WO2022197978A1

WO2022197978A1 - Compositions and methods for modulating mitochondrial function and biogenesis

Info

Publication number: WO2022197978A1
Application number: PCT/US2022/020829
Authority: WO
Inventors: David T. Scadden
Original assignee: President And Fellows Of Harvard College; The General Hospital Corporation
Priority date: 2021-03-17
Filing date: 2022-03-17
Publication date: 2022-09-22

Abstract

The present invention relates to methods of treatment, methods of screening, and compositions related to agents that modulate malic enzyme 2 (ME2) binding to mitochondrial ribosomal protein L45 (MRPL45), ME2 dimerization, and the activation of DUT in a cell that is contacted with the agent. Also disclosed are methods of treating conditions associated with aberrant mitobiogenesis and cancer (e.g., AML).

Description

COMPOSITIONS AND METHODS FOR MODULATING MITOCHONDRIAL

FUNCTION AND BIOGENESIS

RELATED APPLICATION (S )

[0001] This application claims priority to U.S. Provisional Application No. 63/162,493, filed on March 17, 2021, and U.S. Provisional Application No. 63/166,217, filed on March 25, 2021, the entire teachings of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] Dysregulated mitobiogenesis has been implicated in multiple human diseases including cancer and aging. Notably, hyperactivation of mitobiogenesis has been shown to promote leukemia, liver cancer, and breast cancer. Mitobiogenesis regulation is of particular interest in acute myeloid leukemia (AML), a highly lethal hematopoietic neoplasm. AML cells have been reported to have more mitochondria than normal hematopoietic cells and are dependent upon oxidative metabolism. Chemoresistant AML cells shift to higher oxidative phosphorylation (OXPHOS) and are particularly sensitive to inhibition of cellular respiration. Although agents that inhibit mitochondrial translation have shown promising effects in suppressing AML, treatments via regulating mitobiogenesis are not used.

[0003] Therefore, a need exists for identifying metabolite- sensing pathways that regulate mitobiogenesis in normal and malignant cells and developing treatments by manipulating mitobiogenesis.

SUMMARY OF THE INVENTION

[0004] As disclosed herein, Applicants have performed screens for enzymatic dependencies in AML cells not present in their normal cell counterparts. Among the hits was malic enzyme 2 (ME2) that also scored in an independent screen that the inventors conducted on drivers of mitochondrial biogenesis (a distinctive feature of AML cells). Further evaluation of ME2 revealed that its role in mitobiogenesis was distinctive compared with ME1 or ME3 and was independent of its enzymatic activity. [0005] After further analysis of ME2, it has been surprisingly discovered that mitobiogenesis can be manipulated in normal and malignant cells through ME2, an unanticipated governor of mitochondrial biomass production that senses nutrient availability through fumarate and other non-fumarate ME2 dimerization agents.

[0006] Thus, some aspects of the present disclosure are directed to a method of modulating cell proliferation and/or viability, comprising contacting the cell with an agent that modulates malic enzyme 2 (ME2) binding to mitochondrial ribosomal protein L45 (MRPL45). In some embodiments, the agent is not fumarate or a fumarate analog.

[0007] In some embodiments, the agent enhances ME2 binding to MRPL45 and reduces mitobiogenesis in the cell. In some embodiments, the agent suppresses or prevents ME2 binding to MRPL45 and enhances mitobiogenesis in the cell. In some embodiments, the agent blocks a MRPL45 binding site for ME2 or blocks a ME2 binding site for MRPL45. In some embodiments, the agent enhances ME2 dimerization. In some embodiments, the agent suppresses ME2 dimerization. In some embodiments, the agent that suppresses ME2 dimerization reduces fumarate binding to ME2. In some embodiments, the agent causes methylation of the ME2 binding site for fumarate.

[0008] Some aspects of the present disclosure are directed to a method of modulating cell proliferation and/or viability, comprising contacting the cell with an agent that modulates malic enzyme 2 (ME2) dimerization in the cell. In some embodiments, the agent is not fumarate or a fumarate analog.

[0009] In some embodiments, the agent enhances ME2 dimerization. In some embodiments, the agent suppresses ME2 dimerization. In some embodiments, the agent that suppresses ME2 dimerization reduces fumarate binding to ME2. In some embodiments, the agent causes methylation of the ME2 binding site for fumarate.

[0010] In some embodiments, the method modulates the activation of deoxyuridine 5’- triphosphate nucleotidohydrolase (DUT) in the cell. In some embodiments, the method modulates the generation of thymidine and/or mitochondrial DNA (mtDNA) in the cell. In some embodiments, the method modulates mitobiogenesis in the cell. In some embodiments, the cell is contacted with the agent in vivo in a subject in need thereof. In some embodiments, the subject has a condition associated with a mitochondrial defect. In some embodiments, the subject has a cancer (e.g., leukemia such as AML).

[0011] Some aspects of the present disclosure are directed to a method of modulating cell proliferation and/or viability, comprising contacting the cell with an agent that modulates malic enzyme 2 (ME2) activation of deoxyuridine 5 ’-triphosphate nucleotidohydrolase (DUT). In some embodiments, the agent is not fumarate or a fumarate analog.

[0012] In some embodiments, the agent enhances DUT activation. In some embodiments, the agent suppresses DUT activation. In some embodiments, the agent modulates ME2 dimerization. In some embodiments, the agent is a ME2 dimer agonist or antagonist. In some embodiments, the agent is a ME2 dimer mimic. In some embodiments, the method modulates the generation of thymidine and/or mitochondrial DNA (mtDNA). In some embodiments, the method modulates mitobiogenesis in the cell. In some embodiments, the cell is contacted with the agent in vivo in a subject in need thereof. In some embodiments, the subject has a condition associated with a mitochondrial defect. In some embodiments, the subject has a cancer (e.g., leukemia such as AML).

[0013] Some aspects of the present disclosure are directed to a method of screening for a candidate agent that modulates cell proliferation and/or viability, comprising providing a composition comprising malic enzyme 2 (ME2) and mitochondrial ribosomal protein L45 (MRPL45) or a fragment thereof capable of binding ME2 under conditions wherein ME2 and MRPL45 can bind, contacting the composition with a test agent, and assessing binding of ME2 with MRPL45, wherein a test agent that modulates binding of ME2 to MRPL45 as compared to a control composition comprising ME2 and MRPL45 or a fragment thereof capable of binding ME2 without the test agent is identified as a candidate agent that modulates cell proliferation and/or viability. In some embodiments, the MRPL45 fragment capable of binding ME2 comprises or consists of the ME2 binding site.

[0014] Some aspects of the present disclosure are directed to a method of screening for a candidate agent that modulates cell proliferation and/or viability, comprising providing a composition comprising malic enzyme 2 (ME2) under conditions wherein ME2 is capable of forming dimers, contacting the composition with a test agent, and assessing dimerization of ME2, wherein a test agent that modulates dimerization of ME2 as compared to a control composition comprising ME2 without the test agent is identified as a candidate agent that modulates cell proliferation and/or viability.

[0015] Some aspects of the present disclosure are directed to a method of treating a condition associated with a mitochondrial defect and/or a cancer (e.g., AML) comprising administering to a patient in need thereof a composition comprising an effective amount of an agent that modulates malic enzyme 2 (ME2) dimerization. In some embodiments, the agent is not fumarate or a fumarate analog. In some embodiments, the agent modulates the activation of deoxyuridine 5 ’-triphosphate nucleotidohydrolase (DUT). [0016] Some aspects of the present disclosure are directed to a composition comprising an effective amount of an agent for modulating malic enzyme 2 (ME2) dimerization. In some embodiments, the agent is not fumarate or a fumarate analog. In some embodiments, the agent modulates the activation of deoxyuridine 5 ’-triphosphate nucleotidohydrolase (DUT).

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0018] FIG. 1A shows human cord blood CD34+ cells and AML cell lines that were treated with DMF or DEF for 24 hours. The mtDNA was determined by qPCR and normalized to nDNA (left). Cells were stained with MTG and the fluorescent intensity was normalized to cell number (right). All data were normalized to DMSO-treated group. The fold change (FC) was presented on a log2 scale.

[0019] FIG. IB shows Pearson correlation of fumarate and maximum respiration capacity (OXPHOS potential) of AML cell lines.

[0020] FIG. 1C shows mitochondria numbers in 100 Control and DMF-treated MOLM14 cells counted with transmission electron microscopy .

[0021] FIG. ID shows representative images (bar=l pm) of the Control and DMF-treated MOLM14 cells.

[0022] FIG. IE shows mitochondrial fumarate for MOLM14 cells that were treated with fumarate (Fum) and its esters for 24 hours.

[0023] FIG. IF shows mtDNA copies for MOLM14 cells that were treated with fumarate (Fum) and its esters for 24 hours.

[0024] FIG. 1G shows MTG intensity for MOLM14 cells that were treated with fumarate (Fum) and its esters for 24 hours.

[0025] FIG. 1H shows oxygen consumption rate for MOLM14 cells that were treated with fumarate (Fum) and its esters for 24 hours.

[0026] FIG. II shows quantified mitochondrial ATP, NADH, and dNTPs for MOLM14 cells that were treated with fumarate (Fum) and its esters for 24 hours.

[0027] FIG. 1J shows whole cell lysate that were subjected to western blotting wherein b- actin (ACTIN) was included as the loading control. [0028] FIG. IK shows quantified (n=5) mtDNA copies from multiple mice tissues injected intraperitoneally with DMSO (MOCK), MMF, or DMF for seven days.

[0029] FIG. 1L shows mitochondrial proteins in the BM cells from three independent mice determined by western blotting.

[0030] FIG. 1M shows MTG intensity.

[0031] FIG. IN shows oxygen consumption rate of BM cells that were assayed (n=5). All data are shown as mean ± SEM from three independent experiments. *p< 0.05, **p < 0.01, n.s. indicates not significant.

[0032] FIG. 2A shows a schematic overview of fumarate-interacting enzymes.

[0033] FIG. 2B shows a Scrambled control (Scr) or shRNAs targeting fumarate-binding enzymes that were stably expressed in MOLM14 cells. mtDNA was determined by qPCR after DMF treatment.

[0034] FIG. 2C shows a schematic overview of malic enzymes in central carbon metabolism. [0035] FIG. 2D shows mtDNA abundance (left) and MTG intensity (right) that were determined for human CD34+ CB cells and AML cells that were transduced with control short-guide RNA (sgRNA) or sgRNAs targeting malic enzymes.

[0036] FIG. 2E shows cells that were further treated with DMF where mtDNA abundance (left) and MTG intensity (right) were assayed. All data were normalized to the control group. [0037] FIG. 2F shows a panel of solid tumor cell lines was transduced with scrambled control or shRNAstargeting ME2. mtDNA were quantified after DMF treatment. All data were normalized to the scrambled control.

[0038] FIG. 2G shows twenty-one days after transplantation, mice that were injected intraperitoneally with DMSO (MOCK) or DMF for seven days.

[0039] FIG. 2H shows ME2 protein for the transduced mouse BM cells.

[0040] FIG. 21 shows mtDNA abundance for the transduced mouse BM cells.

[0041] FIG. 2J shows MTG intensity for the transduced mouse BM cells.

[0042] FIG. 2K shows oxygen consumption in lin BM cells that were assayed (n=5). All data are shown as mean ± SEM from three independent experiments. *p< 0.05, **p < 0.01, n.s. indicates not significant.

[0043] FIG. 3A shows ME2 dimer (PDB ID: 1PJ4) that was colored green and cyan on each subunit.

[0044] FIG. 3B shows whole cell lysate that was analyzed by crosslinking after treating MOLM14 cells with fumarate or its esters. [0045] FIG. 3C shows MOLM14 cells expressing ME2-Flag and its mutants that were treated with DMF and subjected to crosslinking assay.

[0046] FIG. 3D shows the oxygen consumption rate of ME2-knockdown and re-expression MOLM14 cells that was determined.

[0047] FIG. 3E shows ME2-Flag and its R67F mutant that were stably expressed in MOLM14 cells and numbers of wildtype or mutant ME2-interacting mitochondrial proteins. ME2-interacting proteins were identified by pulldown-mass spectrometry.

[0048] FIG. 3F shows DUT that limits dUTP and enhances dTTP synthesis.

[0049] FIG. 3G shows mitochondrial dUTP and dUMP that were determined after treating MOLM14 cells with fumarate or its esters.

[0050] FIG. 3H shows mitochondrial dUTP and dUMP that were quantified after treating MOLM14 cells with DMF and TAS 114.

[0051] FIG. 31 shows Endogenous ME2 that was immunoprecipitated to determine its interaction with DUT and GOT2 in fumarate-treated MOLM14 cells.

[0052] FIG. 3J shows DUT-Flag that was immunopurified from MOLM14 cells and mixed with recombinant ME2 to determine its activity.

[0053] FIG. 3K shows mitochondria lysate that was subjected to DUT activity assay.

[0054] FIG. 3L shows mitochondrial dUTP, dUMP, and four dNTPs that were quantified. [0055] FIG. 3M shows mtDNA that was determined after treating MOLM14 cells with DMF and TAS 114. All data are presented as mean ± SEM from three independent experiments.

*p< 0.05, **p < 0.01, n.s. indicates not significant.

[0056] FIG. 4A shows ME2 interactors that were functionally grouped; the number on the y- axis indicates total number of wildtype or mutant ME2-binding proteins.

[0057] FIG. 4B shows ME2-interacting proteins that were identified by pulldown-mass spectrometry in three independent experiments. The number of detected interactions of ME2 (wildtype and R67F mutant) with mitoribosomal proteins were determined.

[0058] FIG. 4C shows MOLM14 cells expressing ME2-Flag and its mutants that were treated with DMF. The interaction of ME2 with MRPL45 and MRRF was determined.

[0059] FIG. 4D shows GFP-tagged full-length MRPL45 (FL) and its mutants (N and AC). [0060] FIG. 4E shows those co-expressed with ME2-Flag to determine their association. [0061] FIG. 4F shows ME2-knockdown and re-expression MOLM14 cells that were treated with DMF. Isolated mitochondria were fractionated to determine MRPL45 localization.

[0062] FIG. 4G shows isolated mitochondria that were loaded on a sucrose gradient to fractionate mitoribosome. [0063] FIG. 4H shows mtDNA and nDNA-encoded proteins that were determined. All data are presented as mean ± SEM from three independent experiments. **p < 0.01, n.s. indicates not significant.

[0064] FIG. 5A shows R67 methylation of immunoprecipitated ME2 that was determined in MOLM14 cells after AMI-5 treatment for 24 hours.

[0065] FIG. 5B shows the interaction between ME2 and PRMT1 in AML cells that was assayed.

[0066] FIG. 5C shows recombinant ME2-His that was incubated with PRMT1-HA in the presence of SAM and R67 methylation that was determined.

[0067] FIG. 5D shows subjection to western blotting and enzymatic activity assay.

[0068] FIG. 5E shows whole cell lysate of MOLM14 cells that was subjected to crosslinking assay.

[0069] FIG. 5F shows the melting temperature (Tm) of unmethylated and methylated ME2 (lanes 4 and 5 in FIG. 5C) that was determined.

[0070] FIG. 5G shows the control and PRMT1 -knockdown MOLM14 cells that were treated with DMF. The interaction between ME2 and DUT was determined.

[0071] FIG. 5H shows mitochondrial lysate that was subjected to DUT activity assay. F [0072] IG. 51 shows stable cells that were treated with PRMTli and DMF as indicated.

[0073] FIG. 5J shows the interaction of ME2 and MRPL45 that was assayed.

[0074] FIG. 5K shows MRPL45 protein in inner-membrane and matrix fractions that was quantified.

[0075] FIG. 5L shows the expression of mtDNA and nDNA-encoded proteins that was determined.

[0076] FIG. 5M shows stable MOLM14 cells that were treated with PRMTli and DMF as indicated. MTG intensity was determined.

[0077] FIG. 5N shows AML cells for endogenous ME2 that was immunopurified from CD34+ CB cells.

[0078] FIG. 50 shows representative solid tumor cell lines to determine R67 methylation. Whole cell lysate was used to detect PRMT1 and ME2. All data are presented as mean ±

SEM from three independent experiments. *p< 0.05, **p < 0.01, n.s. indicates not significant. [0079] FIG. 6A shows growth curves of stable MOLM14 cells that were determined. Cells were treated with PRMTli and DMF.

[0080] FIG. 6B shows cell viability that was determined by cell counting after four days of culture. [0081] FIG. 6C shows colonies of MOLM14 cells that were counted 7 days after treatment. [0082] FIG. 6D shows ME2-knockdown and re-expression MOLM14 cells that were transplanted into sublethally irradiated NSG mice to monitor leukemia progression (n=5). [0083] FIG. 6E show ME2 in normal and leukemic human BM samples that were determined.

[0084] FIG. 6F show PRMT1 protein in normal and leukemic human BM samples that were determined.

[0085] FIG. 6G show R67 methylation of immunoprecipitated ME2 that was determined. [0086] FIG. 6H shows ME2 activity that was assayed in the presence of fumarate.

[0087] FIG. 61 shows MRPL45 that was quantified by western blotting.

[0088] FIG. 6J shows MT-COl that was quantified by western blotting.

[0089] FIG. 6K shows MT-ND6 that was quantified by western blotting.

[0090] FIG. 6L shows mtDNA that was quantified by qPCR.

[0091] FIG. 6M shows Pearson's correlation of ME2 protein with MT-COl in AML samples that was determined.

[0092] FIG. 6N shows Pearson's correlation of ME2 protein with MT-ND6 in AML samples that was determined.

[0093] FIG. 60 shows Pearson's correlation of ME2 protein with mtDNA abundance in AML samples that was determined.

[0094] FIG. 6P shows a working model of ME2-mediated fumarate signaling. Data are presented as mean ± SEM from three independent experiments. *p< 0.05, **p < 0.01, n.s. indicates not significant.

[0095] FIG. 7A shows human cord blood CD34+ cells and eight different AML cell lines that were treated with sugars, lipids, amino acids, or metabolic intermediates from glycolysis, Krebs cycle, and lipid metabolism for 48 hours. Total DNA was extracted. Mitochondrial DNA (mtDNA) copy number was determined by quantitative PCR (qPCR) and normalized to nuclear DNA (left). Cells were stained with mitotracker green (MTG), the fluorescent intensity of MTG was normalized to cell number (right). All data were normalized to DMSO- treated group. The fold change (FC) was presented on a log2 scale.

[0096] FIG. 7B shows MOLM14 cells that were treated with 100 DM DMF or DEF at increasing durations up to 48 hours. mtDNA copy number was determined by qPCR and normalized to nuclear DNA.

[0097] FIG. 7C shows cells that were stained with mitotracker green (MTG), the fluorescent intensity of MTG was normalized to cell number. [0098] FIG. 7D shows MOLM14 cells that were treated with DMSO (MOCK) or 100 DM DMF for 24 hours. Cells were collected before and after the treatment. mtDNA copy number was determined by qPCR and normalized to nuclear DNA.

[0099] FIG. 7E shows cells that were stained with mitotracker green (MTG), the fluorescent intensity of MTG was normalized to cell number.

[0100] FIG. 7F shows the intracellular fumarate levels in CD34⁺ cord blood cells and eight different AMLcell lines were determined.

[0101] FIG. 7G shows MOLM14 cells that were treated with DMSO (MOCK), 1 mM fumarate (Fum), or 100 mM fumarate esters (MMF and DMF) for 24 hours. Mitochondria were isolated after treatment, POLRMT, GAPDH, and histone H3 (Histone) were included as markers for mitochondria, cytoplasm, and nucleus, respectively.

[0102] FIG. 7H shows the intracellular fumarate level that was determined and normalized to cell number.

[0103] FIG. 71 shows flag-tagged NRF2 that was stably expressed in MOLM14 cells. Cells were treated with DMSO or 100 pM DMF for 24 hours. mtDNA copy number was determined by qPCR and normalized to nuclear DNA.

[0104] FIG. 7J shows flag-tagged NRF2 that was stably expressed in KG1 cells. Cells were treated with DMSO or 100 p M DMF for 24 hours. mtDNA copy number was determined by qPCR and normalized to nuclear DNA.

[0105] FIG. 7K shows MOLM14 cells that were incubated with increasing concentrations of [U-¹³C] -fumarate for 24 hours. Cellular metabolites were extracted, the isotope distribution in fumarate was quantified by liquid chromatography with tandem mass spectrometry (LC- MS/MS). Shown are two independent experiments (1 and 2).

[0106] FIG. 7L shows MOLM14 cells that were incubated with increasing concentrations of [U-¹³C] -fumarate for 24 hours. Cellular metabolites were extracted, the isotope distribution in malate was quantified by liquid chromatography with tandem mass spectrometry (LC- MS/MS). Shown are two independent experiments (1 and 2).

[0107] FIG. 7M shows MOLM14 cells that were incubated with increasing concentrations of [U-¹³C] -fumarate for 24 hours. Cellular metabolites were extracted, the isotope distribution in citrate was quantified by liquid chromatography with tandem mass spectrometry (LC- MS/MS). Shown are two independent experiments (1 and 2).

[0108] FIG. 7N shows MOLM14 cells that were incubated with increasing concentrations of [U-¹³C] -fumarate for 24 hours. Cellular metabolites were extracted, the isotope distribution in pyruvate was quantified by liquid chromatography with tandem mass spectrometry (LC- MS/MS). Shown are two independent experiments (1 and 2).

[0109] FIG. 70 shows MOLM14 cells that were treated with DMSO (MOCK), 1 mM fumarate (Fum), or 100 mM fumarate esters (MMF and DMF) for 24 hours. Cellular metabolites were extracted, the abundance of ATP, NADH, and dNTPs were quantified.

[0110] FIG. 7P shows the mRNA expression of mtDNA and nDNA-encoded mitochondrial genes that was quantified by qPCR and normalized to actin.

[0111] FIG. 7Q shows mice that were injected intraperitoneally with DMSO (MOCK),

MMF, or DMF for seven days. Tissues were homogenized and metabolites were extracted, the abundance of fumarate was determined and normalized to total protein (n=5).

[0112] FIG. 7R shows the expression of mitochondrial proteins in liver tissues from three independent mice that was determined by western blotting. All data are shown as mean ± SEM from three independent experiments. *p< 0.05, **p < 0.01, n.s. indicates not significant. [0113] FIG. 8A shows the knockdown efficiencies of shRNAs targeting NRF2, ADSL, ASL, FAH, FH, SDHA, and ME2 in MOLM14 cells that were determined by qPCR.

[0114] FIG. 8B shows whole cell lysate of eight different AML cell lines and human CD34+ cord blood (CB) cells from three different donors (D1-D3) that was subjected to western blotting. Protein expression of ME1, ME2, and ME3 was determined and b-actin was included as the loading control.

[0115] FIG. 8C shows Control sgRNA or two different sgRNAs targeting each malic enzyme that were stably expressed in human CD34+ cord blood cells or eight different AML cell lines. The expression of ME1, ME2, and ME3 was determined by western blotting.

[0116] FIG. 8D shows the consumption rate of glucose. All data was normalized to the control group and presented on a log2 scale.

[0117] FIG. 8E shows the consumption rate of glutamine. All data was normalized to the control group and presented on a log2 scale.

[0118] FIG. 8F shows a panel of solid tumor cell lines from glioma, breast cancer, liver cancer, pancreatic ductal adenocarcinoma (PD AC), and melanoma that was transduced with scrambled control or two different shRNAs targeting ME2. The mRNA expression of ME2 was determined by qPCR. # denotes that BxPC3 is a ME2-null cell line. All data are shown as mean ± SEM from three or four independent experiments. **p < 0.01.

[0119] FIG. 9A shows flag-tagged ME2 and AC02 that were transduced into MOLM14 cells. Cells were treated with increasing doses of DMF as indicated. ME2 and AC02 were immunopurified with Flag beads. The succination of proteins were detected by western blotting.

[0120] FIG. 9B shows MOLM14 cells that were treated with increasing doses of DMF as indicated for 24 hours. Cells were lysed and incubated with maleimide-PEG2 -biotin to capture free thiols in cellular protein. Labeled protein was further pulled down with streptavidin agarose beads and subjected to western blotting.

[0121] FIG. 9C shows MOLM14 and KG1 cells that were treated with MMF and DMF for 24 hours, the protein level of ME2 was assayed by western blotting, b-actin was included as the loading control.

[0122] FIG. 9D shows ME2-Flag that was expressed in HEK293 cells, which were treated with fumarate and its esters for 24 hours. Whole cell lysate was crosslinked with glutaraldehyde and subjected to western blotting.

[0123] FIG. 9E shows wildtype ME2-Flag and its mutants that were immunopurified from HEK293 cells. The catalytic activity of ME2 was assayed with or without fumarate and normalized to ME2 protein.

[0124] FIG. 9F shows HA-tagged ME2 that was co-expressed with GFP-tagged wildtype ME2 and its mutants (R67F, E59L, and CM). Cells were treated with DMF (F) for 24 hours. HA-tagged ME2 was immunoprecipitated using an HA antibody. The interaction between differently tagged ME2 was determined by western blotting.

[0125] FIG. 9G shows HA-tagged ME2 that was co-expressed with GFP-tagged wildtype ME2 and its mutants (R67F, E59L, and CM). Cells were treated with fumarate for 24 hours. HA-tagged ME2 was immunoprecipitated using an HA antibody. The interaction between differently tagged ME2 was determined by western blotting.

[0126] FIG. 9H shows MOLM14 that were transduced with shRNA targeting ME2 and re expressed with wildtype ME2 or its mutants. The knockdown and re-expression efficiency of ME2 was determined by western blotting.

[0127] FIG. 91 shows KG1 cells that were transduced with shRNA targeting ME2 and re expressed with wildtype ME2 or its mutants.

[0128] FIG. 9J shows the oxygen consumption rate of KG1 cells was determined by Seahorse flux analyzer.

[0129] FIG. 9K shows wildtype ME2-Flag and its R67F mutant that were stably expressed in MOLM14 cells. ME2 protein was immunoprecipitated by Flag beads and subjected to mass spectrometry analysis to identify ME2 interactors. [0130] FIG. 9L shows MOLM14 cells that were treated with DMF and TAS114 as indicated for 24 hours. Mitochondria were isolated after treatment. POLRMT, GAPDH, and histone H3 (Histone) were included as markers for mitochondria, cytoplasm, and nucleus, respectively. [0131] FIG. 9M shows ME2-Flag that was expressed in HEK293 cells, which were treated with fumarate for 24 hours. The interaction between ME2-Flag and endogenous DUT was determined by co-immunoprecipitation and western blotting.

[0132] FIG. 9N shows His-tagged ME2 and its mutants that were purified from E.coli, resolved on SDS-PAGE, and visualized by Coomassie Blue staining.

[0133] FIG. 90 shows ME2-knockdown and re-expression MOLM14 cells that were treated with DMF for 24 hours. Mitochondria were isolated from MOLM14 cells after DMF treatment. Whole cell lysate and mitochondrial fraction were analyzed by western blotting to determine the isolation efficiency.

[0134] FIG. 9P shows the cellular abundance of dUTP, dUMP and four dNTPs that were quantified and normalized to cell number.

[0135] FIG. 9Q shows ME2-knockdown and re-expression KG1 cells that were treated with DMF and TAS114 for 24 hours. Total DNA was extracted; mtDNA abundance was determined by qPCR. All data are presented as mean ± SEM from three independent experiments. *p< 0.05, **p < 0.01, n.s. indicates not significant.

[0136] FIG. 10A shows HA-tagged MRPL45 that was expressed in HEK293 cells. Cells were treated with or without DMF for 24 hours. HA-tagged MRPL45 was immunopurified and subjected to western blotting to determine its interaction with mitochondrial malic enzymes (ME2 and ME3).

[0137] FIG. 10B shows recombinant ME2 and His-tagged MRPL45 that were incubated with 500 mM fumarate, succinate, or malate for 24 hours in vitro. MRPL45 was pulled down using nickel beads and subjected to western blotting.

[0138] FIG. IOC shows Wildtype ME2 and its mutants (R67F and CM) that were re expressed in ME2-knockdown MOLM14 cells. Cells were treated with or without DMF. Isolated mitochondria were fractionated and subjected to western blotting to determine MRPL45 localization. WCL, whole cell lysate; Cyt, cytoplasm; MT, mitochondria; MP, mitoplast; IM, inner membrane; Mtx, mitochondrial matrix.

[0139] FIG. 10D shows ME2-knockdown and re-expression MOLM14 cells that were treated with DMF for 24 hours, the mRNA expression of multiple mtDNA-encoded genes were quantified by qPCR. [0140] FIG. 10E shows ME2 -knockdown and re-expression KG1 cells that were treated with or without DMF for 24 hours. Whole cell lysate was subjected to western blotting to detect mtDNA and nDNA-encoded proteins. Band intensity was quantified and normalized to b- actin (Ratio).

[0141] FIG. 10F shows shRNAs targeting MRPL45 and NRF2 that were transduced into MOLM14 cells. The knockdown efficiency of MRPL45 and NRF2 were determined by western blotting. Cells were treated with DMF for 24 hours.

[0142] FIG. 10G shows protein expression of mtDNA and nDNA-encoded genes that was detected by western blotting.

[0143] FIG. 10H shows the MTG intensity of treated cells that was quantified.

[0144] FIG. 101 shows the schematic overview of fumarate-induced mitoribosome assembly. Left, ME2 monomer binds to MRPL45 and reduces its inner membrane attachment, leading to mitoribosome disassembly; Right, fumarate promotes the dimerization of ME2 and freeing MRPL45 to enhance mitoribosome activity. All data are presented as mean ± SEM from three independent experiments. **p < 0.01, n.s. indicates not significant.

[0145] FIG. 11A shows R67 is an evolutionarily conserved residue. R67 resides in the fumarate-binding domain, but not the catalytic center (top). Amino acid sequences adjacent to R67 across different species (SEQ ID NOS: 85-95 from top to bottom, respectively) were analyzed with multiple alignments (bottom).

[0146] FIG. 11B shows ME2-Flag that was expressed in HEK293 cells. Cells were treated with AMI-1 or AMI-5 for 24 hours. ME2-Flag protein was immunoprecipitated and subjected to western blotting to detect arginine methylation. ME2 activity was assayed with or without fumarate.

[0147] FIG. llC shows nitrocellulose membrane was spotted with increasing amounts of monomethyl-R67 peptide (R67-me) or unmodified peptide as indicated. The membrane was blotted with site-specific antibody against R67 methylation [a-me-ME2 (R67)] to determine its specificity.

[0148] FIG. 1 ID shows methylated R67 peptide, but not the unmodified peptide, that blocks the recognition of immunopurified ME2 protein by a-me-ME2(R67) antibody.

[0149] FIG. 11E shows Flag-tagged ME2 and its mutants (R67K and R67F) that were expressed in HEK293 cells. Cells were treated with or without AMI-5 for 24 hours. Immunopurified ME2 was subjected to western blotting and enzymatic activity assay.

[0150] FIG. 1 IF shows KG1 cells that were treated with or without AMI-5 for 24 hours. R67 methylation of immunoprecipitated endogenous ME2 was determined by a site-specific methylation antibody [a-me-ME2(R67)]. ME2 activity was assayed in the presence or absence of fumarate.

[0151] FIG. 11G shows ME2-Flag and its mutants (R67K and R67F) that were expressed in HEK293 cells. Cell lysate was cross-linked with glutaraldehyde and analyzed by western blotting.

[0152] FIG. 11H shows HA-tagged ME2 that was co-expressed with GFP-tagged wildtype ME2 or its mutants (R67K and R67F). Interaction between differently tagged ME2 was determined by co-immunoprecipitation and western blotting.

[0153] FIG. Ill shows HA-tagged ME2 that was co-expressed with GFP-tagged wildtype ME2 or R67F mutant. Cells were treated with AMI-5 for 24 hours. Interaction between differently tagged ME2 was determined by co-immunoprecipitation and western blotting. [0154] FIG. 11 J shows ME2-Flag that was co-expressed with GFP-tagged PRMTs in HEK293 cells. GFP-PRMT was immunoprecipitated using a GFP-specific antibody. The interaction between ME2 and PRMTs was determined by western blotting.

[0155] FIG. 11K shows ME2-Flag that was co-expressed with HA-tagged PRMT1 or PRMT4. Immunopurified ME2-Flag was subjected to western blotting and enzymatic activity assay.

[0156] FIG. 11F shows Wildtype ME2-Flag and its mutants that were co-expressed with HA- tagged PRMT1. ME2 protein was purified with Flag beads and subjected to western blotting. [0157] FIG. 11M shows scrambled control or two different shRNAs against PRMT1 that were stably expressed in KG1 cells. Immunopurified endogenous ME2 was subjected to western blotting and enzymatic activity assay

[0158] FIG. 11N shows PRMT1 that was co-expressed with differently tagged ME2 in HEK293 cells. Cells were treated with or without AMI-5 as indicated. HA-ME2 was immunoprecipitated and subjected to western blotting.

[0159] FIG. 110 shows unmethylated ME2 (left) and in vitro methylated recombinant ME2 (right) that were subjected to protein thermal shift assay at the presence of increasing doses of fumarate as indicated.

[0160] FIG. IIP shows Wildtype ME2-Flag and its mutants that were re-expressed in ME2- knockdown MOFM14 cells. Mitochondria were isolated after cells were treated with or without PRMT1 -specific inhibitor. Whole cell lysate and mitochondrial fraction were analyzed by western blotting to determine the isolation efficiency. [0161] FIG. 11Q shows ME2-knockdown and re-expression KG1 cells that were treated with PRMT1- specific inhibitor (PRMTli) and DMF as indicated for 24 hours. Total DNA was extracted and mtDNA copies were quantified by qPCR.

[0162] FIG. 11R shows control and PRMT1 -knockdown MOLM14 cells that were treated with or without DMF. Isolated mitochondria were fractionated and subjected to western blotting to determine MRPL45 localization. WCL, whole cell lysate; Cyt, cytoplasm; MT, mitochondria; MP, mitoplast; IM, inner membrane; Mtx, mitochondrial matrix.

[0163] FIG. 1 IS shows mitochondrial lysate that was loaded on a sucrose gradient to fractionate mitoribosome. MRPL12 and MRPS35 were included as markers for the large subunit and small subunit for mitoribosome, respectively.

[0164] FIG. 11T shows Control and PRMT1 -knockdown KG1 cells that were treated with DMF for 24 hours. Whole cell lysate was subjected to western blotting to determine the expression of mtDNA and nDNA-encoded proteins.

[0165] FIG. 11U shows ME2-knockdown and re-expression KG1 cells that were treated with PRMT1- specific inhibitor (PRMTli) and DMF as indicated. MTG intensity was determined. [0166] FIG. 11V shows protein expression of PRMT1 and CARM1 in the whole cell lysate of eight different AML cell lines that was determined by western blotting. Human CD34+ cord blood cells were included as normal control. All data are presented as mean ± SEM from three independent experiments. *p< 0.05, **p < 0.01, n.s. indicates not significant.

[0167] FIG. 12A shows Wildtype ME2 and its mutants that were re-expressed in ME2- knockdown KG1 cells. Growth curves were determined by cell counting.

[0168] FIG. 12B shows PBS or pyruvate (2 mM) that was added to the culture of scrambled control and ME2-knockdown cells. Growth curves of stable MOLM14 (B) cells that were determined by cell counting.

[0169] FIG. 12C shows growth curves of stable KG1 cells that were determined by cell counting.

[0170] FIG. 12D shows flag-tagged SLC1A3 that was stably expressed in control or ME2- knockdown cells. The expression of SLC1A3-Flag in MOLM14 cells was detected by western blotting. Growth curves were determined by cell counting.

[0171] FIG. 12E shows growth curves for expression of SLC1A3-Flag in MOLM14 cells. [0172] FIG. 12F shows flag-tagged SLC1A3 that was stably expressed in control or ME2- knockdown cells. The expression of SLC1A3-Flag in KG1 cells was detected by western blotting.

[0173] FIG. 12G shows growth curves for expression of SLC1A3-Flag in KG1 cells. [0174] FIG. 12H shows ME2-knockdown and re-expression KG1 cells were treated with PRMT1 inhibitor and DMF. Cell viability was determined by cell counting after four days of culture.

[0175] FIG. 121 shows colonies of KG1 cells that were counted seven days after treatment. [0176] FIG. 12J shows the protein expression of ME2, MT-ND6, MT-COl, MRPL45, and PRMT1 in normal and leukemic human bone marrow samples was determined by western blotting. The ratio indicates relative expression level after normalizing to b-actin. ME2 protein was immunoprecipitated and blotted with site- specific methylation antibody to determine R67 methylation level. The catalytic activity of immunopurified ME2 enzymes was assayed in the presence of fumarate.

[0177] FIG. 12K shows Pearson's correlation of ME2 protein expression with MT-COl protein in AML samples.

[0178] FIG. 12L shows Pearson's correlation of ME2 protein expression with MT-ND6 protein.

[0179] FIG. 12M shows mtDNA abundance in AML samples.

[0180] FIG. 12N shows protein expression of DUT, MRPL45, ME2, and FH that was determined by western blotting for MOLM14 cells.

[0181] FIG. 120 shows protein expression of DUT, MRPL45, ME2, and FH that was determined by western blotting for KG1 cells.

[0182] FIG. 12P shows relative mtDNA/nDNA for MOLM14 cells.

[0183] FIG. 12Q shows relative mtDNA/nDNA for KG1 cells.

[0184] FIG. 12R shows relative MTG intensity for MOLM14 cells.

[0185] FIG. 12S shows relative MTG intensity for KG1 cells.

[0186] FIG. 13 shows a graphical representation of fumarate sensing, mtDNA abundance, and mitoribosome assembly.

DETAILED DESCRIPTION OF THE INVENTION

[0187] Some Definitions

[0188] As used herein, a “subject” means a human or animal. "Subject" and "patient" may be used interchangeably herein. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. In some embodiments, the subject has cancer. In some embodiments, the subject has leukemia (e.g., AML).

[0189] The term “agent” as used herein means any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An “agent” can be any chemical, entity or moiety, including without limitation synthetic and naturally- occurring proteinaceous and non-pro teinaceous entities. In some embodiments, an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof etc. In some embodiments, the agent is selected from the group consisting of a nucleic acid, a small molecule, a polypeptide, and a peptide. In certain embodiments, agents are small molecule having a chemical moiety. For example, chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Compounds can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.

[0190] “Small molecule” is defined as a molecule with a molecular weight that is less than 10 kD, typically less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing an inorganic component, molecules comprising a radioactive atom, synthetic molecules, peptide mimetics, and antibody mimetics. As a therapeutic, a small molecule may be more permeable to cells, less susceptible to degradation, and less apt to elicit an immune response than large molecules.

[0191] As used herein, the term “polypeptide” is used to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The term “polypeptide” refers to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. The term “peptide” is often used in reference to small polypeptides, but usage of this term in the art overlaps with "protein" or "polypeptide." Exemplary polypeptides include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, as well as both naturally and non-naturally occurring variants, fragments, and analogs of the foregoing.

[0192] The term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and should be understood to include double- stranded polynucleotides, single- stranded (such as sense or antisense) polynucleotides, and partially double-stranded polynucleotides. A nucleic acid often comprises standard nucleotides typically found in naturally occurring DNA or RNA (which can include modifications such as methylated nucleobases), joined by phosphodiester bonds. In some embodiments, a nucleic acid may comprise one or more non-standard nucleotides, which may be naturally occurring or non- naturally occurring (i.e., artificial; not found in nature) in various embodiments and/or may contain a modified sugar or modified backbone linkage. Nucleic acid modifications (e.g., base, sugar, and/or backbone modifications), non-standard nucleotides or nucleosides, etc., such as those known in the art as being useful in the context of RNA interference (RNAi), aptamer, CRISPR technology, polypeptide production, reprogramming, or antisense-based molecules for research or therapeutic purposes may be incorporated in various embodiments. Such modifications may, for example, increase stability (e.g., by reducing sensitivity to cleavage by nucleases), decrease clearance in vivo, increase cell uptake, or confer other properties that improve the translation, potency, efficacy, specificity, or otherwise render the nucleic acid more suitable for an intended use. Various non-limiting examples of nucleic acid modifications are described in, e.g., Deleavey GF, et ah, Chemical modification of siRNA. Curr. Protoc. Nucleic Acid Chem. 2009; 39:16.3.1-16.3.22; Crooke, ST (ed.) Antisense drug technology: principles, strategies, and applications, Boca Raton: CRC Press, 2008; Kurreck, J. (ed.) Therapeutic oligonucleotides, RSC biomolecular sciences.

Cambridge: Royal Society of Chemistry, 2008; U. S. Patent Nos. 4,469,863; 5,536,821 ; 5,541,306; 5,637,683; 5,637,684; 5,700,922; 5,717,083; 5,719,262; 5,739,308; 5,773,601; 5,886,165; 5,929, 226; 5,977,296; 6,140,482; 6,455,308 and/or in PCT application publications WO 00/56746 and WO 01/14398. Different modifications may be used in the two strands of a double- stranded nucleic acid. A nucleic acid may be modified uniformly or on only a portion thereof and/or may contain multiple different modifications. Where the length of a nucleic acid or nucleic acid region is given in terms of a number of nucleotides (nt) it should be understood that the number refers to the number of nucleotides in a single- stranded nucleic acid or in each strand of a double- stranded nucleic acid unless otherwise indicated. An “oligonucleotide” is a relatively short nucleic acid, typically between about 5 and about 100 nt long.

[0193] Methods of Modulating Mitobiogenesis

[0194] Some aspects of the present disclosure are directed to a method of modulating cell proliferation and/or viability, comprising contacting the cell with an agent that modulates malic enzyme 2 (ME2) binding to mitochondrial ribosomal protein L45 (MRPL45). In some embodiments, the agent is not fumarate or a fumarate analog.

[0195] As used herein, the cell is not limited and may be any suitable cell. In some embodiments, the cell is isolated (e.g., in vitro or ex vivo). In some embodiments, the cell is in a subject. In some embodiments, the cell is a fibroblast, cells of skeletal tissue (bone and cartilage), cells of epithelial tissues (e.g. liver, lung, breast, skin, bladder and kidney), muscle cells, skeletal muscle cells, cardiac and smooth muscle cells, neural cells (glia and neurons, e.g., motor neurons, cortical neurons, dopaminergic neurons, etc.), endocrine cells (adrenal, pituitary, pancreatic islet cells), melanocytes, and many different types of hematopoietic cells (e.g., cells of B-cell or T-cell lineage, and their corresponding stem cells, lymphoblasts). In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell comprises a mitochondrial defect. In some embodiments, the cell is cancerous.

[0196] In some embodiments of the methods disclosed herein, the cell is a cancer cell. The cancer is not limited and may be any suitable cancer. In some embodiments, the cancer is glycolysis dependent. In some embodiments, the cancer cell is from breast cancer; biliary tract cancer; bladder cancer; brain cancer (e.g., glioblastomas, medulloblastomas); cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic leukemia and acute myelogenous leukemia; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma; adult T- cell leukemia/lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastoma; melanoma, oral cancer including squamous cell carcinoma; ovarian cancer including ovarian cancer arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; neuroblastoma, pancreatic cancer; prostate cancer; rectal cancer; sarcomas including angiosarcoma, gastrointestinal stromal tumors, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; renal cancer including renal cell carcinoma and Wilms tumor; skin cancer including basal cell carcinoma and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullary carcinoma.

[0197] In some embodiments, the agent increases or decrease cell proliferation and/or viability by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, the agent increases or decreases cell proliferation and/or viability by at least about 1.1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, at least a 1,000 fold, at least 10,000 fold, or more relative to a control cell.

[0198] In some embodiments, the agent increases or decrease ME2 binding to MRPL45 by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, the agent increases or decreases ME2 binding to MRPL45 by at least about 1.1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, at least a 1,000 fold, at least 10,000 fold, or more relative to ME2 binding to MRPL45 in a control cell.

[0199] In some embodiments, the agent enhances ME2 binding to MRPL45 and reduces mitobiogenesis in the cell. In some embodiments, mitobiogenesis is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, mitobiogenesis is reduced by at least about 1.1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, at least a 1,000 fold, at least 10,000 fold, or more.

[0200] In some embodiments, the agent suppresses or prevents ME2 binding to MRPL45 and enhances mitobiogenesis in the cell. In some embodiments, mitobiogenesis is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, mitobiogenesis is increased by at least about 1.1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, at least a 1,000 fold, at least 10,000 fold, or more.

[0201] In some embodiments, the agent blocks a MRPL45 binding site for ME2 or blocks a ME2 binding site for MRPL45.

[0202] In some embodiments, the agent enhances ME2 dimerization. In some embodiments, the agent suppresses ME2 dimerization. In some embodiments, the agent increases or decrease ME2 dimerization by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, the agent increases or decreases ME2 dimerization by at least about 1.1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, at least a 1,000 fold, at least 10,000 fold, or more.

[0203] In some embodiments, the agent that suppresses ME2 dimerization reduces fumarate binding to ME2. In some embodiments, the agent causes methylation of the ME2 binding site for fumarate.

[0204] In some embodiments, the agent increases or decrease ME2 dimerization by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, the agent increases or decreases ME2 dimerization by at least about 1.1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, at least a 1,000 fold, at least 10,000 fold, or more. [0205] In some embodiments, the agent that suppresses ME2 dimerization reduces fumarate binding to ME2. In some embodiments, the agent causes methylation of the ME2 binding site for fumarate.

[0206] In some embodiments of the methods disclosed herein, the agent modulate the activation of deoxyuridine 5 ’-triphosphate nucleotidohydrolase (DUT) in the cell. In some embodiments, the agent increases or decrease DUT activation by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, the agent increases or decreases DUT activation by at least about 1.1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, at least a 1,000 fold, at least 10,000 fold, or more.

[0207] In some embodiments of the methods disclosed herein, the agent modulates the generation of thymidine and/or mitochondrial DNA (mtDNA) in the cell. In some embodiments, the agent increases or decrease generation of thymidine and/or mtDNA by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. In some embodiments, the agent increases or decreases generation of thymidine and/or mtDNA by at least about 1.1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold, at least a 1,000 fold, at least 10,000 fold, or more.

[0208] In some embodiments of the methods disclosed herein, the agent modulates mitobiogenesis in the cell.

[0209] In some embodiments, the cell is contacted with the agent in vivo in a subject in need thereof. In some embodiments, the subject has a condition associated with a mitochondrial defect. In some embodiments, the subject has a cancer (e.g., AML).

[0210] Some aspects of the present disclosure are directed to a method of modulating cell proliferation and/or viability, comprising contacting the cell with an agent that modulates malic enzyme 2 (ME2) activation of deoxyuridine 5 ’-triphosphate nucleotidohydrolase (DUT). In some embodiments, the agent is not fumarate or a fumarate analog.

[0211] In some embodiments, the agent enhances DUT activation. In some embodiments, the agent suppresses DUT activation. In some embodiments, the agent modulates ME2 dimerization. In some embodiments, the agent is a ME2 dimer agonist or antagonist. In some embodiments, the agent is a ME2 dimer mimic. In some embodiments, the method modulates the generation of thymidine and/or mitochondrial DNA (mtDNA). In some embodiments, the method modulates mitobiogenesis in the cell. In some embodiments, the cell is contacted with the agent in vivo in a subject in need thereof. In some embodiments, the subject has a condition associated with a mitochondrial defect. In some embodiments, the subject has a cancer (e.g., leukemia such as AML).

[0212] Methods of Treatment

[0213] Some aspects of the present disclosure are directed to methods of treatment with the agents disclosed herein by administration of the agent to a subject. In some embodiments, the subject has a mitochondrial defect or aberrant mitobiogenesis. In some embodiments, the subject has a cancer. In some embodiments, the cancer is AML. In some embodiments, the cancer is dependent upon elevated mitobiogenesis for viability or proliferation.

[0214] Some embodiments of the present disclosure are directed to a method of treating a condition associated with a mitochondrial defect and/or a cancer (e.g., leukemia such as AML) comprising administering to a patient in need thereof a composition comprising an effective amount of an agent that modulates malic enzyme 2 (ME2) dimerization. In some embodiments, the agent is not fumarate or a fumarate analog. In some embodiments, the agent modulates the activation of deoxyuridine 5 ’-triphosphate nucleotidohydrolase (DUT). [0215] Some aspects of the present invention are directed to a method of treating leukemia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that modulates ME2 dimerization and/or ME2 binding to MRPL45 and/or the activation of DUT in the cell. The agent is not limited and may be any agent disclosed herein.

[0216] Acute myeloid leukemia (AML) is a malignancy of hematopoietic stem and progenitor cells that annually affects 20,000 people and claims 13,000 lives in the US alone (National Comprehensive Cancer Network (NCCN), Clinical Practice Guidelines in Oncology (2016)). New therapeutic strategies however have not yet been realized and the survival of AML patients has not improved significantly in decades. Significantly, the present inventors have discovered that inhibition of proton exporters (e.g., MCT4, NHE1) selectively eradicates and reduces the proliferation of leukemia cells, including leukemia initiating cells (LICs). [0217] In some embodiments of the invention, the agent is administered to a subject and reduces or eliminates the likelihood of developing leukemia (e.g., AML). In some embodiments, the subject has an increased risk of developing leukemia (e.g., AML). Several inherited genetic disorders and immunodeficiency states are associated with an increased risk of AML. These include disorders with defects in DNA stability, leading to random chromosomal breakage, such as Bloom's syndrome, Fanconi's anemia, Li-Fraumeni kindreds, ataxia-telangiectasia, and X-linked agammaglobulinemia. In some embodiments, the subject has increased risk of developing leukemia (e.g., AML) due to advanced age (e.g., over about 60, 65, 70, 75, 80, 85 years or more). In some embodiments, the subject has already been treated for leukemia (e.g., AML) and is in relapse. In some embodiments, the subject is treated by the methods of the invention immediately (e.g., within about 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month) after induction chemotherapy.

[0218] In some embodiments, administration of the agent reduces the risk of developing leukemia (e.g., AML) for about 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 7 years, 10 years, 15 years or more.

[0219] As used herein, the term “treating” and “treatment” refers to administering to a subject an effective amount of an agent so that the subject as a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. As used herein, the term “treatment” includes prophylaxis. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0220] As used herein, the term "therapeutically effective amount" means an amount of the agent which is effective to treat a disease (e.g., leukemia, cancer). Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject’s history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other agents that treat the disease (e.g., leukemia, cancer). [0221] As used herein, "administering" is not limited. In some embodiments, the agents described herein are administered, e.g., implanted, e.g., orally, systemically, sub- or trans- cutaneously, as an arterial stent, surgically, or via injection. In some examples, the agents described herein are administered by routes such as injection (e.g., subcutaneous, intravenous, intracutaneous, percutaneous, or intramuscular) or implantation.

[0222] In some embodiments, the agent is administered once every day to once every 10 years (e.g., once every day, once every week, once every two weeks, once every month, once every two months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every year, once every 2 years, once every 3 years, once every 4 years, once every 5 years, once every 6 years, once every 7 years, once every 8 years, or once every 10 years). In other examples, the composition is administered once to 5 times (e.g., one time, twice, 3 times, 4 times, 5 times, or more as clinically necessary) in the subject's lifetime.

[0223] Pharmaceutical Compositions

[0224] In another aspect, the invention is directed to a composition comprising an effective amount of an agent described herein (an agent that modulates ME2 dimerization and/or ME2 binding to MRPL45 and/or the activation of DUT in the cell). In some embodiments, the composition is a pharmaceutical composition.

[0225] In some embodiments, the composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present invention are found in Remington 's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer, Science 249:1527-1533, 1990.

[0226] The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intraarticular injection at areas affected by the vascular or cancer condition. Additional routes of administration include intravascular, intra-arterial, intratumor, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants or components. Thus, the invention provides compositions for parenteral administration that comprise the above mention agents dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, PBS, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. The invention also provides compositions for oral delivery, which may contain inert ingredients such as binders or fillers for the formulation of a tablet, a capsule, and the like. Furthermore, this invention provides compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, and the like.

[0227] These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above mentioned agent or agents, such as in a sealed package of tablets or capsules.

[0228] The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment. The compositions containing an effective amount can be administered for prophylactic or therapeutic treatments. In prophylactic applications, compositions can be administered to a patient with a clinically determined predisposition or increased susceptibility to development of a tumor or cancer. Compositions of the invention can be administered to the patient (e.g., a human) in an amount sufficient to delay, reduce, or preferably prevent the onset of clinical disease or tumorigenesis. In therapeutic applications, compositions are administered to a patient (e.g., a human) already suffering from a cancer in an amount sufficient to cure or at least partially arrest the symptoms of the condition and its complications.

[0229] An amount adequate to accomplish this purpose is defined as a "therapeutically effective dose," an amount of a compound sufficient to substantially improve some symptom associated with a disease or a medical condition. For example, in the treatment of cancer, an agent or compound which decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective. A therapeutically effective amount of an agent or compound is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual.

[0230] Amounts effective for this use may depend on the severity of the disease or condition and the weight and general state of the patient, but generally range from about 0.5 mg to about 3000 mg of the agent or agents per dose per patient. Suitable regimes for initial administration and booster administrations are typified by an initial administration followed by repeated doses at one or more hourly, daily, weekly, or monthly intervals by a subsequent administration. The total effective amount of an agent present in the compositions of the invention can be administered to a mammal as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a more prolonged period of time (e.g., a dose every 4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, 1-2 weeks, once a month). Alternatively, continuous intravenous infusion sufficient to maintain therapeutically effective concentrations in the blood are contemplated.

[0231] The therapeutically effective amount of one or more agents present within the compositions of the invention and used in the methods of this invention applied to mammals (e.g., humans) can be determined by the ordinarily- skilled artisan with consideration of individual differences in age, weight, and the condition of the mammal. The agents of the invention are administered to a subject (e.g. a mammal, such as a human) in an effective amount, which is an amount that produces a desirable result in a treated subject (e.g. the slowing or remission of a cancer or neurodegenerative disorder). Such therapeutically effective amounts can be determined empirically by those of skill in the art.

[0232] The patient may also receive an agent in the range of about 0.1 to 5,000 mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week), 0.1 to 2,500 (e.g., 2,000, 1,500, 1,000, 500, 100, 10, 1, 0.5, or 0.1) mg dose per week. A patient may also receive an agent of the composition in the range of 0.1 to 5,000 mg per dose once every two or three weeks.

[0233] Single or multiple administrations of the compositions of the invention comprising an effective amount can be carried out with dose levels and pattern being selected by the treating physician. The dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in the patient, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.

[0234] The compounds and formulations of the present invention may be used in combination with either conventional methods of treatment or therapy or may be used separately from conventional methods of treatment or therapy. When the compounds and formulations of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to an individual. Alternatively, pharmaceutical compositions according to the present invention include a combination of a compound or formulation of the present invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.

[0235] The formulated agents can be packaged together as a kit. Non-limiting examples include kits that contain, e.g., two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one patient, multiple uses for a particular patient (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple patients ("bulk packaging"). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

[0236] Methods of screening

[0237] Some aspects of the present invention are directed to a method of screening for a candidate agent that modulates cell proliferation and/or viability.

[0238] Some aspects of the present disclosure are directed to a method of screening for a candidate agent that modulates cell proliferation and/or viability, comprising providing a composition comprising malic enzyme 2 (ME2) and mitochondrial ribosomal protein L45 (MRPL45) or a fragment thereof capable of binding ME2 under conditions wherein ME2 and MRPL45 can bind, contacting the composition with a test agent, and assessing binding of ME2 with MRPL45, wherein a test agent that modulates binding of ME2 to MRPL45 as compared to a control composition comprising ME2 and MRPL45 or a fragment thereof capable of binding ME2 without the test agent is identified as a candidate agent that modulates cell proliferation and/or viability. In some embodiments, the MRPL45 fragment capable of binding ME2 comprises or consists of the ME2 binding site.

[0239] Some aspects of the present disclosure are directed to a method of screening for a candidate agent that modulates cell proliferation and/or viability, comprising providing a composition comprising malic enzyme 2 (ME2) under conditions wherein ME2 is capable of forming dimers, contacting the composition with a test agent, and assessing dimerization of ME2, wherein a test agent that modulates dimerization of ME2 as compared to a control composition comprising ME2 without the test agent is identified as a candidate agent that modulates cell proliferation and/or viability .The test agent is not limited and may be any agent described herein. In some embodiments, the agent is a small molecule.

[0240] In some embodiments of the screening methods disclosed herein, a high throughput screen (HTS) is performed. A high throughput screen can utilize cell-free or cell-based assays. High throughput screens often involve testing large numbers of compounds with high efficiency, e.g., in parallel. For example, tens or hundreds of thousands of compounds can be routinely screened in short periods of time, e.g., hours to days. Often such screening is performed in multiwell plates containing, at least 96 wells or other vessels in which multiple physically separated cavities or depressions are present in a substrate. High throughput screens often involve use of automation, e.g., for liquid handling, imaging, data acquisition and processing, etc. Certain general principles and techniques that may be applied in embodiments of a HTS of the present invention are described in Macarron R & Hertzberg RP. Design and implementation of high-throughput screening assays. Methods Mol Biol., 565:1-32, 2009 and/or An WF & Tolliday NJ., Introduction: cell-based assays for high- throughput screening. Methods Mol Biol. 486:1-12, 2009, and/or references in either of these. Useful methods are also disclosed in High Throughput Screening: Methods and Protocols (Methods in Molecular Biology) by William P. Janzen (2002) and High- Throughput Screening in Drug Discovery (Methods and Principles in Medicinal Chemistry) (2006) by Jorg Hhser.

[0241] The term “hit” generally refers to an agent that achieves an effect of interest in a screen or assay, e.g., an agent that has at least a predetermined level of modulating effect on cell survival, cell proliferation, gene expression, protein activity, or other parameter of interest being measured in the screen or assay. Test agents that are identified as hits in a screen may be selected for further testing, development, or modification. In some embodiments a test agent is retested using the same assay or different assays. Additional amounts of the test agent may be synthesized or otherwise obtained, if desired. Physical testing or computational approaches can be used to determine or predict one or more physicochemical, pharmacokinetic and/or pharmacodynamic properties of compounds identified in a screen. For example, solubility, absorption, distribution, metabolism, and excretion (ADME) parameters can be experimentally determined or predicted. Such information can be used, e.g., to select hits for further testing, development, or modification. For example, small molecules having characteristics typical of “drug-like” molecules can be selected and/or small molecules having one or more unfavorable characteristics can be avoided or modified to reduce or eliminated such unfavorable characteristic(s).

[0242] Additional compounds, e.g., analogs, that have a desired activity can be identified or designed based on compounds identified in a screen. In some embodiments structures of hit compounds are examined to identify a pharmacophore, which can be used to design additional compounds. An additional compound may, for example, have one or more altered, e.g., improved, physicochemical, pharmacokinetic (e.g., absorption, distribution, metabolism and/or excretion) and/or pharmacodynamic properties as compared with an initial hit or may have approximately the same properties but a different structure. For example, a compound may have higher affinity for the molecular target of interest, lower affinity for a non-target molecule, greater solubility (e.g., increased aqueous solubility), increased stability, increased bioavailability, oral bioavailability, and/or reduced side effect(s), modified onset of therapeutic action and/or duration of effect. An improved property is generally a property that renders a compound more readily usable or more useful for one or more intended uses. Improvement can be accomplished through empirical modification of the hit structure (e.g., synthesizing compounds with related structures and testing them in cell-free or cell-based assays or in non-human animals) and/or using computational approaches. Such modification can make use of established principles of medicinal chemistry to predictably alter one or more properties. An analog that has one or more improved properties may be identified and used in a composition or method described herein. In some embodiments a molecular target of a hit compound is identified or known. In some embodiments, additional compounds that act on the same molecular target may be identified empirically (e.g., through screening a compound library) or designed.

[0243] Data or results from testing an agent or performing a screen may be stored or electronically transmitted. Such information may be stored on a tangible medium, which may be a computer-readable medium, paper, etc. In some embodiments a method of identifying or testing an agent comprises storing and/or electronically transmitting information indicating that a test agent has one or more propert(ies) of interest or indicating that a test agent is a “hit” in a particular screen, or indicating the particular result achieved using a test agent. A list of hits from a screen may be generated and stored or transmitted. Hits may be ranked or divided into two or more groups based on activity, structural similarity, or other characteristics

[0244] Once a candidate agent is identified, additional agents, e.g., analogs, may be generated based on it. An additional agent, may, for example, have increased cell uptake, increased potency, increased stability, greater solubility, or any improved property. In some embodiments a labeled form of the agent is generated. The labeled agent may be used, e.g., to directly measure binding of an agent to a molecular target in a cell. In some embodiments, a molecular target of an agent identified as described herein may be identified. An agent may be used as an affinity reagent to isolate a molecular target. An assay to identify the molecular target, e.g., using methods such as mass spectrometry, may be performed. Once a molecular target is identified, one or more additional screens maybe performed to identify agents that act specifically on that target.

[0245] Any of a wide variety of agents may be used as a test agent in various embodiments. For example, a test agent may be a small molecule, polypeptide, peptide, amino acid, nucleic acid, oligonucleotide, lipid, carbohydrate, or hybrid molecule. In some embodiments a nucleic acid used as a test agent comprises a siRNA, shRNA, antisense oligonucleotide, aptamer, or random oligonucleotide. In some embodiments a test agent is cell permeable or provided in a form or with an appropriate carrier or vector to allow it to enter cells.

[0246] Agents can be obtained from natural sources or produced synthetically. Agents may be at least partially pure or may be present in extracts or other types of mixtures. Extracts or fractions thereof can be produced from, e.g., plants, animals, microorganisms, marine organisms, fermentation broths (e.g., soil, bacterial or fungal fermentation broths), etc. In some embodiments, a compound collection (“library”) is tested. A compound library may comprise natural products and/or compounds generated using non-directed or directed synthetic organic chemistry. In some embodiments a library is a small molecule library, peptide library, peptoid library, cDNA library, oligonucleotide library, or display library (e.g., a phage display library). In some embodiments a library comprises agents of two or more of the foregoing types. In some embodiments oligonucleotides in an oligonucleotide library comprise siRNAs, shRNAs, antisense oligonucleotides, aptamers, or random oligonucleotides. [0247] A library may comprise, e.g., between 100 and 500,000 compounds, or more. In some embodiments a library comprises at least 10,000, at least 50,000, at least 100,000, or at least 250,000 compounds. In some embodiments compounds of a compound library are arrayed in multiwell plates. They may be dissolved in a solvent (e.g., DMSO) or provided in dry form, e.g., as a powder or solid. Collections of synthetic, semi-synthetic, and/or naturally occurring compounds may be tested. Compound libraries can comprise structurally related, structurally diverse, or structurally unrelated compounds. Compounds may be artificial (having a structure invented by man and not found in nature) or naturally occurring. In some embodiments compounds that have been identified as “hits” or “leads” in a drug discovery program and/or analogs thereof. In some embodiments a library may be focused (e.g., composed primarily of compounds having the same core structure, derived from the same precursor, or having at least one biochemical activity in common). Compound libraries are available from a number of commercial vendors such as Tocris BioScience, Nanosyn, BioFocus, and from government entities such as the U.S. National Institutes of Health (NIH). In some embodiments a test agent is not an agent that is found in a cell culture medium known or used in the art, e.g., for culturing vertebrate, e.g., mammalian cells, e.g., an agent provided for purposes of culturing the cells. In some embodiments, if the agent is one that is found in a cell culture medium known or used in the art, the agent may be used at a different, e.g., higher, concentration when used as a test agent in a method or composition described herein.

[0248] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.

[0249] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0250] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or prior publication, or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. [0251] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0252] The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more active agents, additives, ingredients, optional agents, types of organism, disorders, subjects, or combinations thereof, can be excluded.

[0253] Where the claims or description relate to a composition of matter, it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0254] Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by “about” or “approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by “about” or “approximately”, the invention includes an embodiment in which the value is prefaced by “about” or “approximately”.

[0255] “Approximately” or “about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited.

It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered “isolated”.

EXAMPLES

[0256] Mitochondria are powerhouses of cellular metabolism that are highly integrated into eukaryotic cell bioenergetic requirements (Birsoy et ah, 2015; Chandel, 2015; Sullivan et ah,

2015). Mitochondrial mass is dynamically regulated by both the nuclear and mitochondrial genomes (nDNA and mtDNA) in response to nutrient availability. Mitochondria maintain a dNTP pool to support the replication of mtDNA, which encodes at least 13 proteins, and harbor unique ribosomal proteins for protein generation (Mansueto et ah, 2017; Wallace, 2016; Zong et ah, 2016). The mitoribosomal proteins are synthesized in the cytoplasm and assembled in mitochondria (Bogenhagen et ah, 2018; Brown et ah, 2017; Rackham et ah,

2016) where they are required for mtDNA-encoded protein translation. All of the mtDNA- encoded proteins are synthesized within the mitochondria and function in the electron transport chain (ETC), necessitating their residence in the mitochondrial inner membrane (Richter-Dennerlein et al., 2016). Notably, mitochondrial ribosomal protein L45 (MRPL45) directly binds to the inner membrane and is required for mitoribosome assembly (Kehrein et al., 2015; Zeng et al., 2018).

[0257] Mammalian cells fuel mitochondria with a variety of nutrients (Chen et al., 2016a; Corbet et al., 2016; DeBerardinis and Chandel, 2016; Faubert et al., 2017). Multiple nutrient sensing pathways in the cytoplasm and nucleus have been discovered to govern mitobiogenesis-related transcriptional and translational programs. In contrast, how mitochondria sense nutrients and modulate biomass production is unclear.

[0258] Dysregulated mitobiogenesis has been implicated in multiple human diseases including cancer and aging (Dom et al., 2015; Yambire et al., 2019). Notably, hyperactivation of mitobiogenesis has been shown to promote leukemia, liver cancer, and breast cancer (Carew et al., 2004; Jitschin et al., 2014; LeBleu et al., 2014; Martinez- Outschoorn et al., 2011; Skrtic et al., 2011; Tohme et al., 2017). Mitobiogenesis regulation is of particular interest in acute myeloid leukemia (AML), a highly lethal hematopoietic neoplasm. AML cells have been reported to have more mitochondria than normal hematopoietic cells (Boultwood et al., 1996) and are dependent upon oxidative metabolism (Baccelli et al., 2019; Jones et al., 2018; Konopleva et al., 2016; Molina et al., 2018). More importantly, chemoresistant AML cells shift to higher oxidative phosphorylation (OXPHOS) and are particularly sensitive to inhibition of cellular respiration (Farge et al., 2017). Agents that inhibit mitochondrial translation (Farge et al., 2017) have shown promising effects in suppressing AML (Jones et al., 2018). Therefore, metabolite- sensing pathways that regulate mitobiogenesis were identified. Unexpectedly, fumarate was defined as a signaling metabolite that acts via malic enzyme 2 to regulate mitochondrial biomass production.

[0259] Results

[0260] Fumarate upregulates mitochondrial biomass

[0261] To define metabolites acting as signaling molecules to regulate mitochondrial biomass, a cell-based screen wasconducted. AML cell lines were used due to their sensitivity to OXPHOS (Carew et al., 2004) with normal CD34+ cord blood cells as controls. Cells were exposed to different metabolites for 48h and assayed mtDNA and mitotracker green (MTG) for mitochondrial mass. Glucose mildly increased mitochondrial mass, in agreement with its role as a major carbon source (FIG. 7A). Surprisingly, cell-permeable fumarate (dimethyl fumarate, DMF) strongly elevated mtDNA and MTG staining of AML cells, but not of normal cells. Succinate, the mitochondrial precursor of fumarate, only led to a modest mitochondrial mass increase (FIG. 7A). Notably, treatment with cell-permeable fumarate, including DMF and diethyl fumarate (DEF), increased mitochondrial mass in a dose and time-dependent manner (FIGS. 1A and 7B-C) significantly above control (FIGS. 7D-E). Correspondingly, fumarate levels positively correlate with OXPHOS potential in AML cells (FIGS. IB and 7F). Mitochondria number by transmission electron microscopy was 1.98 fold higher in DMF-treated MOLM14 cells (FIGS. 1C-D). Because DMF is an electrophilic compound that modifies proteins on thiols (Kulkami et al., 2019), it was asked whether DMF rather than fumarate elevated mitochondrial mass. Due to the low cell permeability of fumarate, AML cells were treated with a high dose of fumarate (1 mM) compared with fumarate esters, including monomethyl fumarate (MMF) and monoethyl fumarate (MEF). Fumarate and its esters elevated intracellular and mitochondrial fumarate by approximately 2- fold in MOLM14 cells (FIGS. IE and 7G-H), with a concurrent increase in mtDNA, MTG intensity (FIGS. 1F-G) and mitochondrial respiration (FIG. 1H). Importantly, the mtDNA increase was greater in DMF-treated cells than in cells overexpressing nuclear respiration factor 2 (NRF2), a known nuclear transcription regulator of mitobiogenesis (Guo et al., 2019) (FIGS. 71- J). [U-13C]-fumarate tracing in MOLM14 cells further demonstrated that the products of mitochondrial fumarate metabolism, including malate and citrate, were efficiently labeled (FIGS. 7K-N). These data indicate that exogenous fumarate is capable of entering into mitochondria and increasing mitochondrial mass.

[0262] It was next asked how fumarate modulates mitochondrial mass. As mitochondria have a distinct dNTP pool, multiple metabolites in nucleotide metabolism were quantified. In line with the enhanced respiration, fumarate increased cellular and mitochondrial levels of ATP and NADH, indicating an increase in energy production (FIGS. II, 7G and 70). The substrates for DNA synthesis, dTTP, dCTP, dATP, and dGTP, were also increased in fumarate-treated cells (FIGS. II and 70). These findings suggest that fumarate may promote dNTP anabolism to provide building blocks for mtDNA. This is confirmed by the increased generation of mtDNA after fumarate treatment (FIG. 7D).

[0263] Mitochondrial proteins are encoded by both mtDNA and nDNA. It was tested whether fumarate modulates mitochondrial gene transcription. Notably, fumarate minimally changed mtDNA-encoded mRNAs (FIG. 7P) or nDNA-encoded mitochondrial enzymes including mRNA for SDHA (succinate dehydrogenase A) and GLUD1 (glutamate dehydrogenase 1) (FIG. 7P). Furthermore, fumarate did not modify mitochondrial RNA polymerase (POLRMT) levels (FIG. 1J). mtDNA abundance did not couple with mtDNA transcription, consistent with findings by others (Agaronyan et ah, 2015). However, multiple mitochondrial proteins including ETC proteins encoded by either mtDNA (MT-COl, MT-ND6, and MT- ATP6) or nDNA (NDUFB8, SDHA, and ATP5A) were increased by fumarate treatment (FIG. 1J). This corresponded to the enhanced respiration that was observed. In contrast, nDNA-encoded mitochondrial proteins such as glutamate dehydrogenase 1 (GFUD1), malic enzyme 2 (ME2), and nuclear transcription factors regulating mitochondrial biogenesis such as NRF2 and peroxisome proliferator-activated receptor-g coactivator la (PGCla), were not affected by fumarate and its esters (FIG. 1J). Therefore, fumarate upregulates mitochondrial dNTPs, mtDNA and, selectively, mtDNA-encoded proteins.

[0264] As fumarate esters and high dose fumarate equivalently elevated mitochondrial mass, an esterified form of fumarate in our following experiments was used. Testing fumarate in vivo, daily intraperitoneal administration of fumarate esters (MMF and DMF) led to a remarkable accumulation of fumarate in multiple organs but not kidney (FIG. 7Q). In agreement, mtDNA abundance was significantly increased in the tested organs except kidney (FIG. IK). mtDNA-encoded proteins were elevated by MMF and DMF (FIGS. IF and 7R). Focusing on hematopoietic tissue, MMF and DMF moderately increased MTG staining and mitochondrial respiration of bone marrow (BM) cells (FIGS. 1M-N). Taken together, fumarate upregulates mitochondrial biomass.

[0265] Fumarate relies on ME2 to increase mitochondrial biomass

[0266] Assessing the mechanism by which fumarate alters mitochondria, metabolic enzymes that directly bind fumarate including fumarylacetoacetase (FAH), adenylosuccinate lyase (ADSF), argininosuccinate lyase (ASF), SDHA, fumarate hydratase (FH), and malic enzyme 2 (ME2) were analyzed (Tao et ah, 2003) (FIG. 2A). NRF2 (positive control) and fumarate- binding enzymes with short-hairpin RNAs (shRNAs) were silenced (FIG. 8A). Knockdown of NRF2 and ME2, but not the other enzymes, abolished the DMF-mediated increase of mtDNA (FIG. 2B). These results demonstrate that ME2 and NRF2 are required for fumarate to elevate mitochondrial mass.

[0267] Mammalian malic enzymes have three paralogues. Notably, ME2 is a mitochondrial enzyme coordinating glucose and glutamine metabolism (FIG. 2C) (Jiang et ah, 2013). Interestingly, ME2, but not its mitochondrial paralogue ME3, was highly expressed in AMF cells (FIG. 8B). To evaluate the functions of malic enzymes were depleted with short guide RNAs (sgRNAs) and quantified mitochondrial mass (FIG. 8C). Knockdown of ME2, but not ME1 or ME3, dramatically lowered mtDNA and MTG intensity in AMF cell lines (FIG. 2D), accompanied by decreased consumption of glucose and glutamine (FIGS. 8D-E). Importantly, DMF was incapable of increasing mitochondrial mass in ME2-depleted AML cells (FIG. 2E). To test the cell specificity of fumarate-induced mitobiogenesis, cancer cell lines were collected with different tissues-of-origin and stably expressed ME2-targeting shRNAs (FIG. 8F). DMF increased mtDNA in 27 of 28 cell lines tested. This increment was abolished in ME2-depleted cells (FIG. 2F), Therefore, ME2 is indispensable for fumarate to upregulate mitochondrial mass.

[0268] Examining ME2 in vivo , ME2 with shRNAs in lin BM cells were stably silenced. Three weeks after transplanting control or Me2-knockdown cells, DMF was intraperitoneally injected for seven days (FIGS. 2G-H). Me2 knockdown led to a moderate decrease of mtDNA, MTG intensity, and oxygen consumption in lin- BM cells (FIGS. 21- K).

Importantly, DMF failed to upregulate mitochondria mass or mitochondrial respiration in Me2-knockdown cells (FIGS. 21- K). These observations suggest that Me2 mediates fumarate- induced mitobiogenesis in vivo.

[0269] ME2 responds to fumarate by increasing PUT activity and mtDNA [0270] ME2 mediated fumarate signaling was then investigated. Although ME2 diverts malate from the TCA cycle to produce pyruvate (FIG. 2C), citrate, but not pyruvate, was efficiently labeled in [U-13C]-fumarate tracing assay (FIGS. 7M-N). These data indicate that the catalytic activity of ME2 plays a minor role in mitochondrial carbon flux. Because fumarate is an allosteric activator of ME2 (Tao et ah, 2003), it was hypothesized that fumarate might conjugate with or physically bind to ME2 to regulate mitochondrial mass. [0271] High dose fumarate (>1 mM) was previously reported to modify thiols in the form of succination to destabilize or inactivate thiol-containing proteins, such as aconitase 2 (AC02) (Temette et ah, 2013). MOLM14 cells were transduced with Flag-tagged ME2 and AC02 and treated them with increasing doses of DMF. Western blotting analysis demonstrated that AC02 succination was mildly increased by high dose DMF, while succination of ME2 was undetectable (FIG. 9A). Fumarate conjugation would be expected to decrease free thiols on ME2. Interestingly, the biotin-conjugated maleimide assay for free thiols showed no effect on ME2 from high doses of DMF while the pulldown efficacy of AC02 was decreased (FIG. 9B). Besides, ME2 protein was unchanged by MMF and DMF (FIG. 9C). Together, these data argue against the possibility that the fumarate ester covalently modifies ME2.

[0272] The physical association of fumarate and ME2 were then evaluated. The residence of fumarate in the ME2 dimer interface (FIG. 3A) indicates that fumarate potentially regulates ME2 oligomerization. As anticipated, fumarate and its esters enhanced ME2 dimerization in glutaraldehyde crosslinking assays (FIGS. 3B and 9D). Point mutations at the dimer interface to generate fumarate-binding defective mutants (R67F and E59L) were then introduced (FIG. 3A). A catalytic-inactive mutant (CM) was also generated by mutating three key residues (glutamate 164, argininel65, and isoleucine 166) to alanines in the catalytic center. As anticipated, fumarate-binding defective mutations decreased ME2 activity and abolished fumarate-induced activation, while the CM mutant lost its catalytic activity (FIG. 9E). More importantly, R67F and E59L mutants, but not the CM mutant, existed predominantly as monomers (FIG. 3C). DMF treatment promoted the dimerization of wildtype ME2 and ME2CM, but not fumarate binding-defective mutants (FIG. 3C). Co-immunoprecipitation (co-IP) assay further showed that fumarate enhanced the interaction of ME2-HA with wildtype ME2-GFP and its CM mutant, but not R67F and E59L mutants (FIGS. 9F-G). In addition, ME2 was silenced with shRNA and re-introduced wildtype ME2-Flag and its mutants in MOLM14 and KG1 cells (FIGS. 9H-I). ME2 depletion significantly decreased mitochondrial respiration, which was largely restored by the CM mutant, but not the fumarate binding-defective mutants (FIGS. 3D and 9J). These results suggest that the fumarate-ME2 association is essential to maintain mitochondrial respiration. Importantly, the monomer-to- dimer transition of ME2 then potentially provides a structural basis for fumarate signaling. [0273] Whether ME2 was involved in fumarate-induced upregulation of mtDNA was the next question. Because fumarate increased mitochondrial dNTP, it was hypothesized that fumarate-ME2 interaction may regulate dNTP metabolism. Wildtype ME2-Flag and the R67F mutant from MOLM14 cells were immunopurified to identify ME2 interactors (FIGS. 3E and 9K). Proteomic profiling revealed that two enzymes in dNTP metabolism, deoxyuridine 5'-triphosphate nucleotidohydrolase (DUT) and dCTP pyrophosphatase 1 (dCTPPl), interacted with ME2 (FIG. 3E). DUT converts dUTP to dUMP and enables dTTP production (FIG. 3F). Interestingly, fumarate decreased dUTP and increased dUMP in MOLM14 cells (FIGS. 3G and 7G), suggesting an elevation of DUT activity. DUT was therefore focused on because it not only supports thymidine nucleotide production to maintain mtDNA synthesis but also limits dUTP, which can misincorporate into DNA and destabilize mtDNA (Hirmondo et al., 2017) (FIG. 3F). As expected, TAS114, a DUT-specific inhibitor, elevated dUTP and decreased dUMP in MOLM14 cells (FIGS. 3H and 9L). Importantly, TAS 114 also abrogated the effect of DMF on mitochondrial dUTP and dUMP (FIGS. 3H), suggesting DMF dependency on DUT to modulate dUTP and dUMP levels. Since fumarate mediates ME2 dimerization, the question was whether fumarate affected the association of ME2 and DUT. Fumarate treatment increased the binding of ME2 with DUT, but not with another ME2 interactor, glutamic-oxaloacetic transaminase 2 (GOT2) (FIG. 31). Co-IP assays further showed that R67F and E59L mutations abolished the interaction of ME2 and DUT (FIG. 9M). Notably, fumarate increased the association of DUT with wildtype ME2 and ME2CM, but not the fumarate binding-defective mutants (FIG. 9M). These data demonstrate that dimerization of ME2, which is enhanced by fumarate, increases the interaction between ME2 and DUT.

[0274] To determine whether ME2 modulates DUT activity, the catalytic efficiency of immunopurified DUT-Flag in the presence of fumarate or recombinant ME2 was assayed (FIG. 9N). Fumarate alone was incapable of activating DUT (FIG. 3J). Wildtype ME2 and its CM mutant, but not ME2R67F and ME2E59L, mildly increased DUT’s activity. Importantly, fumarate strongly increased the catalytic efficiency of DUT in the presence of ME2 and its CM mutants, but not fumarate binding-defective mutants (FIG. 3J). Similar results were observed in DUT activity assays using mitochondria lysates (FIGS. 3K and 90). These data indicate that fumarate-induced ME2 dimerization activates DUT.

[0275] Next tested was whether ME2 modulated dNTP levels. DMF mildly elevated dUMP and dTTP, with a concomitant mild increase of dATP, dGTP, and dCTP (FIG. 9P). Mitochondria from treated cells was further isolated and found that the mitochondrial dNTPs showed more dramatic changes (FIGS. 3L and 90). In contrast, DMF treatment of ME2- knockdown cells rescued by fumarate binding-defective mutants failed to increase dUMP and four dNTPs (FIG. 3L). These data demonstrate that fumarate modulates dNTP levels through binding with ME2. Moreover, DMF failed to upregulate mtDNA in ME2-knockdown cells and cells rescued by fumarate binding-defective mutants (FIGS. 3M and 9Q). Importantly, TAS114 strongly decreased mtDNA copy number, which was no longer elevated by DMF (FIGS. 3M and 9Q). These results suggest that DUT activity is indispensable for fumarate signaling to increase mtDNA. Together, fumarate signals through ME2 to activate DUT and elevate mitochondrial dNTP levels, resulting in the upregulation of mtDNA.

[0276] ME2 responds to fumarate by modulating mitoribosome assembly [0277] In addition to mtDNA, fumarate also upregulated mtDNA-encoded proteins (FIG. 1J). Given mitochondrial mRNA levels were modestly affected by fumarate, the question was whether ME2 regulated mitochondrial protein translation. Grouping of ME2 interactors by their annotated functions (www.uniprot.org) revealed a wide distribution across mitochondria biology (FIG. 4A). Notably, the fumarate- sensing defective mutant (R67F) showed a dramatic decrease in the number of ME2-interacting mitoribosomal proteins, but not ETC components (FIGS. 4A-B). This observation led us to question whether ME2 regulates the mitoribosome. [0278] The regulatory proteins of the mitoribosome in the ME2 interactome was a focus, including MRPL45 and mitochondrial ribosome recycling factor (MRRF) (Table 2). While MRPL45 anchors the mitoribosome to the mitochondrial inner membrane (Kummer et ah, 2018), MRRF dissociates mitoribosomes from mRNA upon translation termination to recycle them (Rorbach et ah, 2008). Co-IP assays revealed that wildtype ME2 and R67F mutant bound MRRF at similar levels (FIG. 4C). However, MRPF45 differentially bound ME2R67F compared with wildtype ME2 and ME2CM. More importantly, DMF disrupted the binding of MRPF45 with wildtype ME2 and ME2CM, but not ME2R67F (FIG. 4C). Additionally, semi- endogenous co-IP showed that MRPF45 interacted with ME2, but not ME3 (FIG. 10A). In vitro pulldown further demonstrated that fumarate, but not malate or succinate, dissociated the ME2-MRPF45 complex (FIG. 10B). These results indicate that monomeric ME2 strongly interacts with MRPF45, and that the interaction is disrupted by fumarate.

[0279] MRPF45 has tails on both ends to bind to the mitoribosome large subunit and a core domain that directly interacts with the inner membrane (FIG. 4D). MRPF45 was truncated to map the ME2-binding region. Co-IP assays showed that the full-length MRPF45 and its AC mutant, but not the N-mutant, interacted with ME2 (FIGS. 4D-E), indicating that ME2 binds to the core domain of MRPF45 and potentially regulates its inner membrane attachment. To examine this, mitochondria from ME2-knockdown and re-expression MOFM14 cells was isolated and further fractionated mitochondria into mitoplast (MP, without outer membrane), inner membrane (IM), and matrix (Mtx) (FIG. IOC). Western blotting demonstrated that in cells rescued by wildtype ME2 or ME2CM, approximately 49% of total MRPF45 located on the inner membrane (FIGS. 4F and IOC). In contrast, only 13% of MRPF45 resided on the inner membrane in R67F-rescued MOFM14 cells. Exogenous fumarate dramatically increased inner membrane attachment of MRPF45 in control cells, but not in cells rescued by ME2R67F (FIG. 4F). Therefore, ME2 monomers dissociate MRPF45 from the inner membrane. Fumarate promotes inner membrane attachment of MRPF45 by abolishing ME2- MRPF45 interaction.

[0280] Inner membrane attachment of MRPF45 is a prerequisite for mitoribosome assembly and activity (Kummer et ah, 2018). Mitoribosome assembly using MRPF12 and MRPS35 as the markers for large and small subunits, respectively was then evaluated. While ME2 knockdown reduced the level of mitoribosome assembly, reintroduction of wildtype ME2 or ME2CM, but not ME2R67F, restored mitoribosome complexing in MOFM14 cells (FIG.

4G). Notably, DMF treatment enhanced mitoribosome assembly in control cells and cells that were rescued by wildtype ME2 or ME2CM, but not in ME2-knockdown cells or cells rescued by ME2R67F (FIG. 4G). Therefore, fumarate binding is essential for ME2 to regulate mitoribosome assembly. Mitoribosomes are dedicated to manufacturing proteins in the ETC. Interestingly, mtDNA-encoded ETC genes including MT-ND5, MT-COl, MT-CYB, MT- C02, MT-ATP6, and MT-ND6 showed modest changes in their mRNA expression in ME2- knockdown and re-expression cells (FIG. 10D). However, ME2 knockdown reduced the protein expression of these mtDNA-encoded genes (FIGS. 4H and 10E). Re-introducing wildtype ME2 and ME2CM restored the level of mtDNA-encoded proteins. This did not happen with fumarate- sensing defective mutants (FIGS. 4H and 10E). DMF was unable to enhance mtDNA-encoded protein expression in cells rescued by the R67F and E59F mutants (FIGS. 4H and 10E). Interestingly, nuclear-encoded ETC proteins (NDUFB8, SDHA, and ATP5A) showed a similar expression pattern (FIGS. 4H and 10E). Previously, nDNA and mtDNA have been shown to produce mitochondrial proteins in a synchronized manner (Couvillion et ah, 2016). To examine whether fumarate coordinates nDNA and mtDNA- encoded protein expression, MRPF45 and NRF2 were silenced and observed a decrease of ETC proteins and MTG intensity (FIGS. 10F-H). Moreover, fumarate-induced mitobiogenesis was blocked in these cells (FIGS. 10G-H). Taken together, ME2 functions as a fumarate responsive sensor to modulate mitoribosome assembly and mtDNA-encoded protein expression (FIG. 101).

[0281] PRMT1 methylates ME2 inhibiting fumarate sensing

[0282] Because mitobiogenesis commits a cell to an energetically expensive process, it was hypothesized that there may be more than a simple direct relationship of fumarate levels to ME2 initiation of mitobiogenesis. Specifically, assessed were possible post-translational modifications, a well-defined mechanism of modifying enzyme activity and metabolite binding (Xiong and Guan, 2012), that might modulate the fumarate-ME2 interaction. The focus was on the fumarate-binding site (FIG. 11 A) where arginine 67 (R67) is the only residue reported to be methylated (Farsen et ah, 2016). Whether methylation of it by arginine methyltransferases (PRMTs) regulates fumarate responsiveness was assessed. Notably, treatment with PRMT inhibitors (AMI-1 and AMI- 5) revealed that ME2 arginine methylation associated with downregulated activity (FIG. 11B). To precisely monitor R67 methylation, a site-specific methylation antibody [a-me-ME2(R67)] was generated (FIGS. 11C-D). R67 into lysine (R67K) was also mutated. Notably, wildtype ME2, but not R67K or the R67F mutant, was readily recognized by the site-specific methylation antibody (FIG. 11E). AMI-5 treatment resulted in a 4-fold decrease of R67 methylation, with a concomitant increase of ME2 activity. ME2R67F and ME2R67K were barely recognized by the site-specific methylation antibody and were deficient in catalysis (FIG. 11E). Notably, AMI-5 reduced R67 methylation of endogenous ME2 in MOLM14 and KG1 cells (FIGS. 5A and 11F). Further, both R67K and R67F mutations disrupted ME2 dimerization (FIGS. 11G-H). In addition, AMI-5 treatment enhanced the interaction of ME2-HA with wildtype ME2-GFP, but not ME2R67F-GFP (FIG. 1 II). These data indicate that R67 methylation suppresses ME2 dimerization.

[0283] To identify the ME2 methylase, ME2-Flag was co-expressed with GFP-tagged PRMTs (PRMT1-PRMT9) (Blanc and Richard, 2017). ME2 selectively interacted with PRMT1 and PRMT4 (also named CARM1) (FIG. 11J). PRMT1, but not CARM1, increased R67 methylation and suppressed ME2 activity (FIG. 11K). Importantly, endogenous ME2 readily associated with PRMT1 in AML cells (FIG. 5B). Overexpression of PRMT1 upregulated the methylation of wildtype ME2, but not ME2R67F (FIG. 11L). Furthermore, PRMT1 efficiently methylated recombinant ME2 at R67 in vitro (FIG. 5C). Depletion of PRMT1 prominently reduced R67 methylation of endogenous ME2 (FIGS. 5D and 11M). These results demonstrate that PRMT1 methylates and inhibits ME2.

[0284] Next, we tested whether PRMT1 modulates ME2 dimerization. PRMT1 suppressed the binding between ME2-HA and ME2-GFP, which was restored by AMI-5 treatment (FIG.

1 IN). Silencing PRMT1 induced a monomer-to-dimer transition of ME2 in MOLM14 cells (FIG. 5E). Fumarate markedly increased the thermal stability of recombinant ME2, but not its methylated form (FIGS. 5C, 5F and 110), consistent with methylation directly suppressing fumarate binding. Together, we conclude that PRMT1 methylates ME2 to block fumarate binding and dimerization.

[0285] Next investigated was whether PRMT1 regulated mtDNA levels. Notably, the interaction between ME2 and DUT was increased in PRMT1 -knockdown cells and was further enhanced by DMF treatment (FIG. 5G). These data suggest that PRMT1 potentially inhibits DUT activity through methylating ME2. To test this, ME2-knockdown and re expression cells were treated with the PRMT1 -specific inhibitor TCE5003 (hereafter PRMTli), and isolated mitochondria to determine endogenous DUT activity. PRMTli enhanced DUT activity in control cells and cells re-expressing wildtype ME2 or ME2CM, but not in ME2-knockdown cells or cells rescued by fumarate binding-defective mutants (FIGS. 5H and IIP). Consistently, PRMTli alone increased mtDNA abundance, which was further upregulated by DMF treatment (FIGS. 51 and 1 IQ). mtDNA copy number was not modulated by PRMTi in cells re-expressing fumarate binding-defective mutants (FIGS. 51 and 11Q), suggesting that PRMT1 decreases DUT activity and mtDNA in a manner dependent on the fumarate- sensing activity of ME2.

[0286] It was then asked whether PRMT1 regulated mitochondrial protein expression. PRMT1 depletion weakened the binding between ME2 and MRPL45 (FIG. 5J). Accordingly, PRMT1 knockdown increased the fraction of inner membrane-bound MRPL45 (FIGS. 5K and 11R), enhanced mitoribosome assembly (FIG. 1 IS), and increased mtDNA-encoded protein (FIGS. 5F and 11T). DMF further upregulated these mtDNA-encoded proteins (FIGS. 5F and 11T). MTG staining assay showed that PRMT1 inhibition increased mitochondria mass in control cells and cells re-expressing wildtype ME2 or ME2CM, but not fumarate sensing-defective mutants (FIGS. 5M and 11U). Collectively, PRMT1 suppresses fumarate signaling by decreasing inner membrane attachment of mitoribosome and mtDNA-encoded protein expression.

[0287] The impact of fumarate on mitochondrial mass varies across different cell lines (FIGS. 2D and 2F), it was therefore asked whether ME2 methylation correlates with fumarate-induced mitobiogenesis. While AMF cells and their normal counterparts expressed similar levels of PRMT1 protein (FIG. 1 IV), ME2 methylation was generally lower in AMF cells (FIG. 5N). In addition, cell lines that were sensitive to fumarate-induced mitobiogenesis showed lower R67 methylation (FIGS. 2F and 50). Together, PRMT1 negatively regulates fumarate sensing and mitobiogenesis.

[0288] ME2-mediated fumarate sensing supports leukemia growth

[0289] Oxidative metabolism is a distinctive vulnerability of myeloid malignancies (Pollyea et ah, 2018; Skrtic et ah, 2011; Stevens et ah, 2018). In MOFM14 and KG1 cells, knockdown of ME2 led to a proliferative defect, which was rescued by wildtype ME2 and ME2CM, but not the fumarate binding-defective mutants (FIGS. 6 A and 12A). Activating ETC by exogenous pyruvate or SEC 1 A3, a high affinity glutamate transporter, partially restored the proliferation of ME2-depleted cells (FIGS. 12B-G). Therefore, ETC activity is only part of the growth defect imposed by ME2-knockdown. Further, DMF moderately upregulated cell proliferation and colony formation in control cells, but not cells re-expressing fumarate binding-defective mutants (FIGS. 6B-C and 12H-I). Investigating the role of fumarate sensing in vivo (NSG mice), ME2 depletion delayed MOFM14 leukemia and improved animal survival (FIG. 6D). Wildtype ME2 and ME2CM, but not fumarate binding-defective mutants, restored leukemic aggressiveness (FIG. 6D), showing that fumarate-sensing activity of ME2, but not its catalytic activity, plays a major role in regulating AMF progression in vivo. To evaluate the clinical relevance, collected were 12 human leukemic BM (blast percentage >69%) and six normal human BM samples (FIG. 12J). Leukemic BM cells expressed higher ME2 protein than the normal cells, while the PRMT1 protein was decreased in human leukemia (FIGS. 6E-F). Further, ME2 was hypomethylated at R67 and showed higher activity in leukemic samples (FIGS. 6G-H). MRPL45, MT-COl, and MT-ND6 were also overabundant in AML samples (FIGS. 6I-K). Accordingly, leukemic BM showed a 1.84-fold increase of mtDNA (FIG. 6L). R67 methylation, but not ME2 protein, negatively correlated with MT-ND6, MT-COl, and mtDNA copies (FIGS. 6M-0 and 12K- M). Together, ME2 hypomethylation, which promotes fumarate signaling, is significantly linked to mitobiogenesis in human AML.

[0290] Discussion

[0291] Studies on the control of mitobiogenesis have largely focused on nuclear and cytoplasmic events. However, mtDNA encodes key ETC components critical for mitochondrial function. Mitobiogenesis must, therefore, depend on coordinated generation of both nDNA and mtDNA encoded elements in response to organismal cues. These results show that ME2 regulates mitochondrial aspects of mitobiogenesis, serving as a fumarate sensor to directly link mitochondrial protein synthesis and mtDNA replication with nutrient supply (FIG. 6P). Previously, mitochondrial and nuclear transcription and translation programs have been shown to co-regulate ETC genes (Couvillion et ah, 2016). Mitochondria sense the cytosolic translation efficiency and coordinately generate mtDNA-encoded products (Couvillion et ah, 2016; Richter-Dennerlein et ah, 2016). Here, it was found that multiple mtDNA-encoded proteins were decreased in NRF2-knockdown cells (FIG. 10G), supporting mitochondrial protein production as tightly coupled to the transcription and translation programs in the nucleus and cytoplasm.

[0292] Fumarate has previously been implicated in controlling nDNA participation in mitobiogenesis. DMF suppresses KEAP-mediated clearance of Nrf2, resulting in elevation of mtDNA (Hayashi et ah, 2017). In agreement, it was found that reducing NRF2 suppressed DMF-induced elevation of mtDNA (FIG. 2B). However, more marked effects of DMF on both mtDNA and mitochondrial protein translation that were dependent on ME2 were also found. Therefore, fumarate may act as a metabolic signal in both mitochondria and the nucleus to promote mitochondrial biomass production.

[0293] In addition to DUT and MRPF45, fumarate-ME2 signaling may have other downstream targets. For example, fumarate treatment increased all four dNTPs in mitochondria, implying that fumarate-ME2 axis may modulate enzymes other than DUT in nucleotide metabolism. Two observations further indicate that ME2 may control mitoribosome activity through unknown targets. First, depleting ME2 suppressed mitoribosome assembly (FIG. 4G), which could not be explained by the inhibitory effect of ME2 monomers; Second, wildtype ME2 interacted with more mitoribosomal proteins comparing to ME2R67F (FIG. 4A). Mitoribosomes are complex with the function of most of its components poorly understood. Here, MRPF45 was focused on because of its clear role in attaching mitoribosomes to the inner membrane (Kummer et ah, 2018). It remains possible that ME2 dimers interact with other regulatory proteins of mitoribosome to fulfill fumarate signaling. [0294] Metabolites have gained increasing recognition as signaling molecules (Frezza, 2017; Haas et al., 2016; Husted et al., 2017). Fumarate is involved in oncogenic signaling at multiple levels. Notably, fumarate is a proto-oncometabolite in FH-mutated renal tumors (Tomlinson et al., 2002). Aberrant accumulation of fumarate (millimolar levels or higher) mediates epigenetic reprogramming and hypoxia signaling (Laukka et al., 2016; Sciacovelli et al., 2016) and reduces apoptosis (Bardella et al., 2012). Despite these close links, the mechanism by which cells sense fumarate in its physiological range remains not well understood. It was demonstrated that ME2 serves as a physiological fumarate sensor and regulates mitobiogenesis. While DMF is applied in the treatment of multiple sclerosis (Hayashi et al., 2017), the results disclosed herein suggest that inhibition of ME2 may also be medically useful. ME2-mediated fumarate signaling may be targetable in AML and other mitochondria-related diseases.

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[0353] EXPERIMENTAL MODEL AND SUBJECT DETAILS

[0354] Cell lines and culture conditions

[0355] All cells were cultured at 37°C under 5% C02 humidified atmosphere. Human embryonic kidney cell line (HEK293), human AML cell lines (HL60, KG1, MOLM14, MONOMAC6, MV411, NB4, NOMOl, THP1), human glioma cell lines (A172, LN18, U87MG, U251MG, U118MG), breast cancer cell lines (BT549, HCC1937, HCC38, HS578T, MDAMB231, MDAMB468), liver cancer cell lines (HepG2, SKHEP1, SNU423, SNU387), pancreatic cancer cell lines (MIAPACA2, KP2, AsPCl, SW1990, BxPC3), melanoma cell lines (A375, SKMEL5, SKMEL28), sarcoma cell lines (U20S and HT1080), cervical cancer cell line (HeLa), prostate cancer cell line (DU145), colorectal cancer cell line (HCT116) were maintained in RPMI medium 1640 or Dulbecco's modified Eagle medium (DMEM) (Lonza) supplemented with 10% fetal bovine serum (Invitrogen) in the presence of penicillin, streptomycin, and 2 mM L-glutamine (Corning). Human cord blood CD34+ cells were grown in RPMI 1640 (with serum and glutamine) supplemented with recombinant human growth factors, including 40 ng/mL IL-6 (Peprotech), 50 ng/mL FLT3 ligand (Peprotech), 20 ng/mL stem cell factor (SCF) (Peprotech), and 50 ng/ml TPO (Peprotech).

[0356] Normal and leukemic mouse BM cells were maintained in RPMI 1640 (with serum and glutamine) supplemented with recombinant murine growth factors, including 10 ng/mL IL3 (R&D systems), 10 ng/mL SCF (R&D systems), 100 ng/mL IL6 (Peprotech).

[0357] Microbe strains

[0358] One-shot E. coli BL21 (DE3) (Invitrogen) was grown in LB medium at 37°C and then at 16°C after IPTG induction, for recombinant protein expression.

[0359] Mouse models

[0360] All animal studies were conducted in compliance with NIH guidelines for the care and use of laboratory animals and were approved by the IACUC of Harvard University.

Black 6 (B6) mice (C57BL/6J) and NSG mice (NOD.Prkdcscid.I12rgnull) were purchased from the Jackson Laboratory. Mouse lineage negative (lin-) bone marrow cells were transduced with control short hairpin and two different short hairpins against Me2. Control and Me2 -knockdown leukemic bone marrow cells were transplanted into lethally irradiated animals (9.5 Gy, 6-weeks old, male). In NSG model of human leukemia xenograft, one million human leukemia cells were transplanted into NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ (NSG) mice (6-weeks old, male) 24 hours after sublethal irradiation (2 Gy).

[0361] Human bone marrow samples

[0362] Normal and leukemic human bone marrow samples were collected from diagnostic bone marrow aspirations at Southwest Hospital (Chongqing, China). Bone marrow mononuclear cells were isolated by density gradient centrifugation and stored in liquid nitrogen until further use. Written informed consent was obtained from all patients. The procedures related to human subjects were approved by the Ethics Committee of the Institutes of Biomedical Sciences (Fudan University) and Institutional Ethics Review Board of Southwest Hospital.

[0363] METHOD DETAILS [0364] Chemicals and treatment of cells

[0365] To identify metabolites that regulate mitochondria mass, cells were cultured in RPMI 1640 supplemented with 10% FBS and 2 mM glutamine. The dose of additional nutrient/metabolites was added to physiologically relevant levels. Glucose (10 mM), fructose (5 mM), pyruvate (1 mM), lactate (1 mM), trimethyl citrate (50 mM), dimethyl alpha- ketoglutarate (50 pM), dimethyl succinate (50 pM), dimethyl fumarate (50 pM), dimethyl malate (50 pM), trimethyl oxaloacetate (50 pM), palmitate [bovine serum albumin (BSA)- conjugated, 100 pM], oleate (BSA-conjugated, 100 pM), acetate (5 mM), amino acid mixture (Sigma, #R7131, lx), glutamine (2 mM), non-essential amino acids (NEAA) (Sigma, #M7145, lx), and NH4C1 (1 mM) were added to culture media. Cells were cultured for 48 hours before further analysis. PRMT inhibitor AMI-1 (30 pM), AMI-5 (5 pM), PRMT1- specific inhibitor (PRMTli, also known as TC-E 5003, 2 pM), and DUT inhibitor (TAS114, 10 pM) were added into culture medium 24 hours before harvesting cells, respectively. Fumarate esters including MMF, DMF, MEF, and DEF (100 pM) were added to medium 24 hours or as indicated before harvest.

[0366] Plasmids and transfection

[0367] The cDNAs encoding full-length human ME2, AC02, MRPL45, DUT, NRF2, and SLC1A3 were cloned into Flag, HA, GFP, or His-tagged vectors (pcDNA3.1, pFV-EFla- IRES, pEGFP-Nl, pQCXIH, and pQE-1). Plasmids encoding GFP-PRMTs and NRF2 were generous gifts from Dr. Yanzhong Yang (City of Hope Cancer Center) and Dr. Rong Cai (Shanghai Jiao Tong University School of Medicine), respectively. Point mutations of ME2 were generated by site-directed mutagenesis using the GeneArt Site-Directed Mutagenesis System kit (Invitrogen). Truncated mutants of MRPF45 were cloned into pEGFP-Nl. All expression constructs were verified by DNA sequencing. Plasmid transfection was carried out by using FuGENE 6 (Promega).

[0368] Immunoprecipitation and western blotting

[0369] Cells were lysed in ice-cold NP-40 buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.3% NP-40] containing protease inhibitor cocktail (Sigma). Immunoprecipitation was carried out either by incubating Flag beads (Sigma) at 4°C with lysate for three hours or by incubating HA, GFP or ME2 antibody with cell lysate for one hour, followed by incubating with Protein-A beads (Millipore) for another two hours at 4°C. After the incubation, beads were washed three times with ice-cold NP-40 buffer. Standard western blotting protocols were adopted.

[0370] For proteomic profiling of ME2 interactors, MOFM14 cells expressing vector control, ME2-Flag or the R67F mutant were lysed in NP-40 buffer. ME2 protein was immunoprecipitated with Flag beads and eluted with 0.1 M glycine-HCl (pH 3.0). Mass spectrometric analysis were performed in three independent experiments for vector control (Samples #4553, #4629, and #4630), wildtype ME2 (Samples #4554, #4632, and #4633), and R67F mutant (#4555, #4631, and #4634).

[0371] ME2 enzyme activity assay

[0372] ME2 enzyme activity was determined as described previously (Tronconi et al., 2010). Flag-tagged ME2 proteins were overexpressed in cells, immunoprecipitated with Flag-beads, eluted by Flag peptides (Sigma), and subjected to activity assay with malate and NAD+ as substrates. Reaction mixture consists of 50 mM HEPES (pH 7.4), 10 mM MnC12, 4 mM NAD+, 10 mM malate in a total volume of 200 pF. 200 pM fumarate was added to the mixture to determine allosteric activation. Reactions were initiated by adding the enzyme and analyzed at 25°C. Activities were measured by the conversion of NAD+ to NADH, which was monitored by measuring the increase of fluorescence (Ex. 350nm, Em. 470nm) for NADH generation. Endogenous ME2 proteins were immunoprecipitated by using ME2 antibody, on-beads catalytic activity was assayed.

[0373] PUT enzyme activity assay

[0374] The substrate dUTP (1 mM, final concentration) was added to DUT activity assay buffer [50 mM Tris (pH 7.4), 150 mM NaCl]. Reactions were initiated by adding DUT enzyme into the reaction mixture (100 pL final volume). The reactions were stopped at 0.5 min, 1.0 min, 2.0 min, and 4.0 min by adding 200 pL chloroform and 200 pL methanol, followed by vortexing and centrifugation (5000g, 4°C for 15 minutes). The aqueous phase was dried with nitrogen flow evaporator at 37 °C and subjected to mass spectrometry to quantify the generation of dUMP. Recombinant ME2 was mixed with DUT enzyme at the molar ratio of 1 : 1 before adding to the reaction mixture to determine the effect of ME2 protein on DUT activity. The rate of dUMP accumulation was calculated and normalized to DUT enzyme to determine its catalytic activity.

[0375] Quantification of metabolites

[0376] The abundance of intracellular fumarate (Sigma, #MAK060), NADH (Sigma, #MAK037), and ATP (Abeam, #abl 13849) were determined by using quantification kits, according to the manufacturer’s instruction. Briefly, 1x106 cells were collected and homogenized on ice in assay buffer provided and centrifuged at 4 °C for 10 min at 13,000g. Supernatants were deproteinized using lOkD spin column (Abeam), analyzed and compared to standard curves. The signals obtained were normalized to cell number.

[0377] To quantify intracellular nucleotides, 5x106 cells or isolated mitochondria were washed in 1 mL saline (0.9% NaCl in water) and lysed in ice-cold methanol. Water and chloroform were added to the lysate (methanol: water:chloroform=2: 1:2 final volume), followed by vortexing and centrifugation (5000g, 4°C for 15 minutes). The aqueous phase was dried with nitrogen flow evaporator at 37 °C. For [U-13C]-fumarate tracing, 1x106 MOLM14 cells were cultured in RPMI 1640 supplemented with 10% FBS and 2 mM glutamine. Increasing concentrations of [U-13C]-fumarate was added to the culture medium as indicated, metabolites were extracted after incubation for 24 hours. Extracted metabolites were resuspended in 50% acetonitrile and subjected to mass spectrometry analysis. For the quantification of dATP, dTTP, dGTP, dCTP, dUTP, and dUMP, an Ultimate 3000 UHPLC equipped with a refrigerated autosampler (at 8°C) and a column heater (at 30°C) with a Welch Ultimate AQ-C18 column (2.1x250mm i.d., 5 pm) was used for separations. Solvent A was 10 mM ammonium acetate and 0.075% FA in water and solvent B was acetonitrile. The gradient was as follows: 100% A for 1 min at 0.2 mL/min, 95% A at 7 min with 0.2 mL/min, 5% A at 8 min with 0.2 mL/min, 5% A at 12 min with 0.2 mL/min, 100% A at 12.5 min and 100% A at 20 min with 0.2 mL/min. For MS analysis, the UHPLC was coupled to a 6500 Qtrap mass spectrometer (Sciex, USA). The ion transitions at m/z 307.0— U95.0,

467.0— >-369.0, 490.1 392.1, 506.1 408.1, 481.0 383.0, 466.1 367.9 were selected for monitoring dUMP, dUTP, dATP, dGTP, dTTP, and dCTP, respectively. The operating conditions were as follows: spray voltage -4500 V; Orifice temperature 500 °C; GS1 and GS250; Curtain Gas 40.

[0378] Glucose consumption (Sigma, #GAGO-20) and glutamine consumption (Abeam, #abl97011) was determined by using colorimetric assay kits following the manufacturer’s instructions. Briefly, cells were seeded into six- well plate at 3x105 per well. After three hours of cell culture, the supernatant of the medium was collected, deproteinized using lOkD spin column, and subjected to glucose/glutamine detection. The glucose/glutamine uptake was determined by subtracting the final glucose/glutamine concentration from initial glucose/glutamine concentration in the culture medium.

[0379] In vitro pulldown assay of ME2 and MRPL45

[0380] Recombinant His-tagged MRPL45 and ME2 protein (1 pg each in 0.5 mL NP-40 buffer) were incubated with overhead rotation at 4°C overnight. After the addition of 200 pM fumarate, succinate, or malate, the mixture was further incubated for three hours at 4°C. His- MRPL45 was pulled down with nickel-NTA agarose beads (Invitrogen). Beads were washed three times with 0.3% NP-40 buffer and subjected to western blotting analysis.

[0381] Generation of stable cell pools

[0382] shRNAs targeting ADSL, ASL, FAH, FH, SDHA, ME2, MRPL45, NRF2, and PRMT1 were used to generate stable knockdown cell pools. Lentivirus was produced by using a two-plasmid packaging system (D8.9 and vsvg). Cells were mixed with 8 pg/mL polybrene and spinfected with the lentivirus and selected in 4 pg/mL puromycin for one week.

[0383] To generate ME2-knockdown and re-expression stable cell pools, Flag-tagged human wild-type ME2 or its mutants (R67K, R67F, CM) was cloned into the lentiviral pLV-EFla- IRES-Hygro vector and co-transfected with vectors expressing the D8.9 and vsvg genes in HEK293T cells to produce lentiviruses. After transduction, cells were selected in 200 pg/ml hygromycin B for 1 week.

[0384] CRISPR editing

[0385] Malic enzymes (ME1, ME2, and ME3) were silenced in leukemia cell lines through CRISPR editing using lentiCRISPR v2 with sgRNA sequences targeting ME1, ME2, and ME3 respectively. Oligos were phosphorylated, annealed, and ligated into the lentiCRISPR v2 backbone, which was then transformed into bacteria, isolated, and verified by sequencing. The lentiCRISPR vector expressing sgRNA against GFP was used as control. Lentivirus carrying sgRNA was produced using the two-plasmid packaging system. Leukemia cell lines were transduced and selected in 4 pg/mL puromycin. The depletion effect was verified by western blotting. Targeting sequences for sgRNAs were shown in Table 1.

[0386] Quantitative real-time PCR

[0387] Total RNA was isolated from cultured cells using the RNeasy Kit (Qiagen) and reverse-transcribed with random primers following the manufacturer’ s instructions (RETROscript Kit, Invitrogen). The cDNA was preceded to real-time PCR with gene-specific primers in the presence of SYBR Green PCR Master Mix (Applied Biosystems). PCR reactions were performed in triplicate and the relative amount of cDNA was calculated by the comparative CT method using the b-actin as a control. Primer sequences were listed in Table 1.

[0388] Oxygen consumption rate (OCR)

[0389] OCR was determined using the XFe96 Extracellular Flux Analyzer (Agilent). Briefly, leukemia cells or mouse BM cells were attached to 96-well plates using Cell-Tak (Coming) at the density of 4x104 or 8x104 cells/well, respectively. Cells were incubated with Seahorse XF RPMI medium buffer (without phenol red, with 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate). Cell Mito Stress Test Kit (Agilent) was used to measure cellular mitochondrial function, 180 pL of Seahorse buffer plus 20 pL each of 2 pM oligomycin, 2 pM FCCP, and 0.5 pM rotenone/antimycin A (AA) was automatically injected to determine the oxygen consumption rate (OCR), according to the manufacturer's instructions.

[0390] In vitro methylation assay

[0391] For in vitro methylation assay, HA-tagged PRMT1 protein was overexpressed in HEK293T cells and immunopurified with HA-beads. Recombinant His-tagged ME2 (30 pg) was mixed with PRMT1-HA (on beads) at a molar ratio of approximately 1: 1 in methylation reaction buffer (50 mM Tris-HCl, pH 8.0, 20 mM KC1, 5 mM DTT, 4 mM EDTA). The mixture was incubated with or without 200 pM S-adenosyl-L-methionine (Sigma) at 37°C for 1 hour in a final volume of 500 pL. After centrifugation (500g, 4°C for 3 minutes), the supernatant was transferred to an Amicon filter (Millipore, Amicon Ultra- 15 Centrifugal Filter Device) for buffer exchange and further analysis.

[0392] Protein melting curve analysis

[0393] The thermodynamic stability of ME2 was determined using SYPRO-Orange (Invitrogen). Briefly, 45 pL of 1 mM purified ME2 (in 25 mM HEPES, 150 mM NaCl at pH 8.0) was mixed with 15 pL SYPRO-Orange (20X,). 45 pL buffer (25 mM HEPES, 150 mM NaCl, pH 8.0) mixed with 15 pL of 20X SYPRO-Orange was used as control. The mixture was aliquoted in triplicate (20 pL per well) into a 96-well plate. Data of melting curves were collected by using ABI 7500 (Applied Biosystems). Melting curve fluorescent signal was acquired between 20°C and 70°C using a ramping rate of 0.03 °C/s. Melting temperatures (Tm) were determined by fitting the data with Boltzmann model.

[0394] Cell proliferation and colony formation assay

[0395] To monitor cell proliferation, cells (lxl05/mL) were seeded into six-well plates. Viable cells were visualized by methylene blue staining and counted every day for 4 days. In colony formation assay, AML cells were plated in methylcellulose medium (MethoCult H4434; Stem Cell Technologies) according to the manufacturer’s instructions. Briefly, 4000 cells in 0.5 mL IMDM with 10% FBS were added to 3.5 mL of methylcellulose medium. After thorough vortex mixing, the cell suspension was plated into the six-well plate with 1 mL in each well. Culture plates were incubated at 37°C in a humid atmosphere with 5% C02. Colonies (>50 pm diameter) were counted after seven days of incubation.

[0396] Mitochondria staining and mtDNA quantification

[0397] To quantify mitochondria mass, 1x106 cells were stained with 50 nM MitoTracker Green (Invitrogen) for 30 min at 37°C and analyzed with a fluorescence microplate reader (BioTek, Ex. 491nm, Em. 516nm). To determine mtDNA copy number, total DNA was isolated from cell lines or tissues using DNeasy kits (Qiagen). Samples were adjusted to 1 ng/pL final concentration. Nuclear and mitochondrial DNA content was analyzed by qPCR as described previously (Wang et ah, 2014). mtDNA content was determined by normalizing mitochondrial DNA abundance [tRNA-Leu(UUR) in human and 16S rRNA in mouse] to nuclear DNA (beta-2-microglobulin, B2M) abundance. Primers for qPCR were listed in Table 1. [0398] Mitochondria isolation and fractionation

[0399] Mitochondrial isolation was performed as previously described (Rackham et ah, 2016). Briefly, cells were resuspended in mitochondria isolation buffer (MIB) [310 mM sucrose, 10 mM Tris-HCl (pH7.5) and 0.05 % BSA (w/v), with protease inhibitor cocktail (Sigma)], homogenized with Dounce homogenizer, centrifugated at lOOOg for lOmin at 4°C. The supernatant was further centrifugated at 4500g for 15 min at 4°C, and the pellet was washed once with MIB. Crude mitochondrial pellets were resuspended in MIB with protease inhibitor cocktail.

[0400] Mitochondria subfraction was performed as reported previously (Pallotti and Lenaz, 2007). In brief, isolated mitochondria were resuspended in 1 mL of Mitolysis buffer (3 mM HEPES, pH 7.4, 210 mM mannitol, 70 mM sucrose, 0.2 mM EGTA, and protease inhibitor cocktail) with digitonin (0.2 mg/mL). To achieve mitoplast (MP, inner membrane and matrix), mitochondria were lysed with overhead rotation at 4°C for 15 min. 1 mL of Mitolysis buffer was added to stop digitonin extraction. The lysate was centrifugated at 1 l,000g for 10 min at 4°C to achieve mitoplast pellet. Mitoplast was resuspended in 300 pL Mitolysis buffer and disrupted by a sonicator in ice-cold water bath. Disrupted mitoplast was further centrifugated at 10,000g for 30 min at 4°C, to isolate inner membrane fraction in the pellet and matrix fraction in the supernatant.

[0401] Sucrose gradient fractionation

[0402] Isolated mitochondria (2 mg) were lysed in 2 mL MitoL buffer [10 mM Tris-HCl (pH 7.4), 260 mM sucrose, 100 mM KC1, 20 mM MgC12 and 2% digitonin, RNase inhibitor (40 U/ml) and EDTA-free protease inhibitor cocktail (Sigma)] for 20 min at 4°C. The lysate was centrifuged at 9,200g for 45 min at 4°C. The supernatant was loaded on a continuous 10-30% sucrose gradient (in 10 mM Tris-HCl, pH 7.5, 100 mM KC1, 20 mM MgC12 supplemented with RNase and protease inhibitors) and centrifuged at 20,000g for 6 hours at 4°C in an Optima Beckman Coulter ultracentrifuge. Fractions were collected and precipitated with 20% trichloroacetic acid (final concentration). MRPL12 and MPRS35 were used as markers of the mitochondrial ribosomal subunits. 12S rRNA and 16S rRNA were extracted using the RNeasy kit (Qiagen). The RNA was reverse transcribed and detected by qPCR to determine mitoribosome assembly. Primers for qPCR were listed in Table 1. [0001] QUANTIFICATION AND STATISTICAL ANALYSIS

[0002] Student’s t-tests or one-way ANOVA with Dunnett’s multiple comparisons test were performed to determine statistical significance using GraphPad Prism. All data shown represent the results obtained from three (or as indicated) independent experiments. The p values <0.05 were considered statistically significant.

[0003] TABLE 1: Oligonucleotides used in this study.

TABLE 2: Proteomic profiling of ME2 interactors in mitochondria (1 = interaction; 0 = no interaction).

Claims

CLAIMS The claims are as follows:

1. A method of modulating cell proliferation and/or viability, comprising contacting the cell with an agent that modulates malic enzyme 2 (ME2) binding to mitochondrial ribosomal protein L45 (MRPL45), wherein the agent is not fumarate or a fumarate analog.

2. The method of claim 1, wherein the agent enhances ME2 binding to MRPL45 and reduces mitobiogenesis in the cell.

3. The method of claim 1, wherein the agent suppresses or prevents ME2 binding to MRPL45 and enhances mitobiogenesis in the cell.

4. The method of claim 1 or 3, wherein the agent blocks a MRPL45 binding site for ME2 or blocks a ME2 binding site for MRPL45.

5. The method of claims 1 or 3, wherein the agent enhances ME2 dimerization.

6. The method of claims 1 or 2, wherein the agent suppresses ME2 dimerization.

7. The method of claim 6, wherein the agent that suppresses ME2 dimerization reduces fumarate binding to ME2.

8. The method of claim 7, wherein the agent causes methylation of the ME2 binding site for fumarate.

9. A method of modulating cell proliferation and/or viability, comprising contacting the cell with an agent that modulates malic enzyme 2 (ME2) dimerization in the cell, wherein the agent is not fumarate or a fumarate analog.

10. The method of claim 9, wherein the agent enhances ME2 dimerization.

11. The method of claim 9, wherein the agent suppresses ME2 dimerization.

12. The method of claim 11, wherein the agent that suppresses ME2 dimerization reduces fumarate binding to ME2.

13. The method of claim 12, wherein the agent causes methylation of the ME2 binding site for fumarate.

14. The method of claims 9-13, wherein the method modulates the activation of deoxyuridine 5 ’-triphosphate nucleotidohydrolase (DUT) in the cell.

15. The method of claims 9-14, wherein the method modulates the generation of thymidine and/or mitochondrial DNA (mtDNA) in the cell.

16. The method of claims 9-15, wherein the method modulates mitobiogenesis in the cell.

17. The method of claims 9-16, wherein the cell is contacted with the agent in vivo in a subject in need thereof.

18. The method of claim 17, wherein the subject has a condition associated with a mitochondrial defect.

19. A method of modulating cell proliferation and/or viability, comprising contacting the cell with an agent that modulates malic enzyme 2 (ME2) activation of deoxyuridine 5 ’-triphosphate nucleotidohydrolase (DUT), wherein the agent is not fumarate or a fumarate analog.

20. The method of claim 19, wherein the agent enhances DUT activation.

21. The method of claim 19, wherein the agent suppresses DUT activation.

22. The method of claims 19-21, wherein the agent modulates ME2 dimerization.

23. The method of claims 19-22, wherein the agent is a ME2 dimer agonist or antagonist.

24. The method of claims 19-23, wherein the agent is a ME2 dimer mimic.

25. The method of claim 19-24, wherein the method modulates the generation of thymidine and/or mitochondrial DNA (mtDNA).

26. The method of claim 19-25, wherein the method modulates mitobiogenesis in the cell.

27. The method of claims 19-26, wherein the cell is contacted with the agent in vivo in a subject in need thereof.

28. The method of claim 27, wherein the subject has a condition associated with a mitochondrial defect.

29. A method of screening for a candidate agent that modulates cell proliferation and/or viability, comprising a. providing a composition comprising malic enzyme 2 (ME2) and mitochondrial ribosomal protein L45 (MRPL45) or a fragment thereof capable of binding ME2 under conditions wherein ME2 and MRPL45 can bind, b. contacting the composition with a test agent, and c. assessing binding of ME2 with MRPL45, wherein a test agent that modulates binding of ME2 to MRPL45 as compared to a control composition comprising ME2 and MRPL45 or a fragment thereof capable of binding ME2 without the test agent is identified as a candidate agent that modulates cell proliferation and/or viability.

30. The method of claim 29, wherein the MRPL45 fragment capable of binding ME2 comprises or consists of the ME2 binding site.

31. A method of screening for a candidate agent that modulates cell proliferation and/or viability, comprising a. providing a composition comprising malic enzyme 2 (ME2) under conditions wherein ME2 is capable of forming dimers, b. contacting the composition with a test agent, and c. assessing dimerization of ME2, wherein a test agent that modulates dimerization of ME2 as compared to a control composition comprising ME2 without the test agent is identified as a candidate agent that modulates cell proliferation and/or viability.

32. A method of treating a condition associated with a mitochondrial defect comprising administering to a patient in need thereof a composition comprising an effective amount of an agent that modulates malic enzyme 2 (ME2) dimerization, wherein the agent is not fumarate or a fumarate analog.

33. The method of claim 32, wherein the agent modulates the activation of deoxyuridine 5 ’-triphosphate nucleotidohydrolase (DUT).

34. A composition comprising an effective amount of an agent for modulating malic enzyme 2 (ME2) dimerization, wherein the agent is not fumarate or a fumarate analog.

35. The composition of claim 34, wherein the agent modulates the activation of deoxyuridine 5 ’-triphosphate nucleotidohydrolase (DUT).