat and TFAM-Jurkat released a similar and few cytokine profile in their supernatant, consistent with the idea that TFAM overexpression should affect the mitochondria and not secreted cytokines (FIG. 5C, top and lower plots). Interestingly. GLUTl- Jurkat released many different cytokines including IL-2 (FIG. 5C, middle plot), consistent with GLUT1- induced IL-2 gene expression (FIG. 5B).

[0094] In summary, the results suggest that GLUTl-initated glucose metabolism can coordinate TF AM- regulated mitochondrial adaption to promote T cell activation and proliferation through production / release of IL-2, increase of CDK1. and inhibition of the Nur77-mediated exhaustion pathway in vitro.Example 4: GLUTl-Jurkat and GLUTl / TFAM-Jurkat reduce glucose-uptake and viability of leukemic blasts

[0095] To examine anti-leukemic function of new Jurkat cell lines, direct co-cultures of Jurkat cells and MV4- 11, an AML cell line, were performed. CD15 is a unique biomarkcr for MV4-11 (according to ATCC). Flow cytometry data of direct co-cultures revealed that there was a significant reduction of MV4-11 co-cultured with GLUTl-Jurkat and GLUTl / TFAM-Jurkat in vitro (FIG. 6A, left panel). The percentage of viable CD15+MV4-11 blasts was significantly reduced from 94.9% in untreated control cells (MV4-11 alone) to 25.2% in co-cultures with GLUTl / TFAM-Jurkat or 28.6% in GLUTl-Jurkat, which were superior to 40% in co-cultures with GFP-Jurkat (FIG. 6A, right panel).

[0096] Next, MV4-11 and different Jurkats were co-cultured on transwells. A diagram of the transwell culture setup is also provided with MV4-11 (black) and Jurkat (blue) (FIG. 6C, inset). A cell death kit (MBL #4700) combining both necrosis (Propidium Iodide. PI) and apoptosis (Annexin V) assays was applied to detect early stage of apoptotic MV4-11 cells in transwell co-culture (FIG. 6B). Early apoptotic MV4-11 cells increased from 4.58% in the control to 8.98% with GLUTl-Jurkat and 9.61% with GLUTl / TFAM-Jurkat, both superior to the 7.82% with GFP-Jurkat (FIG. 6B).

[0097] To understand the mechanism underlying the reduction of MV4-11 in co-cultures, transwell cocultures were combined with a glucose (2-NBDG) uptake assay. In contrast to the normal 2-NBDG uptake in MV4-11 control group (without Jurkats) and the minor reduction of 2-NBDG uptake in MV4-11 co-cultured with GFP-Jurkat, the FC histogram data (FIG. 6C) and MFI data (FIG. 6D) revealed that there was a significant reduction of 2-NBDG uptake in MV4-11 co-cultured with GLUTl-Jurkat and GLUTl / TFAM- Jurkat in vitro. Then, the genetic changes of cell division were analyzed through qPCR with the CYCLIN Bl (CCNB 1 gene), a biomarker essential for the initiation of mitosis in tumors. The gene expression of CYCLIN Bl were significantly reduced in MV4-11 co-cultured with GLUTl-Jurkat (1.3-fold decrease) and GLUT 1 / TFAM-Jurkat ( 1.4-fold decrease) when compared to MV4-11 co-cultured with GFP-Jurkat (FIG. 6E).

[0098] Collectively, co-cultures of FACS-sorted Jurkat lines and MV4-11 combined with 2-NBDG-uptake (a fluorescent glucose analog) measurement demonstrated that GLUT1- or GLUTl / TFAM-Jurkat can competitively reduce glucose-uptake in MV4-11, leading to a significant leukemic reduction in vitro.

[0099] AML-T cells exposed to high doses of glucose had reduced proliferative potential, as evidenced by the reduced expression of Ki67 and size of T cell clusters and increased CCR7+expression, a biomarker for quiescent T cells; while, healthy patient control T cells were unaffected. Also, all three patients' derived AML- T cells exhibited decreased percentage of CD8+cytotoxic T cells when exposed to high doses of glucose, while control T cells had no significant change. These molecular phenotype differences suggest that AML-T cells may be more sensitive to the amount of glucose than control T cells in tire leukemic microenvironment.

[0100] As both GLUT1 and TFAM were significantly reduced in primary T cells and Jurkat cells after exposure to high doses of glucose, biological effects that a gain of function of GLUT1 and / or TFAM could execute on T cells were investigated. New Jurkat cell lines with overexpressed transgenes of GLUT1 and / or TFAM were generated, and significantly increased production / re lease of IL-2, reduced exhaustion gene marker Nur77, and enhanced cytotoxicity against blasts by depriving their glucose to reduce leukemic progression in vitro were found (FIG. 7). In summary, modulating glucose metabolism and mitochondrial biogenesis reversed T cell exhaustion / dysfunction.Selection of appropriate T cell source based on glucose sensitivity / tolerance

[0101] CAR-T or CAR-TILs rely on the collection of T cells from patients and then bioengineering them genetically with specific capabilities to eliminate malignant cells. Previous studies have shown that T cells depend on insulin signaling to facilitate glucose metabolism to drive proliferation, differentiation and immune responses. The data provided herein demonstrate that exposure to high doses of glucose results in reduced GLUT1 expression and decreased mitochondrial activities in T cells.

[0102] Particularly, the transcriptomic analyses demonstrated for the first time that in primary AML-T cells. 50mM glucose significantly reduced gene expression of MT-ATP8 while increasing the gene expression of pro-fission FIS1 (FIGs. 1G, 1H). MT-ATP8 loss and mutations have been previously found to be associated with increased oxidative stress, impaired complex V assembly and mitochondrial dysfunction in vivo. More importantly, mitochondrial polymorphisms such as MT-ATP8 gene variants will require increased glycolysis in T cells as a putative compensatory mechanism for maintaining energy homeostasis. It is therefore conceivable that maintaining or enhancing mitochondrial biogenesis might prevent T cell exhaustion caused by the tumor microenvironment (TME). Supportively, human FIS1 was recently found to regulate mitochondrial dynamics through inhibition of the fusion machinery. Also, FIS1 increase is known as a marker for mitochondrial dysfunction since overexpressed FIS1 induces mitochondrial fragmentation and mitophagy. Metabolites such as glucose may induce FIS1 in exhausted T cells in the TME, and accordingly, only a subset of patients may respond to immunotherapy, which limits its use.

[0103] Altogether, in chronic states of hyperglycemia, such as diabetes mellitus (DM), T cells may become tolerant to high levels of glucose and are unable to respond to T cell receptor (TCR) engagement. Notably, a recent study countered conventional theories that T cells are limited by the glucose present in the metabolically poor TME due to out-competition by cancer cells and instead reported that T cells are the main consumers of glucose in the microenvironment, not cancer cells. Consequently, increasing T cell sensitivity to glucose may improve the in vivo outcome. AML-T cells may have decreased competitive capability against blasts for glucose metabolism / energy production and may therefore be less capable of performing anti-leukemic functions due to a developed tolerance to abnormal glucose levels in the TME, analogous to the recent evidence showing hyperglycemia impaired T-cell response to COVID-19 in DM patients. Harvesting T cells from diabetic patients for autologous or allogenic therapy may compromise tire corresponding T cell therapy due to weakened immunity and potential T cell dysfunction.

[0104] T cell activation and growth rely on available environmental nutrients and extracellular signals. Glucose uptake plays an essential part in generating abundant metabolites and ATP energy to support T cell activation, signal transduction and proliferation. Thus, T cell exhaustion and dysfunction occur when they do not adequately uptake glucose due to leukemia-induced suppression of GLUT1 and inhibitory molecules.

[0105] Meanwhile, T cell activation and differentiation also require intracellular metabolic adaptations / energy productions, which are regulated by tire high copy, maternally inherited mitochondrial DNA (mtDNA) and its interaction with other cellular components. TFAM deficiency was found to reduce numbers ofcirculating CD4 / CD8 effector T cells, which can be explained by impaired mtDNA transcription and bioenergetic failure. To reverse the high glucose-induced TFAM deficiency, as provided herein, TFAM with or without GLUT1 was overexpressed in T cells by lentivirus. The data provided herein show that TFAM overexpression can increase gene expression of IL-2 and CDK1 and reduce Nur77 in T cells, supporting previous findings that upregulated TFAM can coordinate the two genomes of mtDNA transcription and nuclear response together to enhance mitochondrial biogenesis. Meanwhile, TFAM knockout in cardiac cells induced a metabolic switch to glycolysis by upregulating glycolytic enzyme genes, demonstrating a conserved interaction between glucose metabolism and mitochondrial biogenesis beyond T cells. Consistently, the data provided herein show GLUT I overexpression can induce TFAM and reveals additive effects of GLUT! and TFAM in T cells, suggesting that there is a previously unnoticed homeostatic balance between GLUTl / glucose metabolism and TFAM / mitochondrial biogenesis in T cells (FIG. 7). Thus, this homeostasis could be a vital therapeutic target to reverse T cell exhaustion / senescence and inhibit leukemic progression.

[0106] Collectively, the data provided demonstrate the importance of glucose sensitivity / tolerance in selection of appropriate T cell sources, and the viability of utilizing glucose metabolism and mitochondrial biogenesis to enhance T cells’ competency and cytotoxicity against leukemic blasts. Bioengineering T cells with GLUTl / or TFAM as provided herein is a novel strategy to enhance the efficacy of adoptive T-cell therapies for AML and other cancers. Bioengineering of GLUT1 and / or TFAM offers one or more of the following advantages: (i) enhance the competitive capability of up-taking glucose (GLUT1), (ii) enhance the mitochondrial biogenesis (TFAM), (iii) facilitate a nutrient-based supply chain of metabolites and ATP energy in T cells (Glucose), (iv) enhance the production and release of cytokines (such as IL-2), (v) accelerate the T cell proliferation (CDK1), and (vi) reverse the exhaustion or senescence in T cells (Nur77).Materials and Methods

[0107] The list of AML patients and controls, as well as reagents including manufacturers and catalogues of antibodies, kits and qPCR primers is found in Tables 1-3. All experiments were performed in replicates (n=3).

[0108] Human samples. Healthy and AML peripheral blood (PB) samples (Table 2) were obtained from Loma Linda University Cancer Center Biospecimen Laboratory (LLUCCBL) and 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 LLUMC and COHNMC in accordance with an assurance fded with and approved by the Department of Health and Human Services, and it met all requirements of the Declaration of Helsinki.

[0109] Isolation and Ex-Vivo Expansion of T cells from primary PB samples. The detailed protocol of isolation and expansion of primary T cells has been previously reported (Cao et al. 2021 Neoplasia 23(12): 1252-1260). Briefly, CD3+ T cells from PB mononuclear cells (BMMNC) were separated by using CD3 microbeads (Miltenyi Biotech, Germany) and a MiniMACS™ Separator with an MS Column according to the manufacturer’s protocol. CD3+ T cells were cultured at 37°C and 5% CO2 in RPMI 1640 culture mediumcontaining 10% fetal bovine serum (FBS, HyClone) with penicillin / streptomycin (lOOpg / ml), IL-2 (lOOOU / ml, Peprotech), and Dynabeads® Human T-Activator CD3 / CD28 (Gibco™) without feeder cells. Around 10-14 days, expanded T cells were treated with different doses of glucose before further analyses.

[0110] Cell culture of V4-ll, Jurkat and new Jurkat cell lines. MV4-11 is a human-derived AML blast cell line with aFLT3 mutation (ATCC CRL-9591). Jurkat is a human-derived T lymphoblast cell line (ATCC TIB- 152™). All cell lines were cultured in RPMI-1640 medium (HyClone) supplemented with 10% heat- inactivated fetal bovine serum (HyClone) and 100 U / ml penicillin / streptomycin. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2.

[0111] Glucose treatment of Jurkat in vitro, a) Short period of glucose treatment: Jurkat were treated with different doses of glucose for 48 hours (48h) before further analyses, b) Extended period of glucose treatment: Jurkat were treated with different doses of glucose for 4 weeks before further analyses. Medium was changed every 2-3 days with glucose.

[0112] Preparation of SLC2A1 (GLUT1) lentivirus, TFAM lentivirus and generation of GLUT1 -Jurkat, TFAM-Jurkat, GLUT 1 / TF 'AM- Jurkat and GFP -Jurkat cell lines in vitro. The lentiviral transfer plasmids contain a full-length open reading frame of human SLC2A1 (GLUT1) (GeneCopoeia catalog: EX-C0124- Lv225) and TFAM (GeneCopoeia catalog: EX-F0774-Lv225). This plasmid expresses GLUT1 or TFAM and GFP or Cherry controlled by the EFl promoter and IRES (FIG. 4A). A GFP empty vector (GeneCopoeia catalog: EX-NEG-Lv225) was used as the vector control. Lentiviruses were prepared as previously described (Xu et al. 2021 IntJMolSci 22(17)). Briefly, HEK-293T cells were cultured in complete Dulbecco’s modified Eagle’s medium containing 10% FBS and 100 U / mL penicillin / streptomycin. When the cells were 70-80% confluent, the culture media was replenished, and a transfection solution containing an envelope, packaging, and transfer plasmid (GLUT 1 or TFAM or GFP) was added dropwise to the cells. After the cells were cultured at 37 °C and 5% CCL for 48h. supernatants were collected, filtered through a 0.45 pm filter, and centrifuged at 4800 g at 4 °C for 24 h. The typical titer of virus was 10s- 109transducing units / ml. Tire Jurkat cells were transduced with lentivirus at a multiplicity of infection of 5. Twent -four hours later, the virus was removed, and the culture media was replenished. The cells were cultured for 24 h and examined for transduction efficiency via fluorescence microscopy and flow cytometry. GLUTl-Jurkat, TFAM-Jurkat and GLUT 1 / TFAM-Jurkat cell lines were purified by Fluorescence-Activated Cell Sorting, (FACS; Research Core Facility. School of Medicine. University of California. Riverside) (FIG. 4B).

[0113] Cytotoxicity Assay and Glucose (P-glucose. 2-[ -(7-nitrobenz-2-oxa-l ,3-diazol-4-yl)amino]-2- deoxy-D-glucose (2-NBDG)) Uptake Assay. Cytotoxicity assays were performed by co-culturing Jurkat cell lines with MV4-11 blasts in 24-well plates. The ratio of Jurkat cells to MV4-11 were 1: 1. After 48h incubation, cells w ere collected, stained, and processed for FACS assay of biomarkers including Viability Dye eFluor™ 780 (eBioscience™), CD3 and CD15 according to manufacturers’ protocols. Glucose (2-NBDG) uptake assays were performed by co-culture of Jurkat cell lines with MV4-11 blasts in 24-w ell plates with transwell inserts(Coming 3422). The ratio of Jurkat cells (outside the transwell insert) to MV4-11 (inside the transwell insert) were 1: 1. After 48h co-culture, 2-NBDG (50pM, MedChemExpress) was added to the medium to treat cells for 2 hours. MV4-11 cells from the transwell insert were collected either for flow cytometry measurement of 2-NBDG uptake, or for RNA isolation and qPCR analyses.

[0114] Flow Cytometry (FC). Experimental groups of T cells were harvested and examined forthe expression of cell surface biomarkers and intracellular proteins by multichromatic flow cytometry, as previously described (Cao et al. 2022 Biomedicines 10(5)). About 0.5-1 x 106cells were resuspended in 100 pl FC buffer (PBS containing 1% FBS and 0.05% sodium azide) and stained with various fluorescent-conjugated antibodies specific for the desired cell surface proteins at 4°C for 30 min. Tire surface-stained cells were then fixed and permeabilized using the appropriate reagents (BD Pharmingen Cytofix / Cytopcnn buffer) and further stained with appropriate fluorescent-conjugated antibodies specific for the desired intracellular proteins such as GLUT1 at 4°C for 2 h in the permeabilizing buffer (BD Perm / Wash buffer). Concentrations of the antibodies were used per the manufacturers’ recommendations (Table 1). Finally, the stained cells were washed twice in permeabilizing buffer and twice in FC buffer before analysis on the BD FACSAria II. Data were analyzed using the FlowJo software (Tree Star Inc., Ashland, OR).

[0115] RNA-sequencing (RNA-seq). High quality total RNA of AML patients-derived T cells treated with or without 50mM glucose was isolated by RNeasy Mini Kit (Qiagen). The service of RNA-seq including libraries preparation, sequencing and data analyses was provided by Beijing Genomics Institute (BGI) Americas. Detailed transcriptomic database of the interaction between high glucoses and primary T cells will be reported separately.

[0116] RNA isolation and qPCR analysis. Cells were collected for RNA isolation and qPCR analysis as previously described (Xu et al. 2022 IntJMol Sci 23(19)). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. First-strand cDNA was synthesized using the SuperScript III Reverse Transcriptase (Invitrogen). The qPCR was performed and analyzed in Applied Biosystems 7900HT Real-Time PCR machine. Sequences of primers used in this study are available in Table 3. Tire PCR conditions were 10 min at 95°C followed by 40 cycles of 10 sec at 95°C and 15 sec at 60°C. Tire relative expression level of a gene was determined using the AACt method and normalized to 0-actin.

[0117] Proteomics Analysis. Cell-free supernatants were collected for human cytokine profile assay as previously described (Cao et al. 2020 JHematol Oncol 13( 1 ): 63) . The Human XL Cytokine Array Kit (Catalog # ARY022B, R&D) w as used according to the manufacturer’s procedures and blot images were acquired from Azure 300 Imager (Azure Biosystems). This kit has 105 different cytokine antibodies and detailed names with their corresponding coordinates arc listed on pages 15-17 of the appendix section of tire manufacturer’s product datasheet (Systems, R D., Human XL Cytokine Array Kit, 2022). Hie reference spots were indicated by blue arrows.

[0118] Imaging Acquisition. Phase-bright images were taken using an Olympus 1X71 inverted microscopeand were processed using an Olympus cellSens Dimension 1.15 Imaging Software.

[0119] Statistical analysis. Statistical analyses were performed with GraphPad software (Prism 5.02). Tire quantitative analyses were analyzed using 1- or 2-way ANOVA followed by a Dunnett’s multiple comparisons test or a Bonferroni post hoc analysis as appropriate, or an unpaired / -test. All values were presented as mean ± SEM. Results were considered statistically significant when the p-value was <0.05.Table 1. List of ReagentsTable 2. List of AML Patients and Healthy ControlsTable 3. List of PrimersTable 4. GLUT1 and TFAM mRNA Sequences

[0120] Other objects, features and advantages of the disclosure will become apparent from the foregoing drawings, detailed description, and examples. These drawings, detailed description, and examples, while indicating specific embodiments of the disclosure, are given by way of illustration only and are not meant to be limiting. In further embodiments, features from specific embodiments may be combined with features from other embodiments. For example, features from one embodiment may be combined with features from any of the other embodiments. In further embodiments, additional features may be added to the specific embodiments described herein. It should be understood that although the disclosure contains certain aspects, embodiments, and optional features, modification, improvement, or variation of such aspects, embodiments, and optional features can be resorted to by those skilled in the art, and that such modification, improvement, or variation is considered to be within the scope of this disclosure.

Claims

CLAIMS1. A nucleic acid construct comprising one or more of a first nucleic acid molecule encoding glucose transporter 1 (GLUT1), operably linked to a first promoter functional in an animal cell and a second nucleic acid molecule encoding mitochondrion transcription factor A (TFAM), operably linked to a second promoter functional in the animal cell.

2. The nucleic acid construct of claim 1, wherein the first nucleic acid molecule encoding GLUT1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT1 function, or a nucleic acid sequence of SEQ ID NO: 15.

3. Tire nucleic acid construct of claim 1, wherein the second nucleic acid molecule encoding TFAM comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 16, wherein the corresponding encoded polypeptide retains a TFAM function, or a nucleic acid sequence of SEQ ID NO: 16.

4. A vector comprising the nucleic acid construct of any one of claims 1-3.

5. The vector of claim 4, wherein tire vector is a lentiviral vector.

6. An engineered T cell overexpressing glucose transporter 1 (GLUT1) and / or mitochondrion transcription factor A (TFAM) relative to a control T cell.

7. Tire engineered T cell of claim 6, wherein the engineered T cell contains a nucleic acid construct comprising one or more of a first nucleic acid molecule encoding GLUT1, operably linked to a first promoter functional in the engineered T cell and a second nucleic acid molecule encoding TFAM, operably linked to a second promoter functional in the engineered T cell.

8. The engineered T cell of claim 7, wherein the first nucleic acid molecule encoding GLUT1 comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 15, wherein a corresponding encoded polypeptide retains a GLUT1 function, or the nucleic acid sequence of SEQ ID NO: 15.

9. Tire engineered T cell of claim 7, wherein the second nucleic acid molecule encoding TFAM comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 16, wherein the corresponding encoded polypeptide retains a TFAM function, or the nucleic acid sequence of SEQ ID NO: 16.

10. The engineered T cell of claim 6, wherein the cell has an increased level or activity of Ki67, IL-2, and / or cyclin-dependent kinase 1 (CDK1), and / or decreased level or activity of nuclear receptor 4A1 (Nur77) relative to the control cell.

11. 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 the engineered T cell of claim 6.

12. A method for treating acute myeloid leukemia (AML) in a patient, the method comprising: administering to the patient an effective amount of the engineered T cell of claim 6.

13. The method of claim 12, wherein administering the engineered T cells decrease mitosis and / or glucose uptake in AML cells in the subject.

14. The method of claim 12, wherein administering the engineered T cells decrease a number of viable mitotic AML cells in the subject.

15. A method of selecting a T cell for a T-cell based immunotherapy for treatment of acute myeloid leukemia, the method comprising: evaluating glucose sensitivity of the T cell.

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