Use of GCN2 Inhibitors in Treating Mitochondrial Myopathies and Diseases Associated with Mitochondrial Dysfunction

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Application Serial No. 63/009,510, filed on April 14, 2020. The entire contents of the foregoing are incorporated herein by reference.

BACKGROUND

Mitochondrial dysfunction is associated with activation of the integrated stress response (ISR), and studies have consistently identified a gene expression program associated with the integrated stress response (ISR) as a signature of mitochondrial dysfunction (Martinus, R. D. et al. Selective induction of mitochondrial chaperones in response to loss of the mitochondrial genome. Eur. J. Biochem. 240, 98-103 (1996); Zhao, Q. et al. A mitochondrial specific stress response in mammalian cells. EMBO J. 21, 4411-4419 (2002); Kiihl, I. et al. Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals. Elife 6, e30952 (2017); Celardo, I, Lehmann, S., Costa, A. C, Loh, S. H. Y & Miguel Martins, L. dATF4 regulation of mitochondrial folate-mediated one-carbon metabolism is neuroprotective. Cell Death Differ. 24, 638 (2017); Quiros, P. M. et al. Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals. J. Cell Biol. 216, 2027 LP - 2045 (2017); Fujita, Y et al. CHOP (C/EBP homologous protein) and ASNS (asparagine synthetase) induction in cybrid cells harboring MELAS and NARP mitochondrial DNA mutations. Mitochondrion 7, 80-88 (2007); Silva, J. M., Wong, A., Carelli, V & Cortopassi, G A. Inhibition of mitochondrial function induces an integrated stress response in oligodendroglia. Neurobiol. Dis. 34, 357-365 (2009); Tyynismaa, H. et al. Mitochondrial myopathy induces a starvation-like response. Hum. Mol. Genet. 19, 3948-3958 (2010); Dogan, S. A. et al. Tissue-Specific Loss of D ARS2 Activates Stress Responses Independently of Respiratory Chain Deficiency in the Heart. Cell Metab. 19, 458-469 (2014); Bao, X. R. et al. Mitochondrial dysfunction remodels one-carbon metabolism in human cells. Elife 5, el 0575 (2016); Magarin, M. et al. Embryonic cardiomyocytes can orchestrate various cell protective mechanisms to survive mitochondrial stress. J. Mol. Cell. Cardiol. 97, 1-14 (2016); Lehtonen, J. M. et al. FGF21 is a biomarker for mitochondrial translation and mtDNA maintenance disorders. Neurology 87, 2290-2299 (2016); Khan, N. A. et al. mTORCl Regulates Mitochondrial Integrated Stress Response and Mitochondrial Myopathy Progression. Cell Metab. 26, 419-428. e5 (2017)). The ISR is triggered by various insults, including nutrient deficiency, unfolded protein stress, and pathogen infection.

Mitochondrial dysfunction is associated with a spectrum of human pathology ranging from individually rare genetic disorders to common conditions, such as neurodegeneration (Schapira, A. H. V Mitochondria in the aetiology and pathogenesis of Parkinson’s disease. Fancet Neurol. 7, 97-109 (2008); Hu, Q. & Wang, G Mitochondrial dysfunction in Parkinson’s disease. Transl. Neurodegener. 5, 14 (2016); Fin, M. T. & Beal, M. F Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787 (2006); Area-Gomez, E., Guardia-Faguarta, C, Schon, E. A. & Przedborski, S. Mitochondria, OxPhos, and neurodegeneration: cells are not just running out of gas. J. Clin. Invest. 129, 34-45 (2019)), diabetes (Kelley, D. E., He, J., Menshikova, E. V & Ritov, V B. Dysfunction of Mitochondria in Human Skeletal Muscle in Type 2 Diabetes. Diabetes 51, 2944 FP - 2950 (2002); Mootha, V K. et al. Erralpha and Gabpa/b specify PGC-1 alpha-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle. Proc. Natl. Acad. Sci. U. S. A. 101, 6570-5 (2004); Ritov, V B. et al. Deficiency of Subsarcolemmal Mitochondria in Obesity and Type 2 Diabetes. Diabetes 54, 8 FP - 14 (2005); Ritov, V B. et al. Deficiency of electron transport chain in human skeletal muscle mitochondria in type 2 diabetes mellitus and obesity. Am. J. Physiol. Metab. 298, E49-E58 (2009)), select forms of cancer (Wallace,

D. C. Mitochondria and cancer. Nat. Rev. Cancer 12, 685 (2012); Reznik, E., Wang, Q., Fa, K., Schultz, N. & Sander, C. Mitochondrial respiratory gene expression is suppressed in many cancers. Elife 6, e21592 (2017); Gaude, E. & Frezza, C. Defects in mitochondrial metabolism and cancer. Cancer Metab. 2, 10 (2014)) and the aging process itself (Sun, N., Youle, R. J. & Finkel, T. The Mitochondrial Basis of Aging. Mol. Cell 61, 654-666 (2016); Bratic, A. & Farsson, N.-G The role of mitochondria in aging. J. Clin. Invest. 123, 951-957 (2013)). Mutations in nearly 300 nuclear or mitochondrial (mtDNA) genes have been implicated in mitochondrial disorders that affect at least 1 in 4,300 live births and lack effective treatments (Schapira, A. H. V. Mitochondrial diseases. Lancet 379, 1825-34 (2012); Vafai, S. B. & Mootha, V K. Mitochondrial disorders as windows into an ancient organelle. Nature 491, 374-383 (2012); Gorman, G S. et al. Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease. Ann. Neurol. 77, 753-759 (2015); Gorman, G S. et al. Mitochondrial diseases. Nat. Rev. Dis. Prim. 2, 16080 (2016); Frazier, A. E., Thorburn, D. R. & Compton, A. G Mitochondrial energy generation disorders: genes, mechanisms, and clues to pathology. J. Biol. Chem. 294, 5386-5395 (2019)). Management of these conditions is complicated by their striking heterogeneity. Patients can develop acutely or progressively and can impact multiple organ systems or just a single cell type. Disease can manifest in post mitotic tissues, such as skeletal muscle or the nervous system, but this pattern does not simply correlate with ATP demand or tissue expression of the causal gene. Thus, better understanding and treatments of mitochondrial dysfunction, ISR activation, and diseases associates with this are needed.

SUMMARY

This disclosure is based, in part, upon the surprising discovery that administering a General Control Nonderepressible 2 (GCN2) inhibitor decreases the ISR response and thereby reduces the concomitant mitochondrial dysfunction.

The present disclosure provides methods for treating or preventing (reducing risk of) mitochondrial myopathy, and/or treating or preventing a sign or a symptom of mitochondrial myopathy in a subject in need thereof by administering to the subject a therapeutically effective amount of an GCN2 inhibitor, thereby resulting in the prevention or treatment of one or more signs or symptoms of mitochondrial myopathy. Provided are methods for treating or preventing a disease or condition associated with mitochondrial dysfunction, and/or treating or preventing a sign or a symptom of a disease or condition associated with mitochondrial dysfunction in a subject in need thereof by administering to the subject a therapeutically effective amount of an GCN2 inhibitor, thereby resulting in the prevention or treatment of one or more signs or symptoms of a disease or condition associated with mitochondrial dysfunction. Described herein, inter alia, are methods for treating or reducing risk of a mitochondrial myopathy or disease associated with mitochondrial dysfunction in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising a GCN2 inhibitor.

Also described herein, inter alia, are methods method of reducing the integrated stress response (ISR) in a mammalian cell, the method comprising administering a composition comprising an effective amount of a GCN2 inhibitor to the mammalian cell.

In some embodiments of any of the methods presented herein, the GCN2 inhibitor is one or more small molecule inhibitors (e.g., GCN2iB); an siRNA, shRNA, or other inhibitory nucleic acid directed against GCN2; a GCN2 blocking peptide; an antibody to GCN2; glutamine or glutamine mimetic; asparagine or asparagine mimetic; aspartate or aspartate mimetic; or an amino acid other than glutamine that inhibits GCN2; or a combination thereof. In some embodiments, the GCN2 inhibitor is a small molecule. In some embodiments, GCN2 inhibitor is GCN2iB.

In some embodiments, the mammalian cell is a human cell. In some embodiments, the cell is cancerous. In some embodiments, the cancer is a leukemia, a lymphoma, a brain tumor, a brain tumor metastases, breast cancer, breast cancer metastases, kidney cancer, kidney cancer metastases, liver cancer, liver cancer metastases, lung cancer, lung cancer metastases, lymphoma, lymphoma metastases, ovarian cancer, ovarian cancer metastases, pancreatic cancer, pancreatic cancer metastases, prostate cancer, prostate cancer metastases, colorectal cancer, colorectal cancer metastases, or any combination thereof. In some embodiments, the cell is noncancerous. In some embodiments, the cell is in a living subject.

In some embodiments of any of the methods presented herein, the subject has or is at risk of developing a mitochondrial myopathy or a disease associated with mitochondrial dysfunction. In some embodiments of any of the methods presented herein, the mitochondrial myopathy or disease associated with mitochondrial dysfunction is any one or more of Kearns-Sayre syndrome (KSS), Leber’s hereditary optic neuropathy (LHON), Leigh syndrome (LS), MEGDEL Syndrome; mitochondrial DNA depletion syndrome (MDS), mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonus epilepsy with ragged-red fibers (MERRF), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), neuropathy, ataxia and retinitis pigmentosa (NARP), OPA1 mutations, Pearson syndrome, progressive external ophthalmoplegia (PEO), Mitochondrial complex I deficiency, Mitochondrial complex P deficiency, Mitochondrial complex III deficiency, Mitochondrial complex IV deficiency, Mitochondrial complex V (ATP synthase) deficiency, Primary coenzyme Q10 deficiency (COQIOD), Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome, Mitochondrial disease resulting from mutations in PolG (e.g. Chronic Progressive External Ophthalmoplegia syndrome (CPEO), Alpers-Huttenlocher syndrome (AHS), Childhood Myocerebrohepatopathy Spectrum (MCHS), Myoclonic Epilepsy Myopathy Sensory Ataxia (MEMSA), Ataxia Neuropathy Spectrum (ANS) (including e.g., mitochondrial recessive ataxia syndrome (MIRAS) and sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO)), Autosomal Recessive Progressive External Ophthalmoplegia (arPEO), Autosomal Dominant Progressive External Ophthalmoplegia (adPEO)), adPEO due to mutations in ANT or due to C10orf2 (twinkle) mutations, Mitochondrial DNA depletion syndrome, Mitochondrial DNA depletion syndrome 1/MyoNeurogenic Gastrointestinal Encephalopathy (MNGIE), Mohr- Tranebjaerg syndrome, 3 -methyl glutaconic aciduria, Combined oxidative phosphorylation deficiency (COXPD), Myopathy, Lactic Acidosis, and Sideroblastic Anemia (MLASA), Hyperuricemia, Pulmonary hypertension, Renal failure, and Alkalosis (HUPRA) syndrome, Leigh Syndrome, Leigh syndrome-French Canadian type,

Friedreich ataxia, Gracile syndrome, Bjo nstad syndrome, Multiple Mitochondrial Dysfunctions Syndrome (MMDS), Early-onset Ataxia with Ocular motor apraxia and Hypoaibuminemia (EAOH), Charcot-Marie-Tooth Disease-2A2, Leber Hereditary Optic Neuropathy (LHON), Sudden Infant Death Syndrome, Myoclonic Epilepsy with Ragged Red Fibers (MERRF), MERRF/MELAS overlap syndrome, Neuropathy, Ataxia, Retinitis Pigmentosa (NARP), Mitochondrial myopathy, Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS), Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL), Mitochondrial disease resulting from SDH mutations (e.g. Pheochromocytoma and Paragangliomas), Optic atrophy type 1, Ethylmalonic encephalopathy, Carnitine-acylca nitine translocase deficiency, Primary systemic carnitine deficiency, Creatine deficiency syndromes (e.g. Cerebral creatine deficiency syndrome- 1 , Cerebral creatine deficiency syndrome-2 or Cerebral creatine deficiency syndrome-3), Carnitine palmitoyltransferase 1 (CPT I) deficiency, Carnitine palmitoyltransferase 2 (CPT II) deficiency, Short-chain acyl-CoA dehydrogenase deficiency, Very long chain acyl -Co A dehydrogenase deficiency, Long-chain 3-hydroxyl- CoA dehydrogenase (LCHAD) deficiency, Pyruvate carboxylase deficiency, Multiple acyl-CoA dehydrogenase deficiency (e.g. Glutaric acidemia PA, Glutaric acidemia PB or Glutaric acidemia EC), Pyruvate dehydrogenase deficiency (e.g. Pyruvate dehydrogenase El -alpha deficiency, Pyruvate dehydrogenase phosphatase deficiency, Pyruvate dehydrogenase E3 -binding protein deficiency, Pymvate dehydrogenase E2 deficiency or Pyruvate dehydrogenase El -beta deficiency), 3 -hydroxyacyl-CoA dehydrogenase (SCHAD) deficiency/(HADH) deficiency, Perrault syndrome, Swedish type myopathy with exercise intolerance, combined mitochondrial complex deficiency, familial myalgia syndrome, myopathy with abnormal mitochondrial translation, myopathy with extrapyramidal signs, myopathy with focal depletion of mitochondria, mitochondrial DNA breakage syndrome, limb-girdle muscular dystrophy type IH (LGMDIH), isolated mitochondrial myopathy (IMMD), a cardiomyopathy, individually rare genetic disorders; neurodegeneration (e.g., Parkinson’s disease), diabetes, or a cancer. In some embodiments, the cancer is a leukemia, a lymphoma, a brain tumor, a brain tumor metastases, breast cancer, breast cancer metastases, kidney cancer, kidney cancer metastases, liver cancer, liver cancer metastases, lung cancer, lung cancer metastases, lymphoma, lymphoma metastases, ovarian cancer, ovarian cancer metastases, pancreatic cancer, pancreatic cancer metastases, prostate cancer, prostate cancer metastases, colorectal cancer, colorectal cancer metastases, or any combination thereof.

In some embodiments of any of the methods presented herein, the cell or subject has a biomarker associated with a mitochondrial myopathy or a disease associated with mitochondrial dysfunction above a reference number or outside a reference range.

In some embodiments of any of the methods presented herein, the reference number or range is an ATF protein (e.g., ATF3 or ATF4) level in a healthy cell not having a condition associated with a mitochondrial myopathy or a disease associated with mitochondrial dysfunction or a control cell. In some embodiments of any of the methods presented herein, the reference number or range is an ATF protein (e.g., ATF3 or ATF4) level in a healthy sample from a subject not having a condition associated with a mitochondrial myopathy or a disease associated with mitochondrial dysfunction.

In some embodiments of any of the methods presented herein, administering the composition comprising the GCN2 inhibitor results the return of ATF protein (e.g., ATF3 or ATF4) levels similar to that of the reference number or range the ATF protein level in a healthy cell not having a condition associated with a mitochondrial myopathy or a disease associated with mitochondrial dysfunction or a control cell or the ATF protein level in a healthy sample from a subject not having a condition associated with a mitochondrial myopathy or a disease associated with mitochondrial dysfunction.

In some embodiments of any of the methods presented herein, the reference number or range is a Ddit3 transcript level in a healthy cell not having a condition associated with a mitochondrial myopathy or a disease associated with mitochondrial dysfunction or a control cell. In some embodiments of any of the methods presented herein, the reference number or range is a Ddit3 transcript level in a healthy sample from a subject not having a condition associated with a mitochondrial myopathy or a disease associated with mitochondrial dysfunction.

In some embodiments of any of the methods presented herein, administering the composition comprising the GCN2 inhibitor results the return of Ddit3 transcript levels similar to that of the reference number or range the Ddit3 transcript level in a healthy cell not having a condition associated with a mitochondrial myopathy or a disease associated with mitochondrial dysfunction or a control cell or the Ddit3 transcript level in a healthy sample from a subject not having a condition associated with a mitochondrial myopathy or a disease associated with mitochondrial dysfunction.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.

All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. All titles and subtitles herein are solely to ease reader comprehension and are meant to be considered together; they do not limit or separate any features or concepts from others disclosed herein.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1N show Complex I inhibition in myoblasts triggers the ISR through the kinase GCN2 due to an aspartate and asparagine deficiency. (FIG 1 A) A bar graph showing secreted lactate concentration (“[lactate]”) following 2 hr piericidin treatment in control (Ctrl), LbNOX {Lb) or NDI1 cells. Data was normalized to DMSO (-) separately for each cell line. Data shown are Mean±SD, N=6 from 3 experiments in control,

LbNOX, or NDI1 cells. Welch’s t-test (two-tailed) was used to compare control with ZANOX and NDI1 cells, followed by Holm’s correction for multiple testing. (FIG IB) A bar graph showing adenylate energy charge following 1 hr piericidin treatment in control, ZANOX or NDI1 cells. Data shown are Mean±SD, N=5-6 from 2 experiments. The Games-Howell test was used to make all pairwise comparisons. Notations with no connecting lines relate to statistical comparison with the equivalent treatment in control cells. (FIG 1C) Scatter plots showing fold-change (x-axis) and statistical significance (y-axis) of metabolite differential abundance following 1 hr piericidin treatment in extracts of control or ZANOX cells. N=3. P-Ser, phosphoserine; Asp, aspartate; Asn, asparagine; Cit, citrate; Sue, succinate; aKQ a-ketoglutarate; IMP, inosine monophosphate; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; R5P, ribose 5-phosphate; G3P/DHAP, glyceraldehyde 3 -phosphate and/or dihydroxyacetone phosphate; P-Cr, phosphocreatine; Pro, proline. (FIG ID) A bar graph showing intracellular aspartate concentration (“[aspartate]”) following 1 hr piericidin treatment in control or Lb NOX cells. Data was normalized to DMSO in control cells. Data shown are Mean±SD, N=6 from 2 experiments (includes samples shown in FIG 4C). The Games- Howell test was used for all pairwise comparisons. (FIG. IE) A bar graph showing peak intensity of intracellular asparagine following 1 hr piericidin treatment in control cells, with or without aspartate, and in LbNOX cells. Data was normalized to DMSO in control cells. Data shown are Mean±SD, N=6 from two experiments. The Games-Howell test was used for all pairwise comparisons. (FIG IF) A bar graph showing qPCR of Ddit3 following 10 hr piericidin treatment in control cells, with or without aspartate, and in /.AN OX cells. Data was presented as fold-change from DMSO in control cells. Data shown are Mean±SD, N=8 from 3 experiments. The Games-Howell test was used to make all pairwise comparisons of AAC _t values. (FIG. 1G) A bar graph showing media [lactate]/[pyruvate] following 2 hr piericidin treatment, with or without aspartate, in control cells. Data was normalized to DMSO without aspartate. Data shown are Mean±SD, N=6 from 3 experiments. Welch’s t-test (two-tailed) was used to compare each treatment with and without aspartate, followed by Holm’s correction. (FIG 1H) A bar graph showing proliferative rate (doublings in 24 hrs) of control cells, with or without aspartate, and of TANOX cells following piericidin treatment. Data was normalized to DMSO in control cells. Data shown are Mean±SD, N=5-6 from 3 experiments. The Games-Howell test was used for all pairwise comparisons. (FIG II) A bar graph showing qPCR of At/3 following 10 hr pyruvate withdrawal, with or without aspartate, in Ndufa9- KO C2C12 myoblasts. Data was presented as fold-change from the condition with pyruvate (+). Data shown are Mean±SD, N=3. (FIG 1 J) A bar graph showing qPCR of GDF15 following 10 hr piericidin treatment, with or without pyruvate or aspartate, in primary human skeletal myoblasts. Data was presented as fold-change from DMSO. Data shown are Mean±SD, N=3. (FIG IK) A bar graph showing qPCR of A If 3 following 10 hr piericidin or tunicamycin (Tuni) treatment, with or without GCN2iB, in control cells. Data was presented as fold-change from DMSO. Data shown are Mean±SD, N=3. Welch’s t-test (two-tailed) was used to compare each treatment with and without GCN2iB, followed by Holm’s correction. (FIG 1L) A western blot of (p-)GCN2, ATF4 and (p-)eIF2a following 6 hr piericidin treatment with the indicated conditions in /.ANOX cells. /.ANOX expression was induced only where indicated. (FIG 1M) Bar graphs of qPCR of At/3 and Gdfl5 in the same cells and conditions shown in FIG 1L. Data was presented as fold-change from DMSO. Data shown are Mean±SD, N=2-3. GiB, GCN2iB. (FIG IN) Schematic depicting the model for ISR activation by complex I inhibition in myoblasts. For FIGS. 1 A-1N: ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. FIGS. 2A-2L. (FIG 2A) A bar graph showing secreted [lactate] following 2 hr inhibitor treatments in control or LbNOX cells. Data was normalized to DMSO separately in each cell line. Data shown are Mean±SD, N=6 from 3 experiments (expanded version of Fig. 1A). (FIG 2B) A bar graph showing adenylate energy charge following 1 hr treatments in control or LbNOX cells. Data shown are Mean±SD, N=5-6 from 2 experiments (expanded version of Fig. 4B). (FIG. 2C) Scatter plot of intracellular metabolite fold-changes between piericidin and DMSO in control cells (x-axis) or ZANOX cells (y-axis). Data shown are N=3 replicates (same data as in Fig. 1C). (FIG 2D) Bar graphs showing intracellular [aspartate] following 1 hr treatments in control or ZANOX cells. Data shown are Mean±SD, N=6 from 2 experiments (expanded version of Fig. ID). (FIG. 2E) Bar graphs showing peak intensity of intracellular asparagine following 1 hr treatments in control or LbNOX cells. Data shown are Mean±SD, N=6 from 2 experiments (expanded version of Fig. IE). (FIG. 2F)-(FIG. 21) Bar graphs showing qPCR of ISR-related transcripts following 10 hr treatments, with or without aspartate, in control cells. Data was presented as fold-change from DMSO without aspartate. Data shown are Mean±SD, N=3. (FIG 2J) A bar graph showing qPCR of A If 3 following 10 hrs piericidin treatment, with or without pyruvate or aspartate, in primary mouse embryonic fibroblasts. Data was presented as fold-change from DMSO (-). Data shown are Mean±SD, N=3. (FIG. 2K) A bar graph showing qPCR of A if 3 following 10 hrs piericidin or tunicamycin treatment, with or without aspartate, GCN2iB or the PERK inhibitor GSK2656157, in control cells. Data was presented as fold-change from DMSO. Data shown are Mean±SD, N=3. (FIG. 2L) A abr graph showing the ratio of p-eIF2a to total eIF2a, measured by western blot, following 6 hr piericidin treatment in LbNOX cells. LbNOX expression was induced only where indicated. Data was normalized separately in each blot to the DMSO-treated sample not expressing LbNOX. Data shown are Mean±SD, N=4 from 4 experiments. A t-test was used to examine whether the mean of DMSO-treated samples expressing LbNOX differed from 1, and Welch’s t-test (two- tailed, paired by blot) was used to compare the piericidin-treated samples with and without LbNOX expression. Holm’s correction was then applied. For FIGS. 2A-2L: ns ns, P > 0.05; **, P < 0.01. DETAILED DESCRIPTION

Mitochondrial dysfunction is associated with activation of the integrated stress response (ISR) but the underlying triggers remain unclear. Experiments herein systematically combined acute mitochondrial inhibitors with genetic tools for compartment-specific NADH oxidation to trace mechanisms linking different forms of mitochondrial dysfunction to the ISR in proliferating myoblasts and in differentiated myotubes. In myoblasts, experiments showed that impaired NADH oxidation upon electron transport chain (ETC) inhibition depletes asparagine, activating the ISR via the eIF2a kinase, GCN2 (also referred to as "General Control Nonderepressible 2," "eIF2AK4," and "eukaryotic translation initiation factor 2 alpha kinase 4"). In myotubes, however, impaired NADH oxidation following ETC inhibition neither depletes asparagine nor activates the ISR, reflecting an altered metabolic state. ATP synthase inhibition in myotubes triggers the ISR via a distinct mechanism related to mitochondrial inner-membrane hyperpolarization. This work dispels the notion of a single, universal path linking mitochondrial dysfunction to the ISR, instead revealing multiple paths that depend both on the nature of the mitochondrial defect and on the metabolic state of the cell. Importantly, the link between GCN2 and the ISR showed that mitochondrial dysfunction (including mitochondrial myopathies and diseases and conditions associated with mitochondrial dysfunction) caused by ISR activation can be treated by administering a GCN2 inhibitor.

GCN2 inhibitors, Compositions, and Administration

General control nonderepressible 2 (GCN2) plays a major role in cellular response to amino acid limitation (Nakamura, A. et al. Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response. Proc.

Natl. Acad. Sci. 115 (33): E7776-E7785 (2018)). Here, experiments show the protein kinase GCN2 plays a role in the integrated stress response (ISR) and mitochondrial dysfunction. The ISR is essential for maintaining cellular homeostasis under a wide range of stressors. Amino acid deficiency is a canonical ISR trigger through the eIF2a kinase GCN2, which is activated due to uncharged tRNA (Hinnebusch, A. G Evidence for translational regulation of the activator of general amino acid control in yeast. Proc. Natl. Acad. Sci. U. S. A. 81, 6442-6446 (1984); Dever, T. E. et al. Phosphorylation of initiation factor 2 alpha by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell 68, 585-596 (1992); Wek, S. A., Zhu, S. & Wek, R. C.

The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids. Mol. Cell. Biol. 15, 4497-4506 (1995); Berlanga, J. J., Santoyo, J. & De Haro, C. Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2alpha kinase. Eur J. Biochem. 265, 754-762 (1999); Zhang, P. et al. The GCN2 eIF2a Kinase Is Required for Adaptation to Amino Acid Deprivation in Mice. Mol. Cell. Biol. 22, 6681 LP - 6688 (2002); Castilho, B. A. et al. Keeping the eIF2 alpha kinase Gcn2 in check. Biochim. Biophys. Acta - Mol. Cell Res. 1843, 1948-1968 (2014); Inglis, A. J. et al. Activation of GCN2 by the ribosomal P-stalk. Proc. Natl. Acad. Sci. 116, 4946 LP - 4954 (2019); Harding, H. P. et al. The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells. Elife 8, e50149 (2019)).

ISR is regulated by four distinct kinases — heme-regulated initiation factor-2a kinase, dsRNA-activated protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2) — all of which phosphorylate eukaryotic initiation factor 2a subunit (eIF2a). The phosphorylation of eIF2a in turn leads to inhibition of global protein synthesis and active translation of specific mRNAs, such as those of activating transcription factor 4 (ATF4). ATF4 functions as a transcriptional activator of genes encoding proteins involved in oxidative stress, nutrient uptake, and metabolism. GCN2, which is activated by uncharged tRNA resulting from amino acid deficiency, serves as a master regulator of the amino acid response (AAR) (Nakamura, A. et al. Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response. Proc. Natl. Acad. Sci.

115 (33): E7776-E7785 (2018)).

A GCN2 inhibitor inactivates or dephosphorylates GCN2. Examples of GCN2 inhibitors, include, for example, one or more small molecule inhibitors (including, for example, those discussed by Muller and Scherle, Nature Reviews Cancer 2006;6:613, discussed in US 2014378431 Al, US 9,409,914, US 20190233425 Al, US 20190375753 Al, US 20190233411 Al, US 20210040083 Al, US 10,793,563, US 2019169166 Al, US 201962832982, and W02020210828A1, and discussed in Nakamura, A. et al. Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response. Proc. Natl. Acad. Sci. 115 (33): E7776-E7785 (2018)).

Examples of small molecule inhibitors, including GCN2iB, as described in Nakamura, A. et al. Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response. Proc. Natl. Acad. Sci. 115 (33) E7776-E7785 (2018), are shown in Table 1.

Table 1: Examples of Small Molecule Inhibitors of GCN2 GCN2 inhibitors can also include inhibitory nucleic acids directed against GCN2.

Inhibitory nucleic acids useful in the present methods and compositions include antisense oligonucleotides, ribozymes, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of the GCN2 nucleic acid and decrease expression of GCN2. In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof. See, e.g., WO 2010040112.

In some embodiments, the inhibitory nucleic acids are 10 to 50, 10 to 20, 10 to 25, 13 to 50, or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having complementary portions of

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,

34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin. In some embodiments, the inhibitory nucleic acids are 15 nucleotides in length. In some embodiments, the inhibitory nucleic acids are 12 or 13 to 20, 25, or 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having complementary portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range therewithin (complementary portions refers to those portions of the inhibitory nucleic acids that are complementary to the target sequence).

An exemplary reference target sequence of human GCN2 can be found at NCBI RefSeq ID NM_001013703.4. The target sequence can be at least 80%, 85%, 90%, 95%, 97%, or 99% identical to a reference sequence. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Other GCN2 inhibitors include GCN2 blocking peptides (commercially available, for example, from Bethyl, Inc., Montgomery, TX); an antibody to GCN2 (commercially available, for example, from Bethyl, Inc., Montgomery, TX), glutamine, and an amino acid other than glutamine that inhibits GCN2. In some embodiments, the one or more small molecule inhibitors can be any one or more of GCN2iB, GCN2iA, Triazolo[4,5- d]pyrimidine derivatives, pyrazolo-pyrimidin-amino-cycloalkyl compounds and derivatives, and any other known GCN2 small molecule inhibitors known in the art.

The methods described herein include the use of compositions comprising or consisting of a GCN2 inhibitor as an active ingredient.

Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions, e.g., one of more of ISR inhibitors (e.g, an ATPase synthase inhibitor or LbNOX; any ISR inhibitors known in the art), one or more amino acid or a mimetic of an amino acid, asparaginase, one or more analogs of a metabolic breakdown product of an amino acid, one or more antigens, one or more antibodies, an antibiotic, one or more antimicrobial agents, one or more antiviral agents (e.g., such as AZT, ddl or ddC), one or more immunotherapies (e.g., CAR T cell therapy); and/or one or more chemotherapeutic agents, and combinations thereof. Chemotherapeutic agents include, but not limited to, cyclophosphamide, methotrexate, fluorouracil, doxorubicin, vincristine, ifosfamide, cisplatin, gemcytabine, busulfan (also known as 1 ,4-butanediol dimethanesulfonate or BU), ara-C (also known as 1 -beta-D-arabinofuranosylcytosine or cytarabine), adriamycin, mitomycin, Cytoxan, methotrexate, or any combination thereof. Additional therapeutic agents also include cytokines, including, but not limited to, macrophage colony stimulating factor, interferon gamma, granulocyte-macrophage stimulating factor (GM-CSF), flt-3, TNFa, TNFP, any mimetics of any cytokine, and combinations thereof.

Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. The methods described herein include the administration of an effective amount of an inhibitor of a GCN2 kinase.

Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.

Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

In some embodiments, compositions comprising a GCN2 inhibitor for transdermal application can further comprise cosmetically-acceptable carriers or vehicles and any optional components. A number of such cosmetically acceptable carriers, vehicles and optional components are known in the art and include carriers and vehicles suitable for application to skin (e.g., sunscreens, creams, milks, lotions, masks, serums, etc.), see, e.g., U.S. Patent Nos. 6,645,512 and 6,641,824. In particular, optional components that may be desirable include, but are not limited to absorbents, anti -acne actives, anti-caking agents, anti-cellulite agents, anti-foaming agents, anti-fungal actives, anti-inflammatory actives, anti-microbial actives, anti-oxidants, antiperspirant/deodorant actives, anti-skin atrophy actives, anti-viral agents, anti-wrinkle actives, artificial tanning agents and accelerators, astringents, barrier repair agents, binders, buffering agents, bulking agents, chelating agents, colorants, dyes, enzymes, essential oils, film formers, flavors, fragrances, humectants, hydrocolloids, light diffusers, nail enamels, opacifying agents, optical brighteners, optical modifiers, particulates, perfumes, pH adjusters, sequestering agents, skin conditioners/moisturizers, skin feel modifiers, skin protectants, skin sensates, skin treating agents, skin exfoliating agents, skin lightening agents, skin soothing and/or healing agents, skin thickeners, sunscreen actives, topical anesthetics, vitamin compounds, and combinations thereof.

The GCN2 inhibitor compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal or vaginal delivery. Such suppositories can be used particularly for the treatment of conditions associated with the loss of in elastic fibers that affect the pelvic organs, e.g., pelvic organ prolapse and/or urinary incontinence, inter alia.

Therapeutic compounds that are or include nucleic acids can be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine. These methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Patent No. 6,168,587. Additionally, intranasal delivery is possible, as described in, inter alia, Hamajima et al., Clin. Immunol. Immunopathol., 88(2), 205-10 (1998). Liposomes (e.g., as described in U.S. Patent No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Patent No. 6,471,996).

In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Mitochondrial Myopathy, Diseases Associated with Mitochondrial Dysfunction, and Methods of Treatment

The methods described herein include methods for the treatment of disorders associated with mitochondrial dysfunction. In some embodiments, the disorder is a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction. Generally, the methods include administering a therapeutically effective amount of a GCN2 inhibitor as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.

In some embodiments, a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction can be any one or more of the following: Kearns-Sayre syndrome (KSS), Leber’s hereditary optic neuropathy (LHON), Leigh syndrome (LS), MEGDEL Syndrome; mitochondrial DNA depletion syndrome (MDS), mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonus epilepsy with ragged-red fibers (MERRF), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), neuropathy, ataxia and retinitis pigmentosa (NARP), OPA1 mutations, Pearson syndrome, progressive external ophthalmoplegia (PEO), Mitochondrial complex I deficiency, Mitochondrial complex II deficiency, Mitochondrial complex III deficiency, Mitochondrial complex IV deficiency, Mitochondrial complex V (ATP synthase) deficiency, Primary coenzyme Q10 deficiency (COQIOD), Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome, Mitochondrial disease resulting from mutations in PolG (e.g. Chronic Progressive External Ophthalmoplegia syndrome (CPEO), Alpers-Huttenlocher syndrome (AHS), Childhood Myocerebrohepatopathy Spectrum (MCHS), Myoclonic Epilepsy Myopathy Sensory Ataxia (MEMSA), Ataxia Neuropathy Spectrum (ANS) (including e.g., mitochondrial recessive ataxia syndrome (MIRAS) and sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO)), Autosomal Recessive Progressive External Ophthalmoplegia (arPEO), Autosomal Dominant Progressive External Ophthalmoplegia (adPEO)), adPEO due to mutations in ANT or due to C10orf2 (twinkle) mutations, Mitochondrial DNA depletion syndrome, Mitochondrial DNA depletion syndrome 1/MyoNeurogenic Gastrointestinal Encephalopathy (MNGIE), Mohr-Tranebjaerg syndrome, 3-methylglutaconic aciduria, Combined oxidative phosphorylation deficiency (COXPD), Myopathy, Lactic Acidosis, and Sideroblastic Anemia (MLASA), Hyperuricemia, Pulmonary hypertension, Renal failure, and Alkalosis (HUPRA) syndrome, Leigh Syndrome, Leigh syndrome-Prench Canadian type, Lriedreich ataxia, Gracile syndrome, Bjo nstad syndrome, Multiple Mitochondrial Dysfunctions Syndrome (MMDS), Early-onset Ataxia with Ocular motor apraxia and Hypoaibuminemia (EAOH), Charcot-Marie-Tooth Disease-2A2, Leber Hereditary Optic Neuropathy (LHON), Sudden Infant Death Syndrome, Myoclonic Epilepsy with Ragged Red Libers (MERRF), MERRF/MELAS overlap syndrome, Neuropathy, Ataxia, Retinitis Pigmentosa (NARP), Mitochondrial myopathy, Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS), Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL), Mitochondrial disease resulting from SDH mutations (e.g. Pheochromocytoma and Paragangliomas), Optic atrophy type 1, Ethylmalonic encephalopathy, Carnitine-acylca nitine translocase deficiency, Primary systemic carnitine deficiency, Creatine deficiency syndromes (e.g. Cerebral creatine deficiency syndrome- 1 , Cerebral creatine deficiency syndrome-2 or Cerebral creatine deficiency syndrome-3), Carnitine palmitoyltransferase 1 (CPT I) deficiency, Carnitine palmitoyltransferase 2 (CPT II) deficiency, Short-chain acyl-CoA dehydrogenase deficiency, Very long chain acyl-CoA dehydrogenase deficiency, Long-chain 3- hydroxyl-CoA dehydrogenase (LCHAD) deficiency, Pyruvate carboxylase deficiency, Multiple acyl -Co A dehydrogenase deficiency (e.g. Glutaric acidemia II A, Glutaric acidemia PB or Glutaric acidemia IIC), Pyruvate dehydrogenase deficiency (e.g.

Pyruvate dehydrogenase El -alpha deficiency, Pyruvate dehydrogenase phosphatase deficiency, Pyruvate dehydrogenase E3 -binding protein deficiency, Pymvate dehydrogenase E2 deficiency or Pyruvate dehydrogenase El -beta deficiency), 3- hydroxyacyl-CoA dehydrogenase (SCHAD) deficiency/(HADH) deficiency, Perrault syndrome, cardiomyopathies, individually rare genetic disorders; neurodegeneration (e.g., Parkinson’s disease)(Schapira, A. H. V. Mitochondria in the aetiology and pathogenesis of Parkinson’s disease. Lancet Neurol. 7, 97-109 (2008); Hu, Q. & Wang, G. Mitochondrial dysfunction in Parkinson’s disease. Transl. Neurodegener. 5, 14 (2016); Lin, M. T. & Beal, M. F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787 (2006); Area-Gomez, E., Guardia-Laguarta, C, Schon, E. A. & Przedborski, S. Mitochondria, OxPhos, and neurodegeneration: cells are not just running out of gas. J. Clin. Invest. 129, 34-45 (2019)); diabetes (Kelley, D.

E., He, J., Menshikova, E. V & Ritov, V. B. Dysfunction of Mitochondria in Human Skeletal Muscle in Type 2 Diabetes. Diabetes 51, 2944 LP - 2950 (2002); Mootha, V. K. et al. Erralpha and Gabpa/b specify PGC-1 alpha-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle. Proc. Natl. Acad. Sci. U. S. A. 101, 6570-5 (2004); Ritov, V. B. et al. Deficiency of Subsarcolemmal Mitochondria in

Obesity and Type 2 Diabetes. Diabetes 54, 8 LP - 14 (2005); Ritov, V. B. et al. Deficiency of electron transport chain in human skeletal muscle mitochondria in type 2 diabetes mellitus and obesity. Am. J. Physiol. Metab. 298, E49-E58 (2009)); cancer (Wallace, D. C. Mitochondria and cancer. Nat. Rev. Cancer 12, 685 (2012); Reznik, E., Wang, Q., La, K., Schultz, N. & Sander, C. Mitochondrial respiratory gene expression is suppressed in many cancers. Elife 6, e21592 (2017); Gaude, E. & Frezza, C. Defects in mitochondrial metabolism and cancer. Cancer Metab. 2, 10 (2014)); and the aging process (Sun, N., Youle, R. J. & Finkel, T. The Mitochondrial Basis of Aging. Mol. Cell 61, 654-666 (2016); Bratic, A. & Larsson, N.-G. The role of mitochondria in aging. J. Clm. Invest. 123, 951-957 (2013)).

Mutations in nearly 300 nuclear or mitochondrial (mtDNA) genes have been implicated in mitochondrial disorders that affect at least 1 in 4,300 live births and lack effective treatments (Schapira, A. H. V. Mitochondrial diseases. Lancet 379, 1825-34 (2012); Vafai, S. B. & Mootha, V. K. Mitochondrial disorders as windows into an ancient organelle. Nature 491, 374-383 (2012); Gorman, G. S. et al. Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease. Ann. Neurol. 77, 753-759 (2015); Gorman, G. S. et al. Mitochondrial diseases. Nat. Rev. Dis. Prim. 2, 16080 (2016); Frazier, A. E., Thorburn, D. R. & Compton, A. G. Mitochondrial energy generation disorders: genes, mechanisms, and clues to pathology. J. Biol. Chem. 294, 5386-5395 (2019)). In some embodiments, mitochondrial myopathies and/or diseases or conditions associated with mitochondrial dysfunction are a result of point mutations in tRNA genes selected from the group consisting of: tRNA ^Leu (T3250C; A3302G; A12320G, A3288G); tRNA ^Pro (G15990A; A16002G; G15995A); tRNA ^phe (T618C; G622A); tRNA ^Met (T4409C; T5543C); tRNA ^Ser (G7497A; A7480G); tRNA ^Asp (A7526G); tRNA ^Gln (4366msA); tRNA ^Ala ; tRNA ^Glu (T14709C); tRNA ^Tlp (G5521A); and tRNA ^Tyr . In some embodiments, mitochondrial myopathies and/or diseases or conditions associated with mitochondrial dysfunction are a result of one or more point mutations in mtDNA selected from the group consisting of: G15243A, T9185C, G3421A, G10197A, T12148C, and G6570A, and/or one or more nuclear mutations linked to disease including, but not limited to, NDUF mutations in Leigh syndrome, SC02 mutations in infantile cardiomyopathy, POLG mutations (mutations in gene that codes for DNA polymerase gamma) in many disorders such as SANDO (sensory ataxic neuropathy, dysarthria, and ophthalmoparesis), ataxia, and Alper Syndrome, SPG7 mutations in hereditary spastic paraparesis, MFN2 mutations in peripheral neuropathy, amongst others (www.mitomap.org/MITOMAP). In some embodiments, mitochondrial myopathies and/or diseases or conditions associated with mitochondrial dysfunction can also include myopathies selected from the group consisting of: Swedish type myopathy with exercise intolerance; combined mitochondrial complex deficiency; familial myalgia syndrome; myopathy with abnormal mitochondrial translation; myopathy with extrapyramidal signs; myopathy with focal depletion of mitochondria; mitochondrial DNA breakage syndrome; limb-girdle muscular dystrophy type IH (LGMDIH); and isolated mitochondrial myopathy (IMMD).

In some embodiments, the signs or symptoms of a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction include any one or more of abnormal breathing rhythm, abnormal choroid plexus function, accumulation of metabolites, acidosis, asymmetric vascular dilatation, ataxia, basal ganglia calcifications, basal ganglia lesions, bilateral striatal necrosis, borborygmi, brainstem events with oculomotor palsies, brisk tendon reflexes, cachexia, carbohydrate intolerance, cardiac arrhythmia, cardiac hypertrophy, cerebellar atrophy, cerebral atrophy, muscle atrophy, chorea, choreoathetosis, chronic partial denervation, constipation, COX deficiency in muscle, dementia, demyelinization of corticospinal tracts, developmental delay, diarrhea, diffuse leukoencephalopathy, distal arthrogryposis, distal renal tubular acidosis, dysarthria, dysmorphic facies, dysphagia, dystonia, elevated plasma deoxyuridine and deoxythymidine levels, elevated plasma thymidine levels, elevated serum creatine kinase levels, encephalopathy, epigastralgia, episodic encephalopathy, exercise intolerance, exocrine insufficiency, gait impairment, gastrointestinal dysmotility, glucose intolerance, heart block, hemiplegia, hereditary spastic paraparesis, high CSF protein levels, high homovanillic acid (HVA) in CSF, high lactate levels in CSF, hypertelorism, hypertension, hypertrophic cardiomyopathy, hyperventilation, hypoacusis, hypoplasia of the corpus callosum, hypotonia, incomplete right bundle branch block, increased tendon reflexes, lactic acidosis, limb athetosis, limb spasticity, limitation or absence of movement in all fields of gaze, lordosis, loss verbal milestones, low 5- methyltetrahydrofolate (5-MTHF) in CSF, mental retardation, mitochondrial capillary angiopathy, mitochondrial proliferation in muscle, motor retardation, motor spasticity, mtDNA depletion, myelopathy, nausea, nephrotic syndrome, neuronal hyperexcitability, nystagmus, occasional fatigue or pain on exertion, pancreatitis, paralysis, paresthesias, Parkinsonism, peripheral neuropathy, Pes cavus, pigmentary degeneration of retina (retinitis pigmentosa), progressive encephalopathy, progressive or acute encephalopathy, proximal renal tubular acidosis, pseudoathetosis, ptosis, Purkinje dendrite cactus formations with increased mitochondria, pyramidal features, ragged-red fibers, reduced cardiopulmonary capacity, reduced respiratory drive, renal cysts, respiratory failure, rhabdomyolysis, reduced maximal whole body oxygen consumption (V02 max), seizures, sensory neuropathy, sensory- motor polyneuropathy, sialoadenitis focal segmental glomerulosclerosis, small fiber modality loss, spasticity, status spongiosis in gray and white matter, recurrent apnea, stroke, subacute necrotizing encephalomyelopathy, tetany, tonic-clonic seizures, tubular dysfunction, variation in muscle fiber size, vascular narrowing, vertebral anomalies, vomiting, weakness, weight loss, and white matter atrophy.

In some embodiments, a sign or symptom of a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction include activation of the ISR. The ISR is triggered by various insults, including nutrient deficiency, unfolded protein stress and pathogen infection. In mammalian cells, it is typically activated by a family of four kinases, each of which can phosphorylate the alpha subunit of translation initiation factor 2 (eIF2a) in response to distinct stimuli. eIF2a phosphorylation acutely inhibits global protein synthesis, but promotes translation of transcription factors, such as activating transcription factor 4 (ATF4) and DNA damage-inducible transcript 3 protein (DDIT3), that then engage their downstream targets (Harding, H. P. et al. An Integrated Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress. Mol. Cell 11, 619-633 (2003); Palam, L. R, Baird, T. D. & Wek, R. C. Phosphorylation of eIF2 Facilitates Ribosomal Bypass of an Inhibitory Upstream ORF to Enhance CHOP Translation. J. Biol. Chem. 286, 10939-10949 (2011); Harding, H. P. etal. Regulated Translation Initiation Controls Stress-Induced Gene Expression in Mammalian Cells.

Mol. Cell 6, 1099-1108 (2000); Vattem, K. M. & Wek, R. C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 101, 11269-74 (2004); Wek, R. C. Role of eIF2a Kinases m Translational Control and Adaptation to Cellular Stress. Cold Spring Harb. Perspect. Biol. 10, (2018); Pakos-Zebrucka, K. et al. The integrated stress response. EMBO Rep. 17, 1374 LP - 1395 (2016); Taniuchi, S., Miyake, M., Tsugawa, K., Oyadomari, M. & Oyadomari, S. Integrated stress response of vertebrates is regulated by four eIF2a kinases. Sci. Rep. 6, 32886 (2016)). Without being bound by theory, the transcriptional program induced by the ISR varies across species and cell types and depends on the underlying trigger. Nevertheless, it frequently encompasses amino acid transport and biosynthesis genes, cytosolic tRNA synthetases and translation factors, pro-apoptotic factors, and genes involved in antioxidant defense, proteostasis and organelle quality control (Harding, H. P. et al. An Integrated Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress. Mol. Cell 11, 619-633 (2003); Han, J. et al. ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol 15, 481-490 (2013)). In tissues affected by mitochondrial disease, the ISR activates the mitochondrial 1 -carbon pathway (Kiihl, I. et al. Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals.

Elife 6, e30952 (2017); Tyynismaa, H. et al. Mitochondrial myopathy induces a starvation-like response. Hum. Mol. Genet. 19, 3948-3958 (2010); Bao, X. R. et al. Mitochondrial dysfunction remodels one-carbon metabolism in human cells. Elife 5, el0575 (2016); Nikkanen, J. et al. Mitochondrial DNA Replication Defects Disturb Cellular dNTP Pools and Remodel One-Carbon Metabolism. Cell Metab. 23, 635-648 (2016)) and is thought to underlie secretion of the circulating cytokines fibroblast growth factor 21 (FGF21) and growth/differentiation factor 15 (GDF15) that are under consideration as disease biomarkers (Lehtonen, J. M. et al. FGF21 is a biomarker for mitochondrial translation and mtDNA maintenance disorders. Neurology 87, 2290-2299 (2016); Fujita, Y. et al. GDF15 is a novel biomarker to evaluate efficacy of pyruvate therapy for mitochondrial diseases. Mitochondrion 20, 34-42 (2015); Yatsuga, S. et al. Growth differentiation factor 15 as a useful biomarker for mitochondrial disorders. Ann. Neurol. 78, 814-823 (2015); Chung, H. K. et al. Growth differentiation factor 15 is a myomitokine governing systemic energy homeostasis. J. Cell Biol. (2016); Miyake, M. et al. Skeletal muscle-specific eukaryotic translation initiation factor 2alpha phosphorylation controls amino acid metabolism and fibroblast growth factor 21- mediated non-cell-autonomous energy metabolism. FA SEE J. Off. Publ. Fed. Am. Soc. Exp. Biol. 30, 798-812 (2016); Restelli, L. M. etal. Neuronal Mitochondrial Dysfunction Activates the Integrated Stress Response to Induce Fibroblast Growth Factor 21. Cell Rep. 24, 1407-1414 (2018)). Experiments disclosed herein show that in proliferating myoblasts, electron transport chain (ETC) inhibition elevates the mitochondrial and cytosolic NADH/NAD ⁺ ratios, hindering aspartate synthesis and ultimately depleting asparagine, which activates the ISR via the eIF2a kinase GCN2.

In some embodiments, the subject with a mitochondrial myopathy or disease associated with mitochondrial dysfunction displays abnormal levels of one or more biomarkers compared to a normal control subject or control cell. In some embodiments, the biomarker is selected from the group consisting of Activating Transcription Factor (ATF) protein levels (e.g., ATF3 (exemplary sequence at GenBank RefSeq ID NP 001025458.1) or ATF4 (exemplary sequence at GenBank RefSeq ID NP 001666.2)), lactic acid (lactate) levels; pyruvic acid (pyruvate) levels; lactate/pyruvate ratios; phosphocreatine levels; NAHD/(NAD ⁺ ) levels; NADH (NADH+H+) or NADPH (NADPH+H+) levels; NAD or NADP levels; ATP levels; reduced coenzyme Q (CoQred) levels; oxidized coenzyme Q (CoQox) levels; total coenzyme Q (CoQtot) levels; oxidized cytochrome C levels; reduced cytochrome C levels; oxidized cytochrome C/reduced cytochrome C ratio; acetoacetate levels; beta -hydroxy butyrate levels; acetoacetate/ beta- hydroxy butyrate ratio; 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels; levels of reactive oxygen species; oxygen consumption (V02), carbon dioxide output (VC02), respiratory quotient (VCO2/VO2), and to modulate exercise intolerance/tolerance and to modulate anaerobic threshold.

As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction. Often, a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction results in any one or more of the signs and symptoms described above; thus, a treatment can result in a reduction in at least one of the signs and symptoms a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction. Administration of a therapeutically effective amount of a compound or composition (e.g., comprising a GCN2 inhibitor) described herein for the treatment of a condition associated with a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction will result in a reduction of at least one of the signs or symptoms of a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction as described herein. Administration of a therapeutically effective amount of a compound or composition (e.g., comprising a GCN2 inhibitor) described herein for the treatment of a condition associated with a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction will also result in a return of the levels of one or more energy biomarkers of a subject having or at risk of having a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction to the levels of a normal control subject.

An “effective amount” or a “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect or amelioration or reduction of a sign or symptom of a mitochondrial myopathy and/or a disease or condition associated with mitochondrial dysfunction. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.

Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Methods and Materials

Cell culture

C2C12 cells were obtained from ATCC (CRL-1772) and used as-is for differentiation. The same cells were infected with lentivirus to generate stable cell lines expressing luciferase (control), T6NOX, mito/ANOX or NDI1 under a doxycycline- inducible promoter (TRE3G; Clontech, CA), as previously described (Titov, D. V et al. Complementation of mitochondrial electron transport chain by manipulation of the NAD(+)/NADH ratio. Science 352, 231-235 (2016)). A7/»/a9-knockout C2C12 cells were previously generated using CRISPR/Cas9 (Vafai, S. B. et al. Natural Product Screening Reveals Naphthoquinone Complex I Bypass Factors. PLoS One 11, eO 162686 (2016)). Primary mouse embryonic fibroblasts were obtained from Lonza (Morristown, NJ; M-FB-481) and primary human skeletal myoblasts from Thermo Fisher Scientific (San Jose, CA; Al 1440). Cells were kept in a 37C, 5% CO2 incubator except where otherwise indicated.

All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM). The base media formulation (Thermo Fisher Scientific; 11966-025) lacked glucose and sodium pyruvate and contained 4mM /.-Glutamine. Glucose (Sigma, St. Louis, MO; G8769) was always supplemented to a concentration of lOmM.

Doxycycline-inducible myoblasts were cultured in media supplemented to 10% dialyzed fetal bovine serum (dFBS) (Thermo Fisher Scientific; 26400-044). Up until cells were seeded for experiments, the media also contained 1 pg/ml puromycin (Thermo Fisher Scientific; All 138-03) and 0.5mg/ml geneticin (Thermo Fisher Scientific; 10131- 035) to maintain selection of expression vectors. Ndufa9- knockout C2C12 myoblasts, primary mouse embryonic fibroblasts, and primary human myoblasts not intended for differentiation were also cultured in media supplemented to 10% dFBS. For the Ndufa9- knockout cells, the standard culture media contained lmM sodium pyruvate (Thermo Fisher Scientific; 11360-070). C2C12 myoblasts intended for differentiation were grown in media supplemented to 20% dFBS and kept sparse until seeded for experiments. When seeded cells had grown to confluence, they were switched to media supplemented to 2% dialyzed horse serum (Valley Biomedical, Winchester, VA; AS3053-DI) to induce differentiation. Human myoblasts intended for differentiation were seeded immediately after thaw directly at a confluent density in media supplemented to 2% horse serum, New Zealand origin (Thermo Fisher Scientific; 16050-130).

Cell treatments for RNA isolation

Doxycycline-inducible myoblasts were seeded for most treatments at 20,000 cells per well in 24- well plates with 0.5ml/well of media without selection antibiotics. Only 2,000 cells per well were seeded for chloramphenicol treatment. Approximately 3 hours after initial seeding, an additional 0.5ml/well of media supplemented with doxycycline (Sigma; D9891; dissolved in water) was dispensed to induce protein expression, such that the final concentration was 300ng/ml.

24 hours after doxycycline addition, inhibitor treatments were started with complete replacement of the media to a final volume of 1 ml/well (still containing 300ng/ml doxycycline). Piericidin A (Santa Cruz Biotechnology, Santa Cruz, CA; sc- 202287) was used at 0.5mM, Antimycin A (Sigma; A8674) at 0.5mM, Oligomycin A (Sigma; 75351) at ImM, Tunicamycin (Sigma; T7765) at lpg/ml, Chloramphenicol (Sigma; C0378) at 20pg/ml. All small-molecule inhibitors were dissolved in dimethyl sulfoxide (DMSO) (Sigma; D2650). Final DMSO concentrations did not exceed 1:1,000. When nutrients were supplemented during inhibitor treatments, they were introduced with the media at the start of treatment. /.-Aspartic acid (Sigma; A9256) was supplemented to a concentration of lOmM. Media supplemented with aspartate was titrated back to pH 7.4 with NaOH. /.-Asparagine monohydrate (Sigma; A8381) was supplemented to a concentration of 0.5mM. Uridine (Sigma; U3003) was supplemented to a concentration of 200mM.

When eIF2a kinase inhibitors were used, they were introduced with the media at the start of treatment. The GCN2 inhibitor GCN2iB was custom synthesized (Acme Bioscience, Palo Alto, CA) based on the published recipe and used at 0.5mM (Nakamura, A. et al. Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response. Proc. Natl. Acad. Sci. (2018)). The PERK inhibitor GSK2656157 (Cayman Chemical, Ann Arbor, MI; 17372) was used at 0.25mM. eIF2a kinase inhibitors were also dissolved in DMSO.

Primary mouse embryonic fibroblasts were seeded for treatments at 15,000 cells per well in 24- well plates with 1 ml/well of media, and primary human myoblasts (not intended for differentiation) were seeded at 30,000 cells per well in 12-well plates with 2ml/well of media. Treatments were started 24 hours later with complete replacement of the media. Inhibitor and aspartate concentrations were as described above. Sodium pyruvate was supplemented to a concentration of ImM.

Ndufa9- knockout myoblasts were seeded at 20,000 cells per well in 24-well plates with 1 ml/well of media. 24 hours later, reductive stress was induced by replacing the culture media with media lacking sodium pyruvate. The aspartate concentration was as described above.

C2C12 myoblasts intended for differentiation were seeded at 50,000 cells per well in 24-well plates with 1 ml/well of media. Cells became fully confluent ~48 hours after seeding and were then switched to low-serum media. The media was fully replenished 48 hours after the switch to low-serum. If protein expression was required, the cells were transduced at this time with adenovirus expressing eGFP alone (Vector Biolabs, Malvern, PA; 1060-HT; 0.05pl/well of -5E12 VP/ml), or eGFP in combination with /ANOX (0. 125pl/well of -2E12 VP/ml) or with mito/ANOX (0.05pl/well of -2E12 VP/ml). Adenovirus production was previously described (Titov, D. V et al. Complementation of mitochondrial electron transport chain by manipulation of the NAD(+)/NADH ratio. Science 352, 231-235 (2016)). The media was fully replenished every 24 hours thereafter. After a total of 4 days in low-serum, differentiated myotubes were treated with mitochondrial inhibitors as described above. BAM15 (TimTec, Tampa, FL; ST056388) was used at 0.5mM except where otherwise indicated. For hypoxic pre-conditioning, myotubes were placed in a 37C, 5% O2, 5% CO2 incubator the night prior to the start of treatment and during treatment.

Human myoblasts intended for differentiation were seeded directly at a confluent density of 250,000 cells per well in 24-well plates with lml/well of low-serum media. The media was fully replenished 48 hours later and every 24 hours thereafter. After a total of 4 days, inhibitor treatments were started with complete replacement of the media. Inhibitor concentrations were as above.

Treatments intended for RNA isolation lasted 10 hours, except where otherwise indicated. At the end of the treatment, cells were lysed in 150pl/well buffer RLT from the RNeasy kit (Qiagen, Germantown, MD; 74106). The lysates were immediately frozen at - 80C until RNA isolation. RNA isolation was performed using the RNeasy kit or RNeasy96 kit (Qiagen; 74181) following the manufacturer’s protocol.

RNA sequencing and data analysis

The integrity of RNA intended for sequencing was assayed using Agilent Bioanalyzer 2100 or Advanced Analytical Fragment Analyzer. All tested RNA samples yielded optimal (10) or near optimal (>9) RIN/RQN values. 50ng RNA per sample were submitted for preparation of sequencing libraries at the Broad Technology Labs based on the Smart-seq2 method (Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171 (2014)). The protocol included selection for polyadenylated RNA and was not strand-specific. Libraries were sequenced on 2 flow- cells of an Illumina NextSeq 500 instrument, generating 2x37bp paired-end reads. Demultiplexed sequencing data has been deposited in the Gene Expression Omnibus (GEO) under accession GSE132234.

Sequencing reads were pseudo-aligned using kallisto (v. 0.44, with bias correction) (Bray, N. L., Pimentel, FL, Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525 (2016)) to an index consisting of the mouse transcriptome (ENSEMBL v. 93; GRCm38.p6) and the sequences of the transgenes luciferase, ZANOX, NDI1 and eGFP (previously reported; Titov, D. V et al. Complementation of mitochondrial electron transport chain by manipulation of the NAD(+)/NADH ratio. Science 352, 231-235 (2016)). Estimated counts were aggregated across the 2 flow-cells and summarized to the gene level in R using the tximport package (Soneson, C, Love, M. L & Robinson, M. D. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. FlOOOResearch 4, 1521 (2015)). Raw (un-normalized) gene level counts for all samples are available through the GEO record. Counts were then processed further in R using the DESeq2 package (Love, M. I, Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)). Gene fold-changes derived from RNA-seq were calculated after normalization for sequencing depth, as implemented in DESeq2.

Within a given subset of experimental conditions analyzed together, a gene was retained if there was at least one condition for which each replicate sample had at least 16 pre-normalization counts associated with the gene. Principal components analysis (PCA) was performed on the gene count data after regularized log2 transformation, as implemented in DESeq2, and mean centering. For the PCA visualization of all doxycycline-inducible myoblasts the data was also scaled. The jackstraw method (10,000 resampling iterations, 10% of variables permuted in each iteration) was used to assign statistical significance for the association of each gene with each of the top principal components (Chung, N. C. & Storey, J. D. Statistical significance of variables driving systematic variation in high-dimensional data. Bioinformatics 31, 545-554 (2015)). Differential expression analyses based on GLM regression were performed in DESeq2. The design formulas and results are reported in Supp. File 1. P-values are derived from the Wald test and were adjusted within each analysis with the method of Benjamini and Hochberg. Log2 fold-changes were shrunk using the apeglm shrinkage estimator (Zhu, A., Ibrahim, J. G. & Love, M. I. Heavy -tailed prior distributions for sequence count data: removing the noise and preserving large differences. Bioinformatics 35, 2084-2092 (2018)), as implemented in DESeq2.

( V.s-regulatory analysis was performed with iRegulon (v. 1.3) in Cytoscape (v.

3.7) (Janky, R. etal. iRegulon: From a Gene List to a Gene Regulatory Network Using Large Motif and Track Collections. PLOS Comput. Biol. 10, el003731 (2014)). For control myoblasts, the input was the 500 genes with the most positive PCI weights among genes with a PCI jackstraw p- value < 0.01, or, separately, the 500 genes with the most negative weights. For all doxycycline-inducible myoblasts, the input was the 500 genes with the most positive PC2 weights among genes with a PC2 jackstraw p-value < 0.0001. For enrichment of transcription factor binding motifs, the mouse gene list was input as-is. For enrichment of ChIP-seq binding peaks, the gene list was converted to the equivalent human genes, as this feature is not available directly for mouse genes. In all cases, the regulatory regions interrogated comprised 500bp upstream of the transcription start site, as annotated in iRegulon. The 7 species option was used in the rankings of transcription factor targets. The ROC threshold for AUC calculation was set at 1%.

Gene set enrichment analysis (GSEA; Subramanian, A. et al. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. 102, 15545 LP - 15550 (2005)) was performed for REACTOME pathways with a minimal size of 15 genes and a maximal size of 1,000 genes (Croft, D. et al. The Reactome pathway knowledgebase. Nucleic Acids Res. 42, D472-D477 (2014)). The input consisted of all genes with a PCI jackstraw p-value < 0.01 and pre-ranked by their PCI weights. The gene list was converted to the equivalent human genes since REACTOME pathways are defined for human genes. GSEA was run using the fgseaMultilevel function in the R package fgsea (Sergushichev, A. A. An algorithm for fast preranked gene set enrichment analysis using cumulative statistic calculation. bioRxiv 60012 (2016). doi:l 0.1101/060012). The output was subset to pathways with a Benjamini-Hochberg corrected p-value < 0.05. The overlap in leading edge genes between each pair of pathways was calculated and the overlap matrix was hierarchically clustered based on Euclidean distance with complete linkage. The clustering guided manual selection of minimally redundant, representative gene sets to include in the visualization. qPCR

Isolated RNA was annealed to random primers (Thermo Fisher Scientific; 48190- 011) for 5 minutes at 70C, then reverse transcribed for 1 hour at 37C using M-MLV reverse transcriptase (Promega, Madison, WI; Ml 705) in the presence of RNase inhibitor (Thermo Fisher Scientific; 10777-019). The resulting cDNA was subjected to qPCR using TaqMan gene expression master mix (Thermo Fisher Scientific; 4369514) and TaqMan gene expression probes (see Key Resources Table for details) on a CFX96 instrument (Bio-Rad, Hercules, CA). Raw amplification cycle data was produced by the accompanying analysis software using default parameters. Cycle differences between tested conditions and the baseline condition were normalized against the reference gene Ubr3 (for mouse samples) or TBP (for human samples), yielding AAC\. Fold-changes were calculated as 2 ^DDa Statistical testing was performed on the underlying AAC\ values.

[Lactate] and [pyruvate] in spent media

Cells were cultured and treated in the same way as for RNA isolation, except the final treatment media volume for myoblasts was 0.5ml/well. IOOmI of the media in each well was collected 2 hours after the start of treatment and immediately frozen at -80C. 30m1 of the spent media was extracted by adding 117m1 acetonitrile and 20m1 water containing ImM Sodium /)/.- Lactate-3 , 3 , 3 -d3 (CDN Isotopes, Pointe-Claire, Quebec, Canada; D6556) and 50mM Sodium pyruvate- ¹³ C ₃ (Sigma; 490717). The samples were vortexed and left on ice for 10 minutes before being centrifuged at 21.1k x g for 10 minutes. Finally, IOOmI of the supernatant was transferred to a glass vial and 10m1 was subjected to LC/MS analysis.

Media samples were separated using an Xbridge amide column (2.1x100mm, 2.5pm particle size) (Waters, Milford, MA), with mobile phase A: 5% acetonitrile, 20 mM ammonium acetate, 0.25% ammonium hydroxide, pH 9.0 and mobile phase B: 100% acetonitrile. The separation gradient was as follows: 85% B for 0.5 minutes, ramp to 35% B for 8.5 minutes, ramp to 2% B for 1 minute, hold for 1 minute, ramp to 85% B for 1.5 minutes, hold for 4.5 minutes. The flow rate was 220pl/min for the first 14.6 minutes, then increased to 420pl/min for the last 3.4 minutes. Mass spectrometry analysis was performed on a QExactive Plus instrument (Thermo Fisher Scientific) with polarity switching mode at 70,000 resolving power (at 200 m/z), a scan range of 70-1,000 m/z and an AGC target of 3E6.

Absolute concentrations for lactate and pyruvate were obtained based on a standard curve and used to report media [lactate]/[pyruvate] ratios and secreted [lactate]. For myoblasts, secreted [lactate] values were adjusted within each (transgene X treatment) combination based on the median [pyruvate] value, to account for variation in cell number. Cell extract metabolite profiling

Doxycycline-inducible myoblasts were seeded at 200,000 cells per dish in 6cm dishes with 4ml/dish of media without selection antibiotics. Approximately 3 hours later, 2ml/dish were removed and replaced with 2ml/dish of media containing doxycycline (final concentration 300ng/ml). 24 hours after doxycycline addition, the media was fully replaced with 3ml/dish fresh media (including doxycycline). 2 hours after the media replenishment, 1.5ml/dish was removed and replaced with 1.5ml/dish of media containing inhibitors at 2x the final concentrations, which were the same as described for RNA isolation.

Beginning at 1 hour after the start of treatments, dishes were individually removed from the incubator, placed on ice, the media was aspirated, cells were washed in lml/dish ice-cold PBS and this was aspirated. Finally, 800pl/dish metabolite extraction buffer was introduced, which consisted of 40% methanol:40% acetonitrile:20% water + 0.1M formic acid (Thermo Fisher Scientific; A117-50) to rapidly quench metabolism ¹⁶¹ . The extraction buffer included 0.5mM Adenosine- ^{I 5} N _J 5 '-Monophosphate (Sigma; 900382), ImM Adenosine- ^|3 C _? 5 ’ -Diphosphate (Toronto Research Chemicals, North York, Ontario, Canada; A281697), 5mM Adenosine- ¹³ Cio 5 '-Triphosphate (Sigma; 710695) and 2.5mM /.-Aspartic acid-1, 4- ¹³ C ₂ (Cambridge Isotope Laboratories, Tewksbury, MA; CLM-4455). The cells were thoroughly scraped, the lysate was transferred to a fresh tube, vortexed briefly and placed on ice. 2 minutes later, 70m1 of 15% w/v ammonium bicarbonate (Sigma; 5.33005) was pipetted into the tube to neutralize the pH, the tube was vortexed and placed at -20C until all extractions were completed. Then, all the tubes were spun at 4C at a speed of 21. Ik x g for 10 minutes to pellet cellular debris. IOOmI of the supernatant was transferred into a glass vial and 10m1 was immediately subjected to LC/MS analysis.

For myotubes, 200,000 cells were seeded per dish in 35mm dishes with 3ml/dish of media. The cells were differentiated for 4 days in low-serum media upon becoming confluent. Treatments were started as described for myoblasts. Metabolite extraction was performed as described for myoblasts except 500pl/dish extraction buffer was used; the extraction buffer contained ImM labeled AMP, 5mM labeled ADP, 25mM labeled ATP and 1 OmM labeled /.-Aspartic acid; and 44m1 ammonium bicarbonate was used to neutralize the pH.

Extracted samples were separated using a ZIC pHILIC column (2. lxl 50mm, 5pm particle size) (EMD Millipore, Burlington, MA), with mobile phase A: 20mM ammonium bicarbonate, and mobile phase B: 100% acetonitrile. The gradient began at 80% B, held for 0.5 minutes, ramped to 20% B for 20 minutes, held for 0.8 minutes, ramped to 80% B for 0.2 minutes, then held for 8.5 minutes. The flow rate was 150pl/minute.

Progenesis QI software (Waters) was used to perform peak picking, peak alignment across samples, deconvolution of adduct peaks, peak intensity integration and intensity normalization across samples. An in-house metabolite retention time library of reference standards was used to identify the peaks based on accurate mass within a 2 ppm tolerance window and retention time within a 0.25 min tolerance window. Metabolite differential abundance analysis upon inhibitor treatment was performed on log2- transformed intensities using the R package limma. P-values are based on a moderated t- statistic.

Absolute concentrations for AMP, ADP, ATP and aspartate were obtained based on a standard curve. The adenylate energy charge was calculated as:

[ATP] + ½ [ADP]

[ATP] + [ADP] ÷ [AMP]

Oxygen consumption rate (OCR) measurements

Doxycycline-inducible myoblasts were seeded at 15,000 cells per well in Seahorse 24- well cell culture microplates with 0.5ml/well of media without selection antibiotics. 24 hours later, the media was replenished including doxycycline at a final concentration of 300ng/ml. 24 hours after doxycycline addition, the media was replaced with 0.5ml/well of the same media formulation except containing 25mM HEPES-KOH instead of sodium bicarbonate. The cells were placed for 1 hour into a non-CCh controlled 37C incubator and then transferred to the XF24 Extracellular Flux Analyzer for OCR measurement. Each measurement was performed over 4 minutes after a 2- minute mix and a 2-minute wait. Inhibitors were introduced into the wells from the XF24 ports in 50m1 of media to the same final concentrations as described above for RNA isolation.

For myotube OCR measurement, myoblasts were seeded at 25,000 cells per well, became confluent within 24 hours and differentiated in low-serum media for 4 days. The rest of the protocol was as described above except the media volume in each well during the assay was 1ml.

Western blotting

Doxycycline-inducible T6NOX cells were seeded at 200,000 cells per dish in 6cm dishes with 4ml/dish of media without selection antibiotics. Approximately 3 hours later, 2ml/dish were removed and replaced with 2ml/dish of fresh media supplemented with either water, to avoid T6NOX expression, or doxycycline at a final concentration of 300ng/ml, to induce expression. Inhibitor treatments were started 24 hours after water or doxycycline addition with complete replacement of the media (still including 300ng/ml doxycycline where needed).

After 6 hours of treatment, each dish was individually removed from the incubator and handled as follows: the media in the dish was aspirated, the dish was briefly washed with 2ml of ice-cold PBS and this was aspirated, IOOmI ice-cold IX Laemmli SDS-sample buffer (Boston BioProducts, Ashland, MA; BP-111R) supplemented with protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA; 5872S) was introduced, the dish was thoroughly scraped and the lysate was transferred into a tube and placed on ice. Because sample buffer is incompatible with standard assays for protein concentration, each experiment also included two spare, untreated dishes that were set up identically but lysed in IOOmI RIPA buffer (Boston BioProducts; BP- 115) supplemented with protease/phosphatase inhibitor cocktail and nuclease (Thermo Fisher Scientific; 88701). When all samples were collected, the tubes were vortexed, sample buffer lysates were boiled for 5 minutes at 95 C and then all samples were frozen at -80C.

When lysates were thawed, protein concentration was determined in the RIPA lysates using the DC protein assay kit (Bio-Rad; 5000112). Volumes of sample buffer lysates corresponding to 30pg of protein were boiled for 5 minutes at 95C, resolved on 4- 12% Tris-Glycine mini gels (Thermo Fisher Scientific; XP04120BOX) and transferred to nitrocellulose membranes (Bio-Rad; 1704159) using a 2-hour wet transfer. Membranes were blocked for 30 minutes with Intercept TBS blocking buffer (LI-COR Biosciences, Lincoln, NE; 927-60001).

To probe the complete ISR pathway on the same membrane, as in Fig. 4L, the membrane was first probed overnight at 4C with an anti-GCN2 antibody (rabbit, polyclonal; Cell Signaling Technology; 3302) at a dilution of 1:500 in Intercept T20 TBS antibody diluent (LI-COR Biosciences; 927-65001). The following day, the membrane was washed 3 times with TBST for 5 minutes each and then incubated for 1 hour at room temperature (RT) with IRDye 800CW goat anti-rabbit IgG secondary antibody (LI-COR Biosciences; 926-32211) at a dilution of 1:10,000 in the antibody diluent. The membrane was again washed 3 times for 5 minutes each and then scanned for infrared signal on the Odyssey imaging system (LI-COR Biosciences). The membrane was then incubated for 15 minutes at RT with Restore western blot stripping buffer (Thermo Fisher Scientific; 21059), blocked for 30 minutes and probed overnight at 4C with an anti-Phospho-GCN2 (Thr899) antibody (rabbit, monoclonal; Abeam; ab75836) at a dilution of 1:500. The following day, the membrane was washed, incubated with anti-rabbit secondary antibody and imaged. After this, the membrane was probed for 2 hours at RT with an anti-ATF4 antibody (rabbit, monoclonal; Cell Signaling Technology; 11815) at a dilution of 1:500 along with an anti-Actin antibody (mouse, monoclonal; Sigma; A4700) at a dilution of 1 :3,000. The membrane was then washed, incubated with both the anti -rabbit secondary antibody and an IRDye 680RD goat anti-mouse IgG secondary antibody (LI-COR Biosciences; 926-68070) at a dilution of 1:10,000 and imaged. The membrane was then probed overnight at 4C with an anti-phospho-eIF2a (Ser51) antibody (rabbit, monoclonal; Cell Signaling Technology; 3597) at a dilution of 1 :500. The next day, the membrane was washed, incubated with anti-rabbit secondary antibody and imaged. Finally, the membrane was probed for 2 hours at RT with an anti-eIF2a antibody (mouse, monoclonal; Cell Signaling Technology; 2103) at a dilution of 1:500, washed, incubated with anti-mouse secondary antibody and imaged. Thus, the exact same bands were probed for both total- and phospho-eIF2a, and the corresponding signals obtained at separate wavelengths. Note, when GCN2 was not examined, the procedure began directly with probing for ATF4 and Actin. Band intensities were quantified using Image Studio Lite (LI-COR Biosciences).

Cell proliferation

Doxycycline-inducible myoblasts were seeded at 5,000 cells per well in 24-well plates with 0.5ml/well of media without selection antibiotics. Approximately 3 hours later, an additional 0.5ml/well of media supplemented with doxycycline was dispensed in each well, such that the final concentration was 300ng/ml. Inhibitor treatments were started 24 hours later with complete replacement of the media (still including 300ng/ml doxycycline). Baseline counts for each condition were collected immediately after the start of treatment from separate wells. Final counts were collected 24 hours after the start of treatment. Counts were obtained using a Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter, Brea, CA). The proliferative rate was calculated as the number of cell doublings between final and baseline counts.

Example 1. Complex I inhibition in myoblasts activates the eIF2a kinase GCN2 due to an asparagine deficiency

To investigate the functional consequence of complex I inhibition that triggered the ISR, as the response was most effectively blunted by oxidizing cytosolic NADH, experiments were performed to determine if glycolysis was inhibited by elevated cytosolic NADH/NAD ⁺ , which would limit the cells’ ability to defend their adenylate energy charge in the absence of OXPHOS. Experiments were performed utilizing proliferating C2C12 mouse myoblasts, which can be induced to exit the cell cycle and then terminally differentiate into myotubes (Yaffe, D. & Saxel, O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270, 725-727 (1977); Blau, H. M. et al. Plasticity of the differentiated state. Science (80- ). 230, 758 LP - 766 (1985)). The myoblasts were acutely treated with inhibitors of complex I (piericidin), complex PI (antimycin) or ATP synthase (oligomycin) as shown in FIGS. 1A and 2A media lacking pyruvate and uridine, to avoid masking known metabolic vulnerabilities of ETC-compromised cells75,76 (Methods; Morais, R., Gregoire, M., Jeannotte, L. & Gravel, D. Chick embryo cells rendered respiration- deficient by chloramphenicol and ethidium bromide are auxotrophic for pyrimidines. Biochem. Biophys. Res. Commun. 94, 71-77 (1980; King, M. P. & Attardi, G. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Sci. 246, 500-503 (1989)). Using lactate secretion as a proxy for glycolytic flux, experiments showed that

Lb NOX did modestly stimulate glycolysis (FIGS. 1A and 2A). However, this did not translate into an improved energy charge, and in fact, the opposite was true (FIGS. IB and 2B).

Experiments were then performed to investigate if elevated mitochondrial and cytosolic NADH/NAD ⁺ depleted a critical nutrient, which then triggered the ISR.

Experiments were performed for intracellular metabolite profiling on control cells or Lb NOX cells acutely treated with piericidin, a complex I inhibitor (FIGS. 1C, 2C). The amino acids aspartate and its derivative asparagine, alongside related TCA cycle intermediates, emerged among the top metabolites depleted by piericidin in a manner responsive to oxidizing the cytosol. This result is consistent with the contribution to aspartate synthesis of the mitochondrial and cytosolic NAD ⁺ -linked malate dehydrogenase (MDH) reactions that yield its precursor, oxaloacetate (Birsoy, K. et al. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell 162, 540-551 (2015); Sullivan, L. B. et al. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells. Cell 162, 552-563 (2015); Chen, W. W., Fremkman, E., Wang, T., Birsoy, K. & Sabatim, D. M. Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism. Cell 166, 1324-1337.ell (2016)). Absolute quantification confirmed a -16- fold drop in aspartate within 1 hour of piericidin treatment in control cells compared with only a 2.5-fold drop in LbNOX cells (FIGS. ID and 2D).

Amino acid deficiency is a canonical ISR trigger through the eIF2a kinase GCN2, which is activated due to uncharged tRNA (Hinnebusch, A. G. Evidence for translational regulation of the activator of general amino acid control in yeast. Proc. Natl. Acad. Sci.

U. S. A. 81, 6442-6446 (1984); Dever, T. E. et al. Phosphorylation of initiation factor 2 alpha by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell 68, 585-596 (1992); Wek, S. A, Zhu, S. & Wek, R. C. The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids. Mol.

Cell. Biol. 15, 4497-4506 (1995); Berlanga, J. J., Santoyo, J. & De Haro, C. Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2alpha kinase. Eur. J. Biochem. 265, 754-762 (1999); Zhang, P. etal. The GCN2 eIF2a Kinase Is Required for Adaptation to Amino Acid Deprivation in Mice. Mol. Cell. Biol. 22, 6681 LP - 6688 (2002); Castilho, B. A. et al. Keeping the eIF2 alpha kinase Gcn2 in check. Biochim. Biophys. Acta - Mol. Cell Res. 1843, 1948-1968 (2014); Inglis, A. J. et al. Activation of GCN2 by the ribosomal P-stalk. Proc. Natl. Acad. Sci. 116, 4946 LP - 4954 (2019); Harding, H. P. et al. The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells. Elife 8, e50149 (2019)). If amino acid deficiency triggers the ISR following complex I inhibition in myoblasts, then correcting it should abrogate the response. Experiments were conducted by adding aspartate since the magnitude of its depletion was greater and its addition also allowed the cells to partially restore asparagine (FIGS. IE and 2E). Experiments showed that lOmM aspartate significantly attenuated ISR activation upon complex I inhibition, though not to the same extent as Lb NOX, as LbNOX expression was sufficient to almost completely ablated ISR activation, represented by the Ddit3 transcript, upon complex I inhibition (FIGS. IF and 21). This concentration was required since most mammalian cells do not efficiently take up aspartate (Birsoy, K. et al. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell 162, 540-551 (2015)). Importantly, aspartate addition did not indirectly oxidize cytosolic NADH/NAD ⁺ (FIG. 1G) and was insufficient to appreciably restore cell proliferation (FIG. 1H). Aspartate also attenuated ISR-r elated gene expression in C2C12 myoblasts engineered with a genetic defect in complex I (afai, S. B. et al. Natural Product Screening Reveals Naphthoquinone Complex I Bypass Factors. PLoS One 11, e0162686 (2016)), where reductive stress was induced by pyruvate withdrawal (FIG. II), as well as in piericidin- treated primary human myoblasts (FIG. 1 J) and primary mouse embryonic fibroblasts (FIG. 2J).

To test whether ISR activation by complex I inhibition involved GCN2 activity, experiments were performed using a small-molecule, ATP-competitive inhibitor of the kinase (GCN2iB) (Nakamura, A. et al. Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response. Proc. Natl. Acad. Sci. (2018)). Experiments showed that co-treatment with GCN2iB significantly suppressed ISR gene expression in response to complex I inhibition but had no effect on ISR activation in response to endoplasmic reticulum (ER) stress induced by tunicamycin (FIG. IK). The converse was true when the ER-resident eIF2a kinase, PERK was inhibited (FIG. 2K) (Harding, H. P., Zhang, Y. & Ron, D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397, 271 (1999);

Axten, J. M. et al. Discovery of GSK2656157: An Optimized PERK Inhibitor Selected for Preclinical Development. ACS Med. Chem. Lett. 4, 964-968 (2013)).

Finally, experiments were performed to examine how GCN2 sensed the amino acid deficiency. Using autophosphorylation at Threonine-898 as a readout of kinase activation (Romano, P. R. et al. Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2. Mol. Cell. Biol. 18, 2282-2297 (1998)), 6 hours of complex I inhibition resulted in marked GCN2 activation, which was partially attenuated by supplementing aspartate (FIG. 1L). Strikingly, asparagine alone abrogated GCN2 activation with no discernible additive effect of aspartate, suggesting the drop in asparagine was the most proximal activating signal. As expected, ZANOX expression, which enables synthesis of both amino acids during complex I inhibition, effectively prevented GCN2 activation, while GCN2iB suppressed autophosphorylation both at baseline and upon piericidin treatment.

The effects of the same interventions on eIF2a phosphorylation, the event downstream of GCN2 activation, were notably less pronounced, especially given the narrow dynamic range of this signal. Experiments showed clear attenuation of eIF2a phosphorylation upon piericidin treatment in the case of LbNOX expression, though the rescue was still incomplete (FIGS. 1L and 2L). ATF4 protein levels largely tracked the degree of GCN2 activation (FIG. 1L) and this was reflected at the level of transcriptional ISR targets at the same time-point (FIG. 1M). GCN2iB completely prevented ATF4 protein accumulation despite some residual eIF2a phosphorylation (FIG. 1L), suggesting GCN2 was required to attain the threshold phosphorylation level that elicits ATF4 translation. Collectively, these experiments indicate that in myoblasts, the rise in the NADH/NAD ⁺ ratio is a major driver of ISR-related gene expression following complex I inhibition, as it limits biosynthesis of aspartate, depletes asparagine, and activates GCN2

(FIG. IN)

These experiments interrogated the ISR response in two cell states, proliferating myoblasts and differentiated myotubes, by employing a battery of small -molecule inhibitors of mitochondria in combination with genetic and chemical tools for selectively buffering their effects.

The present results in proliferating myoblasts mechanistically relate two recently recognized consequences of ETC dysfunction: a defect in aspartate biosynthesis and ISR activation. Aspartate is largely produced from TCA cycle-derived oxaloacetate (OAA) but elevated mitochondrial NADH/NAD ⁺ blocks the oxidative TCA cycle when complex I is inhibited (Chen, W. W., Freinkman, E., Wang, T., Birsoy, K. & Sabatini, D. M. Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism. Cell 166, 1324-1337.el 1 (2016)). OAA must then be obtained by other means, which in myoblasts depended on correcting the concomitant rise in cytosolic NADH/NAD ⁺ , thus tilting the MDH1 reaction toward OAA (Birsoy, K. et al. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell 162, 540-551 (2015); Sullivan, L. B. et al. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells. Cell 162, 552-563 (2015)). Absent this, experiments showed the aspartate deficiency triggered the ISR, at least initially due to depletion of its derivative asparagine that was sensed by the eIF2a kinase GCN2 (FIGS. 1C-1M). The cytosolic aspartate and asparagine biosynthesis enzymes are both ISR targets (Fujita, Y. et al. CHOP (C/EBP homologous protein) and ASNS (asparagine synthetase) induction in cybrid cells harboring MELAS and NARP mitochondrial DNA mutations. Mitochondrion 7, 80-88 (2007); Harding, H. P. et al. An Integrated Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress. Mol. Cell 11, 619-633 (2003); Han, J. et al. ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol 15, 481-490 (2013)), suggesting a homeostatic logic to the response. Supplementation with asparagine alone, which cannot be converted to aspartate in most mammalian cells (Pavlova, N. N. et al. As Extracellular Glutamine Levels Decline, Asparagine Becomes an Essential Amino Acid. Cell Metab. 27, 428-438. e5 (2018)), was sufficient to abrogate GCN2 activation (Fig. 1L). That GCN2 did not directly sense the profound aspartate depletion seems surprising. This result may be explained by the fact that while the K _M values for aspartate and asparagine of their respective cytosolic tRNA synthetases are similar (~15-30mM) (Bour, T. et al. Plasmodial Aspartyl-tRNA Synthetases and Peculiarities in Plasmodium falciparum. J. Biol. Chem. 284, 18893- 18903 (2009); Messmer, M. et al. Peculiar inhibition of human mitochondrial aspartyl- tRNA synthetase by adenylate analogs. Biochimie 91, 596-603 (2009); Andrulis, I. L., Chiang, C. S., Arfin, S. M., Miner, T. A. & Hatfield, G. W. Biochemical characterization of a mutant asparaginyl-tRNA synthetase from Chinese hamster ovary cells. J. Biol. Chem. 253, 58-62 (1978)), the cellular concentrations of these amino acids are orders of magnitude apart. Aspartate is estimated at ~5-10mM whereas asparagine is much closer to the K _M , at ~100-200mM (Chen, W. W., Freinkman, E., Wang, T., Birsoy, K. &

Sabatini, D. M. Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism. Cell 166, 1324-1337.el 1 (2016); Park, J. O. et al. Metabolite concentrations, fluxes and free energies imply efficient enzyme usage. Nat. Chem. Biol. 12, 482 (2016)). Thus, even a large fold-change drop in aspartate would not compromise its cognate tRNA charging but a smaller drop in asparagine would, activating GCN2. The K _M for aspartate of asparagine synthetase is much higher (~lmM) (Horowitz, B. & Meister, A. Glutamine-dependent Asparagine Synthetase from Leukemia Cells: Chloride Dependence, Mechanism of Action, and Inhibition. J. Biol. Chem. 247, 6708-6719 (1972); Ciustea, M., Gutierrez, J. A., Abbatiello, S. E., Eyler, J. R. & Richards, N. G. J. Efficient expression, purification, and characterization of C-terminally tagged, recombinant human asparagine synthetase. Arch. Biochem. Biophys. 440, 18-27 (2005)), aspartate deficiency readily depletes asparagine (Fig. IE). Based on our experiments, the asparagine tRNA charging is a sensitive sensor of NADH reductive stress in proliferating cells.

Experiments showed that oxidizing cytosolic NADH/NAD ⁺ using Lb NOX attenuated the ISR better than aspartate or asparagine (FIGS. IF, 1L, 1M), suggesting eIF2a kinases may be attuned to additional consequences of reductive stress. Moreover, ZANOX cells still showed residual ISR activation (FIGS. 1L and 2L), likely due to reasons beyond reductive stress. However, these results suggest threshold and non-linear effects along the ISR pathway so that NADH reductive stress was the primary driver of ISR-related gene expression.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.