IACS-010759

An inhibitor of oxidative phosphorylation exploits cancer vulnerability
Jennifer R. Molina1,2,16, Yuting Sun1,2,16, Marina Protopopova1,2, Sonal Gera1,2, Madhavi Bandi1,2, Christopher Bristow1,2, Timothy McAfoos1, Pietro Morlacchi1,15, Jeffrey Ackroyd 3,
Ahmed-Noor A. Agip4, Gheath Al-Atrash5, John Asara6, Jennifer Bardenhagen1, Caroline C. Carrillo7, Christopher Carroll1, Edward Chang1,2, Stefan Ciurea5, Jason B. Cross1, Barbara Czako1,
Angela Deem1,2, Naval Daver8, John Frederick de Groot7, Jian-Wen Dong7, Ningping Feng1,2, Guang Gao1,2, Jason Gay1,2, Mary Geck Do1, Jennifer Greer1, Virginia Giuliani1,2, Jing Han1,2,
Lina Han8, Verlene K. Henry7, Judy Hirst4, Sha Huang1, Yongying Jiang1, Zhijun Kang1, Tin Khor1,2, Sergej Konoplev9, Yu-Hsi Lin 3, Gang Liu1, Alessia Lodi10, Timothy Lofton1, Helen Ma8,
Mikhila Mahendra1,2, Polina Matre8, Robert Mullinax1,2, Michael Peoples1,2, Alessia Petrocchi1, Jaime Rodriguez-Canale11, Riccardo Serreli4, Thomas Shi1,2, Melinda Smith1,2, Yoko Tabe8,12,
Jay Theroff1, Stefano Tiziani10, Quanyun Xu1, Qi Zhang8, Florian Muller 3, Ronald A. DePinho13, Carlo Toniatti1,2, Giulio F. Draetta 1,2,14, Timothy P. Heffernan1,2, Marina Konopleva8, Philip Jones1,16,
M. Emilia Di Francesco1,16 and Joseph R. Marszalek1,2,16*
Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncol- ogy drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of com- plex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

etabolic reprogramming is a well-appreciated hallmark of cancer, and there has been extensive drug discovery research in this area. Coordinated upregulation of glycoly-
sis, known as the Warburg effect1, is a phenomenon that arises as tumor cells adapt to increased demands for energy and biomass pro- duction. Elevated glycolysis is currently being clinically exploited using positron emission tomography with 2-deoxy-2-fluoro-D-glu- cose (FDG-PET) to detect metabolically active tumors2; it is also being therapeutically exploited through the development of inhibi- tors of enzymes essential for glucose metabolism3. Recent reports

emphasize that, in addition to a strong dependence on glycolysis, many tumors or cancer cell subpopulations rely on OXPHOS4 for bioenergetic5–15 and biosynthetic processes16,17. Biguanides, such as metformin, have been evaluated for the treatment of diabetes and metabolic disorders, providing rationale that targeting OXPHOS for clinical benefit can be done safely. However, metformin and other drugs targeting oxidative metabolism possess pharmacologi- cal limitations, including inadequate potency (i.e., biguanides)18,19, transport-mediated accumulation (i.e., OCT1 for metformin)20, ‘off-target’ pharmacology (i.e., rotenone)21, or lack of a suitable

1Institute for Applied Cancer Science, University of Texas MD Anderson Cancer Center, Houston, TX, USA. 2Center for Co-Clinical Trials, University of Texas MD Anderson Cancer Center, Houston, TX, USA. 3Department of Cancer Imaging Systems, University of Texas MD Cancer Center, Houston, TX, USA. 4Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Wellcome Trust/MRC Building, Cambridge Biomedical Campus, Cambridge, UK. 5Department of Stem Cell Transplantation and Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, TX, USA. 6Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. 7Department of Neuro-Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA. 8Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX, USA. 9Department of Hematopathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA. 10Department of Nutritional Sciences, University of Texas
at Austin, Austin, TX, USA. 11Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA. 12Department of Next Generation Hematology Laboratory Medicine, Department of Laboratory Medicine, Juntendo University School of Medicine, Tokyo, Japan. 13Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA. 14Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, TX, USA. 15Present address: Agilent Technologies Inc., Lexington, MA, USA. 16These authors contributed equally: Jennifer R. Molina, Yuting Sun, Philip Jones, M. Emilia Di Francesco, Joseph R. Marszalek. *e-mail: [email protected]

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Fig. 1 | iACS-010759 is a potent inhibitor of mitochondria complex i. a, The structure of IACS-010759. b, An illustration of the mitochondria electron transport chain. c, The OCR of permeabilized H460 cells grown in medium supplemented with pyruvate and malate to measure complex I activity that were then treated with IACS-010759 (Injection 1) and 10 mM succinate (Injection 2) (mean from n = 2 cultures plotted, performed one time). d, OCR of H460 cells engineered to ectopically express Saccharomyces cerevisiae NDI1 (complex I equivalent) or GFP grown in Seahorse medium (mean from n = 6 cultures plotted, data shown are from 1 representative experiment out of 2 experiments) and relative viability (cell confluence) in galactose medium (mean from n = 2 cultures plotted, performed one time) following treatment with either DMSO or 14 nM IACS-010759 for 1 h (for OCR) or 72 h (for viability).
***P < 0.0001 by two-sided Student's t-test. e, Complex I isolated from mouse mitochondria was treated with 60 nM IACS-010759 to measure effects on ubiquinone reduction (decylubiquinone) (mean from n = 9 technical replicates plotted), flavin site activity as determined via 3-acetylpyridine adenine dinucleotide (APAD+) expression (mean from n = 3 technical replicates plotted), and H2O2 production (mean from n = 3 technical replicates plotted).
Results with IACS-010759 are normalized to those from DMSO treatment. f, Dose-response curves for a H292-Parental cell line and an H292 clone resistant to IACS-010759 (H292-RES) (mean from n = 2 cultures plotted, performed 1 time). g, Sanger sequencing chromatogram of PCR-amplified MT- ND1 DNA from an H292-RES cell resistant to IACS-010759. h, The structure of complex I with the location of the ND1 subunit highlighted in red. i, The location of the L55F mutation relative to the proposed ubiquinone-binding channel (shown as a surface) in complex I. Residues and structures that form the binding site are shown; the redox-active ubiquinone headgroup moves through the channel and is reduced next to cluster N2. h and i were created using the highly homologous structure of bovine complex I (5LC5.pdb)29. j, H460 cells were treated with IACS-010759, and OCR (mean from n = 6 cultures plotted) and viability (mean from n = 2 cultures plotted) were measured after 1 or 72 h, respectively. c, d, and j were repeated once; e and f were repeated ≥2 times, and each replicate had comparable results.

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pharmacokinetic (PK) profile (i.e., oligomycin), that restrict their use as oncology therapeutics. Here, we report the discovery of IACS-010759 (Fig. 1a), a clinical-grade, highly potent and selective small-molecule inhibitor of complex I of the mitochondrial elec- tron transport chain (ETC; Fig. 1b) and its mechanism of antitumor activity in acute myeloid leukemia (AML) and genetically defined subsets of glioblastoma and neuroblastoma.
Results
IACS-010759 was identified through an extensive medicinal chem- istry campaign of lead optimization initially seeded with known modulators of Hypoxia-inducible factor 1-α (HIF-1α) that act via inhibition of OXPHOS22–24. Consistent with IACS-010759 acting solely at complex I, treatment of detergent-permeabilized cells with IACS-010759 in medium supplemented with pyruvate and malate (to generate NADH for use by complex I) resulted in an attenuated oxygen consumption rate (OCR; Fig. 1c), whereas the OCR was not affected by treatment with IACS-010759 when medium was supplemented with succinate to feed complex II, thus bypassing the requirement for complex I function. This mechanism was further
supported by the finding that ectopic expression of Saccharomyces cerevisiae NDI1, the yeast complex I ortholog25,26, completely restored cell viability and OCR to baseline levels in the presence of IACS-010759 (Fig. 1d and Supplementary Fig. 1a,b). Similar results were obtained with rotenone, a well-established but less-specific inhibitor of complex I (Supplementary Fig. 1c); however, only a partial rescue of viability was observed, likely because of off-target toxicity (Supplementary Figs. 2b,d,g,i,j,l). Further, IACS-010759 treatment of complex I isolated from mouse mitochondria resulted in decreased catalysis owing to inhibition at the ubiquinone-bind- ing site versus the flavin site, with no effect on H2O2 generation (Fig. 1e). To define the interaction of IACS-010759 with complex I, clones with reduced sensitivity to IACS-010759 were generated by growing cells for 12 weeks in the presence of increasing amounts of IACS-010759 in galactose medium, wherein cells were rendered dependent on OXPHOS for survival27,28. Half-maximal inhibitory concentration (IC50) values for resistant clones ranged from 3.7 to 74 nM as compared to 1.1 nM for parental cells (Supplementary Fig. 1d). In contrast to IACS-010759, sensitivity to rotenone was consistent in most clones (Supplementary Fig. 1d), indicating that the reduced sensitivity to IACS-010759 was not likely due to a gen- eral decrease in complex I dependence (see Fig. 1f for representa- tive dose-response curves for clone DC4 and the parental cell line). Total and mitochondrial RNA from 12 clones were subjected to next-generation sequencing, which confirmed that 9 of the clones contained an identical nucleotide change (m.C3469T) in their mitochondrial DNA (mtDNA) yielding an amino acid substitution (p.L55F) in the ND1 subunit of complex I (Fig. 1g). This residue resides close to the entrance of the proposed ubiquinone-10 bind- ing channel of complex 1 (Fig. 1h,i)29, but numerous attempts to ectopically express mutant ND1-L55F through multiple strategies were unsuccessful owing to the technical challenges of ectopically expressing proteins encoded by mtDNA. Taken together, these data are consistent with IACS-010759 binding in or at the entrance to the ubiquinone channel and thus blocking ubiquinone binding or function to inhibit complex I activity.
To correlate phenotypic response directly with OXPHOS inhi- bition, the effect of IACS-010759 on the OCR of cells grown in Seahorse medium was compared to the viability of cells grown in galactose-containing medium. IACS-010759 robustly inhibited both OCR and galactose-dependent cell viability and had nearly identical IC50 values of 1.4 nM in both assays (Fig. 1j). By comparison, IC50 values for rotenone were 0.24 nM and 0.87 nM in assays for OCR and galactose-dependent cell viability, respectively (Supplementary Fig. 1e). IC50 values in these assays were similar for both IACS- 010759 and for rotenone across several human cell line models

(Supplementary Table 1). To examine the potency of IACS-010759 across species that are widely used for preclinical safety analysis, the response of representative cell lines from mouse, rat, dog, and cyno- molgus monkey to the inhibitor was assessed using the highly quan- titative galactose growth assay. IACS-010759 was similarly active in mouse (average IC50 = 5.6 nM), rat (IC50 = 12.2 nM), and cynomol- gus monkey (IC50 = 8.7 nM), thus making them appropriate pre- clinical models for further safety studies (Supplementary Table 1). In contrast, IACS-010759 was minimally active in canine cell lines (IC50 = 180–360 nM). Rotenone potency was very similar across all species (Supplementary Table 1).
The effect of IACS-010759 on cell growth and viability was evalu- ated across a panel of cancer cell lines and normal diploid cells using standard culture medium containing glucose and glutamine, which provides multiple energy sources to the cells. IACS-010759 yielded a maximal reduction of growth of > 50% in the majority of cancer cell lines (24 of 30 pancreatic (PDAC), 19 of 20 ovarian, 13 of 16 tri- ple-negative breast (TNBC), 8 of 10 non-small-cell lung (NSCLC)) and a subset (11of 30 PDAC, 10 of 20 ovarian, 5 of 16 TNBC, 2 of 10 NSCLC) exhibited > 100% growth inhibition (Supplementary Fig. 3a–d). All diploid cell lines were insensitive to IACS-010759 and exhibited little or no growth inhibition (Supplementary Fig. 2a,c,g,i,k), whereas rotenone exposure reduced viability in these models, consistent with rotenone possessing nonspecific off-target toxicities (Supplementary Fig. 2b,d,h,j,l). These data establish dif- ferential sensitivity of normal and cancer cells to OXPHOS inhibi- tion by IACS-010759.
In addition to its direct antigrowth effects, OXPHOS inhibition has been reported to reduce hypoxia and HIF pathway activity4,22,24,30. In accordance with this notion, exposure of cell line spheres to IACS-010759 eliminated hypoxia, most likely as a consequence of increased intracellular oxygen (Supplementary Fig. 4a,b). This coin- cided with decreased HIF pathway activity (Supplementary Fig. 4c) via oxygen-, prolyl hydroxylase– and VHL-dependent degradation of HIF-1α (Supplementary Fig. 4d–h).
IACS-010759 targets glycolysis-deficient tumor cells. Glycolysis is under negative control by OXPHOS and is induced by tricarbox- ylic acid cycle (TCA)-mediated allosteric inhibition of glycolytic enzymes (the ‘Pasteur effect’31); therefore, genetic or pharmacologi- cal OXPHOS inhibition should result in compensatory upregulation of glycolysis to maintain ATP levels and redox balance, resulting in modest antiproliferation effects32. Thus, we hypothesized that tumor cells with a reduced capacity for compensatory glycolysis would be more sensitive to OXPHOS inhibition.
As previously reported, a subpopulation of brain tumor cell lines with homozygous deletion of Enolase 1 (ENO1) are glycoly- sis-deficient owing to a > 90% reduction of cellular enolase enzy- matic activity33,34. Consistent with glycolysis inhibition at the step of enolase enymatic activity, the levels of glycolysis metabolites upstream of enolase were elevated in ENO1-null cell lines, such as D423 and Gli56, whereas metabolites downstream of enolase were reduced relative to glioblastoma multiforme (GBM) cell lines with a heterozygous ENO1 mutation or wild-type ENO1 (Supplementary Fig. 5a). Ectopic expression of wild-type ENO1 in Gli56 and D423 (Supplementary Fig. 5b) robustly increased lactate production, con- sistent with restoration of glycolysis (Fig. 2a and Supplementary Fig. 5c). Further, there was minimal lactate production upon treatment of parental Gli56 cells with IACS-010759, whereas the baseline level and induction of lactate production were substantially increased in Gli56 cells ectopically expressing ENO1 (Supplementary Fig. 5d). Similar to ENO1-null cells, phosphoglycerate dehydrogenases (PGD)-null cells, such as the NB1 cell line, are glycolysis-defi- cient, as these cells accumulate 6-phosphogluconate, an allosteric inhibitor of glucose-6-phosphate isomerase35–37. We evaluated the metabolic profile of NB1 cells by determining the extracellular

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Fig. 2 | Glycolysis-deficient and AML tumor cells are sensitive to OXPHOS inhibition. a, Extracellular lactate levels (indicating glycolysis endpoint) in NB1 (PGD (6PGD)–/–), Gli56 (ENO1–/–), and D423 (ENO1–/–) cells and their counterparts that were each engineered to ectopically express either PGD (NB1) or ENO1 (Gli56 and D423). The mean and individual data points for n = 6 cultures for NB1 and D423 and n = 3 cultures for Gli56 are plotted. For each cell line, the vector control was defined as 1. b, Results from Seahorse analysis measuring the ratio of glycolysis (proton production rate) to OXPHOS (OCR) in NB1 cells and H460 cells (mean and individual data points for n =8 cultures are plotted). c, Percent change in viability of the same matched-pair cell lines as in a that were cultured for 3 d in medium containing 100 nM IACS-010759 (mean and individual data points for n = 2 cultures for NB1 and D423; n = 3 cultures for Gli56 are plotted) relative to cells treated with DMSO. d, Percentage of annexin V+ NB1, Gli56, and D423 cells cultured in medium containing DMSO or 100 nM IACS-010759 for 3 d (mean and individual data points for n = 3 cultures are plotted). e, Viability of AML cell lines treated with indicated concentrations of IACS-010759 for 3–7 d. Data represent mean ± s.d. from n = 3 cultures. f,g, Viability of primary AML (f) or normal bone marrow (g) samples treated ex vivo with 0, 10, 30, or 100 nM IACS-010759 for 4 or 5 d. Data from IACS-010759-treated samples were normalized to corresponding DMSO-treated controls. h, Percentage of apoptotic cells (propidium iodide (PI)+ and annexin V+) for each AML cell line (mean and individual data points for n = 2 cultures are plotted; a replicate study was performed only for OCI-AML3 with comparable results) after culture for 72 h in medium containing DMSO or 123 nM IACS-010759. i,j, Percentage of apoptotic (annexin V+) cells in primary AML (i) or normal bone marrow (j) cells after culture for 4
or 5 d in medium containing DMSO or IACS-010759. For f, g, i, and j, each point is the mean of three cultures from a single patient sample analyzed in
one experiment; the bars represent the mean value for all of the samples. Patient sample (UPIN) characteristics are included in Supplementary Table 2.
***P < 0.0001, **P < 0.001,*P < 0.05 by two-sided Student's t-test. Experiments in a–e were performed twice with comparable results.

acidification rate (ECAR):OCR ratio (Fig. 2b), and we observed both an extremely low glycolysis:OXPHOS ratio and restored lac- tate production following ectopic expression of PGD (Fig. 2a).

Furthermore, IACS-010759 treatment resulted in > 70% reduction in viability and a two- to fivefold increase in apoptosis in NB-1, Gli56, or D423 cells, whereas ectopic expression of either ENO1 or

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PGD (Fig. 2c,d and Supplementary Fig. 6a–c) substantially attenu- ated the response. Similarly, the viability of ENO1−/− or ENO1+/− glioma sphere–derived cell lines was reduced upon IACS-010759 exposure (Supplementary Fig. 6d–f). These data are consistent with our hypothesis that glycolysis deficiency renders cell lines highly sensitive to OXPHOS inhibitors, such as IACS-010759.
AML tumor cells are sensitive to IACS-010759. Previous reports have suggested that leukemia cells are highly dependent on OXP HOS6,7,11–13,38–40, warranting evaluation of IACS-010759 in AML models. Established AML cell lines were exposed to a range of IACS-010759 concentrations for 3–7 d, resulting in reduced viability with half-maximal effective concentration (EC50) values of < 3 nM (Fig. 2e and Supplementary Fig. 7a). Although the inflection point of response (at the EC50 point on the dose-response curve) was equivalent across cell lines (Supplementary Fig. 7a), the effect of IACS-010759 on viability varied, with MOLM-13 (in which FLT3 has an internal tandem duplication (ITD) mutation) being rela- tively insensitive (Fig. 2e). Treatment with IACS-010759 uniformly reduced OCR, indicating that the differential effects on viability are not due to the lack of OXPHOS inhibition. This is consistent with our finding of similar EC50 values across cell lines (Supplementary Fig. 7b). In most cell lines, IACS-010759 treatment modestly increased apoptosis by up to twofold (Fig. 2h). Cells responded more robustly to IACS-010759 treatment when glucose availabil- ity was restricted, whereas glutamine restriction did not enhance the response (Supplementary Fig. 7c,d), which is consistent with glucose utilization being the primary compensatory response to OXPHOS inhibition. Interestingly, MOLM-13 had the highest base- line OCR and largest glycolytic reserve of the evaluated cell lines, and both of these factors may contribute to its relative insensitivity to IACS-010759 (Supplementary Fig. 7b). This might represent a potential mechanism underlying treatment resistance and is con- sistent with results reported for OXPHOS inhibition in melanoma models41. We extended our studies to primary AML blasts isolated from peripheral blood of patients with relapsed/refractory AML by treating blasts ex vivo with multiple concentrations of IACS-010759 for up to 5 d. In nearly all primary AML samples, but not in non- transformed mononuclear cells isolated from normal bone marrow, IACS-010759 reduced viability and induced apoptosis (Fig. 2f,g,i,j and Supplementary Table 2), consistent with our observations in AML cell lines and supporting the existence of a therapeutic win- dow in which IACS-010759 can selectively target leukemic versus normal hematopoietic cells. The patient-derived xenograft (PDX- 4030094) also responded robustly to IACS-010759 (approximate IC50 < 1.5 nM) upon ex vivo treatment (Supplementary Fig. 7e). Further, we evaluated IACS-010759 response in cell lines established from two previously described murine AML models genetically engineered to ectopically coexpress the clinically relevant translo- cation between KMT2A (also known as mixed-lineage leukemia, MLL) and MLLT1 (ENL) in a KRASG12D background with or without TP53 deletion42. Viability and OCR were equally inhibited in both cell lines (Supplementary Fig. 7f,g) with approximate IC50 values of 20 nM and 55 nM, respectively. These results, taken together with those from ENO1- and PGD-null GBM models, define two biologi- cal tumor contexts with striking sensitivity to IACS-010759.
IACS-010759 safely targets glycolysis-deficient tumors in vivo. To determine whether the observed in vitro and ex vivo effects pre- dicted in vivo responses in preclinical models at tolerated doses, we evaluated IACS-010759 in mouse models of glioblastoma and/ or neuroblastoma and AML. The PK profile of IACS-010759 was determined in mice following intravenous (0.3 mg per kg body weight (mg/kg)) and oral (1 mg/kg) administration (Supplementary Fig. 8a). IACS-010759 was characterized by low plasma clearance with a high volume of distribution, resulting in a prolonged terminal

half-life (>24 h) of IACS-010759 with sustained levels of compound in the plasma following oral dosing. Conversion of IACS-010759 free base into the corresponding HCl salt (IACS-010759.HCl) resulted in a ~10-fold increase in plasmatic exposure after oral dosing (Supplementary Fig. 8b). We did not observe changes in blood glucose level with single or repeated doses of IACS-010759 (Supplementary Fig. 8c), which is consistent with previous reports for metformin43. However, at 2 h after the first or fifth dose, plasma insulin levels transiently decreased and returned to control levels by 24 h postdose (Supplementary Fig. 8d).
To assess the tolerability and antitumor activity of IACS-010759 in a glycolysis-deficient context, mice bearing NB-1 (PGD-null) subcutaneous xenografts received daily oral doses of 0, 5, 10, or 25 mg/kg IACS-010759 free base per day for 21 d. Treatment with IACS-010759 at the 5 or 10 mg/kg dose resulted in tumor regres- sion with minimal body weight loss (Fig. 3a and Supplementary Fig. 8e), whereas IACS-010759 at the 25 mg/kg dose was not tolerated, and body weight loss (Supplementary Fig. 8e), lethargy and hypo- thermia were observed. At the tolerated doses, IACS-010759 was equally effective and well tolerated when administered in various intermittent dosing schedules (Supplementary Fig. 8f,g), provid- ing flexibility for dosing. Transient, intermittent treatment of Gli56 intracranial tumors with 5 mg/kg IACS-010759.HCl using a 5 d on and 2 d off (5 on/2 off) schedule for 4 weeks reduced tumor size as measured through magnetic resonance imaging (MRI) (Fig. 3b,c) and extended median survival from 84 d to 130 d (Supplementary Fig. 9a). Tumor hypoxia was eliminated in Gli56 and D423 intra- cranial tumors after two daily doses of 5 mg/kg IACS-010759. HCl (Supplementary Fig. 9b,c), whereas tumor hypoxia persisted in vehicle-treated control animals, supporting the occurrence of OXPHOS inhibition within the tumor. We also observed reduc- tions of 84% and 43% in the number of cells positive for the mitotic marker phosphorylated histone H3 in IACS-010759.HCl-treated Gli56 and D423 tumors, respectively, compared to control animals (Supplementary Fig. 9d–g), which is indicative of reduced tumor cell proliferation in the inhibitor-treated animals. Collectively these data provide evidence of profound antitumor activity of IACS- 010759 at well-tolerated doses in the context of glycolysis-deficient brain tumors.
Immunohistochemistry (IHC) analysis of tumor samples from 92 patients treated at The University of Texas MD Anderson Cancer Center was conducted to determine the prevalence of ENO1- and PGD-null GBM with the aim of defining a potential path for clini- cal treatment. Eight tumors (8.6%) were either unambiguously ENO1-null (3.3%) or showed very low ENO1 staining (five tumors (5.3%)), which represented a slightly higher percentage of ENO1- null tumors compared to reports from The Cancer Genome Atlas (TCGA) sequencing data (Fig. 3d and Supplementary Fig. 9h). Whether tumors with weak staining represent a nonspecific back- ground or very low expression of ENO1 could not be ascertained and will require independent validation with orthogonal assays. Regardless, a clinically relevant population with at least 3.3% of GBM tumors being ENO1-null was clearly identified. It should be noted that the strong, residual signal represents ENO1 expression in nontumor stromal cells, such as microglia, lymphoid cells, and blood vessel endothelial and smooth muscle cells (Supplementary Figs. 9i,j and 10a–d). No PGD-null tumors were identified. On the basis of this analysis, we conclude that ENO1-null GBM tumors can be detected and therefore represent a viable, clinically relevant population expected to benefit from treatment with IACS-010759.

IACS-010759 is tolerated and extends survival in mouse mod- els of AML. In vivo AML models were evaluated to confirm the antitumor activity and tolerability of IACS-010759 in this con- text. The OCI-AML3 cell line, PDX-4030094 (MLL-rearranged AML PDX model refractory to standard-of-care treatment),

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Fig. 3 | Glycolysis-deficient and AML xenografts are sensitive to OXPHOS inhibition. a, Tumor volume of subcutaneous NB1 tumors in mice that received daily oral dosing of IACS-010759 or vehicle (n = 10 per group; data represents mean ± s.e.m.). b,c, Mice that received intracranial implantation of ENO1-null Gli56 cells were treated with vehicle or 5 mg/kg IACS-010759 for 4 weeks following a 5 on/2 off dosing schedule. Representative pre- and post-treatment magnetic resonance imaging (MRI) coronal images of brain (vehicle, n = 5 mice; IACS-010759, n = 7 mice; experiment repeated once with similar results) (b) and quantitation of tumor volumes calculated from MRI images at Day 33 (c) are shown. For box plots, midlines indicate the median, upper and lower perimeters indicate first and third quartiles, and tails indicate the minimum and maximum. Each point is an independent mouse.
*P = 0.025 by two-sided Student’s t-test. a.u., arbitrary units. d, H&E and immunohistochemistry staining for 6PGD and ENO1 protein expression in an array of tumors from patients with GBM. Representative images from wild-type ENO1 (ENO1 WT; n = 84) and ENO1-null (n = 8) tumors are shown. Scale bars, 100 µm. e, Kaplan–Meier survival analysis of mice inoculated with OCI-AML3 cells and treated for 5 weeks (starting on Day 7) with oral vehicle or with 10 mg/kg IACS-010759 daily (QD), 5 on/2 off (QD×5), every other day (Q2D), or every third day (Q3D) (n = 9 mice per group). P = 0.0007 (Q3D), 0.0008 (Q2D), and < 0.0001 (QD and QD×5) by Mantel–Cox log-rank test. f, Kaplan–Meier survival analysis of mice inoculated with primary patient sample 4030094 and orally treated with 1, 2.5, or 7.5 mg/kg IACS-010759 daily starting on Day 25 (n = 9 mice per group). P = 0.0002 (1 mg/kg) and
P < 0.0001 (2.5 and 7.5 mg/kg) by Mantel–Cox log-rank test.

PDX-S6-AP (PDX with complex cytogenetics and unfavorable prognosis) and three genetically defined murine leukemia syn- geneic models42 were grown orthotopically in mice. Seven days after inoculation with OCI-AML3 cells, mice were orally dosed

for 35 d with 10 mg/kg IACS-010759.HCl or vehicle using sev- eral intermittent schedules (Fig. 3e) and doses that were all well tolerated (Supplementary Fig. 11a,c). Daily dosing schedules, QD (daily) or QD×5 (5 d on/2 d off), were most effective and
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increased median survival from 28 d to longer than 60 d, whereas less-frequent dosing schedules (Q2D or Q3D) enhanced survival to a lesser extent (Fig. 3e). In the PDX-4030094 model, 2.5 or
7.5 mg/kg IACS-010759.HCl per day nearly doubled median survival from 35 d to almost 70 d (Fig. 3f and Supplementary Fig. 11b), whereas 1 mg/kg IACS-010759.HCl per day extended survival to 55 d. Additionally, disease burden, as measured by splenic hCD45 abundance at treatment day 21, was significantly reduced in IACS-010759-treated groups relative to vehicle- treated groups (Supplementary Fig. 11d). IACS-10759 similarly improved survival in the PDX-S6-AP model (Supplementary Fig. 11e). Treatment of the highly aggressive, matched-pair MLL– ENL translocation syngeneic models with IACS-010759 resulted in modest but statistically significant prolonged survival in the TP53−/− model, but not the TP53+/+ model (Supplementary Fig. 11f,g). In contrast, the mouse syngeneic model, AML1/ETO9a (KRAS-G12D, p53-null), was more sensitive to IACS-010759. In this model, there were significant decreases in disease burden at days 8 (42%) and 15 (24%) following treatment (Supplementary Fig. 11h), and median survival was statistically significantly extended from 17 d to 21 d (24%) (Supplementary Fig. 11i). Similarly to the xenograft models, all three syngeneic models experienced transient loss of body weight during the first week of dosing (Supplementary Fig. 11j–l). Overall, the consistent anti- leukemic responses observed in vitro and in vivo provided the preclinical rationale to evaluate IACS-010759 in a phase 1 study in subjects with relapsed/refractory AML (NCT02882321), which enrolled its first subject in October 2016.
OXPHOS inhibition leads to depletion of energy and nucleo- tide biosynthesis. To better understand the molecular mecha- nism through which IACS-010759 exerts antitumor activity, we performed metabolomic analyses, stable-isotope tracing experi- ments, and functional characterization on a subset of AML cell lines. IACS-l010759 induced modest increases in reactive oxygen species (ROS) levels, consistent with previous reports examin- ing OXPHOS inhibition41,44 (Supplementary Fig. 12a). To evaluate metabolic reprogramming, OCI-AML3 cells were exposed to IACS- 010759 for 6, 24, or 72 h, and the resultant cell lysates were sub- jected to liquid chromatography–mass spectrometry (LC–MS) analysis to measure metabolites from central carbon metabolism and amino acids. IACS-010759 treatment elevated the intracel- lular steady-state level of the complex I substrate, NADH, as well as that of nucleotide monophosphates (NMPs), whereas it reduced levels of nucleotide triphosphates (NTPs)); all of these changes are consistent with complex I inhibition and reduced energetic status (Fig. 4a). This was further confirmed by elevated pT172-AMPK lev- els (Fig. 4j and Supplementary Fig. 12m), a well-established readout of energetic stress. Stable isotope–tracing experiments using uni- formly labeled [13C]glucose (Fig. 4b) revealed that IACS-010759 significantly increased incorporation of [13C]glucose into the gly- colysis endpoints lactate and alanine, as evaluated by M + 3 isoto- pologue fractions (Fig. 4c), and decreased incorporation into the tricarboxylic acid (TCA) cycle intermediates and mitochondria- produced metabolites (M + 2 isotopologue fractions), including aspartate and glutamate (Fig. 4d), suggesting that glucose utiliza- tion through these pathways was substantially reduced. Although IACS-010759-treated cells uptake less glutamine than control cells do (Supplementary Fig. 12b), tracing experiments with uniformly labeled [13C]glutamine confirmed that treatment with IACS-010759 increased incorporation of [13C]glutamine carbons into the TCA cycle to fuel both oxidative and reductive metabolism, likely to support fatty acid biosynthesis, but failed to productively contrib- ute toward aspartate synthesis (Supplementary Fig. 12c–f)45. These observations suggest an increased utilization of glutamine as an alternative compensatory mechanism to IACS-010759 treatment.

Targeted metabolomics in OCI-AML3 cells showed that, among all amino acids measured, aspartate was the only other amino acid reduced by IACS-010759 (Fig. 4e), which is consistent with other published findings16,17. To assess whether aspartate was incorpo- rated into nucleotides, as has been suggested16,17, OCI-AML3 cells were grown with uniformly labeled [13C]aspartate. After treatment with IACS-010759, an increased M + 3 isotopologue fraction of sev- eral nucleotides was observed (Fig. 4f,g) that was consistent with increased utilization of exogenous aspartate for nucleotide biosynthe- sis, which suggests that mitochondria-produced aspartate becomes rate limiting upon OXPHOS inhibition. Although aspartate supple- mentation partially restored cell growth (Fig. 4h and Supplementary Fig. 12g,h), cell cycle progression, and incorporation of BrdU into cycling cells (Fig. 4i and Supplementary Fig. 12i), it failed to influ- ence pT172-AMPK activation, indicating persistent energetic stress (Fig. 4j and Supplementary Fig. 12m). Interestingly, AMPK activa- tion does not appear to be essential for the reduced tumor cell via- bility observed upon IACS-010759 exposure, as shRNA-mediated knockdown of AMPK in OCI-AML3 cells minimally impacted cell viability (Supplementary Fig. 12j,k). Aspartate also abrogated IACS- 010759-induced accumulation of DNA damage, as measured by pS139-γH2AX levels (Fig. 4j). Interestingly, aspartate supplementation was not able to rescue the proliferation phenotype in the relatively insensitive MOLM-13 cell line, suggesting that the hypersensitive cell lines may rely on OXPHOS for energy as well as aspartate production (Supplementary Fig. 12h). As an additional response mechanism, we found that IACS-010759 markedly increased expression of the CD14 myeloid differentiation marker in OCI-AML3 cells (Supplementary Fig. 12l), but not in MOLM-13 cells (data not shown). Taking these data together, we propose that the IACS-010759-mediated reduction in cell viability and induction of differentiation or apoptosis result from a combination of energy depletion and reduced aspartate pro- duction (Fig. 5a,b).

Therapeutic targeting of OXPHOS in AML. On the basis of mech- anisms defined by in vitro studies, we evaluated the effects of IACS- 010759 on OCR, aspartate, proliferation, and differentiation status in blast cells from PDX-4030094 in vivo. Blast cells were harvested from mouse spleens 2 h after the first dose or 24 h after a second dose of IACS-010759.HCl (representing Cmax and Cmin, respectively; Fig. 5b). At each dose and time point, OCR, aspartate, and an IACS- 010759-regulated transcriptomic signature (Supplementary Fig. 13a–f) were measured to establish a relationship among PK, phar- macodynamic (PD), and efficacy outcomes. At 2 h, OCR was equally inhibited at all doses (Fig. 5c and Supplementary Fig. 14a). By 48 h, OCR (Fig. 5d and Supplementary Fig. 14b), aspartate (Fig. 5f), and the gene expression signature (Fig. 5g) were suppressed in a dose- dependent manner at both 2.5- and 7.5-mg doses of IACS-010759, and these measures correlated with increased survival (see Fig. 3f). At both time points, aspartate levels were unaltered at a dose of 1 mg/ kg IACS-010759 but were suppressed in a dose-dependent manner at the top two doses (Fig. 5e,f), suggesting that aspartate level is a less-sensitive measure than OCR or the gene expression signature. Blasts isolated from mice that received two doses of IACS-010759 (7.5 mg/kg) exhibited reduced proliferation (Fig. 5h) and increased apoptosis (Fig. 5i). Additionally, IACS-010759 treatment reduced the fraction of CD34+ stem and progenitor AML cells (Fig. 5j) in a dose-dependent manner, most likely owing to induction of leuke- mia cell differentiation, as suggested by enrichment of AML cells expressing the myeloid differentiation antigens myeloid cell nuclear differentiation antigen (MNDA), CD14, CD11b, and CD68 (Fig. 5k and Supplementary Fig. 15a–k). These data support employing OCR, aspartate levels, gene expression changes, and markers of dif- ferentiation as PD biomarkers to assess the biology of response in clinical samples from patients with relapsed/refractory AML treated with OXPHOS inhibitors, such as IACS-010759.

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a b
NAD NADH ATP UTP CTP GTP AMP

GOT

LAC (M + 3)
PC

OAA (M + 3)

[13C ]glucose
6

PYR (M + 3)

AcCoA (M + 2)

OAA

SER (M + 2)

ALA (M + 3)

GLY (M + 2)

UMP CMP
GMP

ASP (M + 3)

GOT

Second round

to second round
TCA

CIT (M + 2)

GLUD1

Fold change (log2)

ASP (M + 2)

+ GOT
First round

+ OAA cycle
(M + 2)

a-KG (M + 2)

GLU (M + 2)

GSH

–2.0

0.001

0.0
P value

0.05

2.0

n.s.

FUM (M + 2)

SUC (M + 2)

CO2

(M + 2) (M + 4)

c DMSO d
IACS-010759

DMSO
IACS-010759

6 h 24 h 48 h

Aspartate GSH

Fold change (log2)

80
***
60

20
**** 15

Taurine Proline Alanine Asparagine Glutamate Glycine Histidine Lysine

–2.0

0.001

0.0
P value 0.05

2.0

f
[13C6]aspartate

g 100
80

DMSO ([U-13 C]ASP)
IACS-010759 ([U-13 C]ASP)

h
3.0

DMSO
IACS-010759

Phenylalanine Methionine Tryptophan Isoleucine Leucine Threonine Valine Tyrosine Serine Glutamine

IACS-010759 + aspartate
60 2.0

Pyrimidine (M + 3)

0
Uracil UPM

UTP CMP

1.0

OCI-AML3

24 h 48 h 72 h

DMSO IACS-010759 IACS-010759 + Asp

10 mM Asp – – +

– – + – – +

105

104

103

G1 63.7

S 22.0

G2 7.74

105

104

103

G1 77.2

S 2.48

G2
13.0

105

104

103

IACS-010759 – + + p-AMPK
(T172) AMPK
p-4H2AX (S139)
HSP90

– + + – + +

7-AAD

Fig. 4 | inhibition of OXPHOS by iACS-010759 leads to energy deprivation and impairs nucleotide biosynthesis. a, Targeted metabolomic analysis of nucleotide biomolecules in OCI-AML3 cells treated with 100 nM IACS-010759 for 6, 24, or 72 h. The heat map depicts alterations of metabolites
associated with complex I and energy production as fold change (log2) of IACS-010759-treated cells versus DMSO-treated controls (n = 4 cultures; the experiment was repeated once with comparable results). Data are shown as mean values. P values were derived by two-sided Welch's t-test accounting for unequal variance; n.s., nonsignificant. b, Illustration of [U-13C]glucose metabolism in OCI-AML3 cells. White circles, 12C carbons; blue circles, 13C carbons. M + 2 and M + 3 refer to the number of 13C carbons. c, Cells were grown in culture medium containing [U-13C]glucose and were treated with DMSO or 100 nM IACS-010759 for 24 h. Incorporation of [U-13C]glucose into glycolysis endpoints (lactate (extracellular) and alanine (intracellular)) is shown. Data are plotted as mean values and individual data points from n = 4 cultures. ***P = 0.0002, ****P < 0.0001 by two-sided Student's t-test. d, TCA intermediates (isocitrate, α-ketoglutarate (α-KG), succinate, and fumarate (intracellular)) and mitochondria metabolites (glutamate, glutathione, and aspartate (intracellular)) in OCI-AML3 cells after treatment with DMSO or 100 nM IACS-010759 for 24 h. Data are plotted as mean values and individual data points from n = 4 cultures; the experiment was performed twice with comparable results. ****P < 0.0001 by two-sided Student's t-test. e, Targeted metabolomic analysis of amino acid biomolecules in OCI-AML3 cells treated with DMSO or 100 nM IACS-010759 for 6, 24, or 72 h. The heat map depicts alterations of metabolite levels noted as fold-change (log2) of IACS-010759-treated cells versus DMSO-treated controls. P values were derived using two- sided Welch's t-test accounting for unequal variance. Data are plotted as mean values and individual data points from n = 4 cultures; the experiment was repeated once with comparable results. f, Illustration of the conversion of [U-13C]aspartate carbon into pyrimidines. Blue circles depict carbons derived from aspartate. g, Incorporation of carbons derived from [U-13C]aspartate ([U-13C]ASP) into representative pyrimidine metabolites in OCI-AML3 cells treated with DMSO or 100 nM IACS-010759 for 72 h. Data are plotted as mean values and individual data points from n = 4 cultures; the experiment
was performed twice with comparable results. h, OCI-AML3 cells were cultured in medium treated with DMSO, 100 nM IACS-010759, or 100 nM IACS-
010759 supplemented with 10 mM aspartate, and the number of cells was measured after 72 h. Data are plotted as mean values and individual data points from n = 2 cultures; the experiment was performed twice with comparable results. i, Incorporation of BrdU into DNA was measured through flow
cytometry, in OCI-AML3 cells treated for 72 h with DMSO, 123 nM IACS-010759, or 123 nM IACS-010759 supplemented with 10 mM aspartate. Cells were also stained with 7-AAD to detect DNA. j, Immunoblot for activated AMPK (phosphorylated AMPL (p-AMPK), T172) and γ-H2AX (p-γ-H2AX, S139) in OCI-AML3 cells that were treated as described in the Methods. See Supplementary Fig. 12m for blots with molecular weight markers. Experiments in i and j were repeated once with comparable results.

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DNA/RNA
Proteins

Glucose

Glutamine

Glucose

ATP
Aspartate

Aspartate

Nucleotides Proteins DNA repair

Disease burden

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Glutamine

Analysis

Vehicle 1 mg/kg

c
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>90% hCD45 PK/PD

d
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7.5 mg/kg

2 h (Cmax)
48 h daily (Cmin)

1.0
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2.5 mg/kg
7.5 mg/kg

f 1.0
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IACS-010759 concentration (nM) IACS-010759 concentration (nM) IACS-010759 concentration (nM) IACS-010759 concentration (nM)

g 0.5
0.0
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–2.5

h i j k
60 100 80 80
80
60 60
40
60
40 40
40
20
20 20 20

0 0 0 0

IACS-010759 concentration (nM)

Fig. 5 | iACS-010759 modulates several clinically translatable pharmacodynamic biomarkers. a, Model showing glucose and glutamine utilization by the TCA cycle to produce ATP and aspartate, which are used for nucleotide biosynthesis (left), and the changes that occur when complex I is inhibited by
IACS-010759 (right). b, Schematic of workflow for assessment of PD markers of target inhibition and biology of response. After irradiation and inoculation, tumor burden was monitored. Upon reaching 90% tumor burden in the spleen (high human CD45+ cells compared to mouse CD45+ cells), mice received two doses of IACS-010759 that were administered 24 h apart. Tumor cells were isolated from the spleen at 2 h after the first dose or at 24 h after the second dose (2-h (Cmax) and 48-h (Cmin) time points, respectively). c,d, OCR of leukemia cells normalized to the cell number at the 2-h (c) and 48-h
(d) time points. e,f, Aspartate levels of leukemia cells at the 2-h (FC = fold-change) (e) and 48-h (f) time points. g, Change in expression of a 19-gene (pharmacodynamic (PD)) score for leukemia cells at the 48-h time point. h–k, Average number of phosphorylated Histone H3 (p-H3)+ cells (h), apoptotic cells measured by cleaved caspase 3 (i), CD34+ cells (j), or MNDA+ cells (k) per field in the spleens of mice treated with vehicle or IACS-010759 at the 48-h time point. In c–g, each symbol represents the mean value for three technical replicates from a single mouse. In h–k, each symbol represents the mean of five random fields from a single mouse, and the overall mean ± s.e.m. is shown. *P = 0.02, ***P < 0001by two-sided Student's t-test.

Advancing IACS-010759 into clinical evaluation. The antitumor activity of IACS-010759 in glycolysis-deficient GBM and/or neuro- blastoma tumors and relapsed/refractory AML justified evaluation of IACS-010759 in clinical studies. During preclinical development, we evaluated IACS-010759 PK in mouse, rat, dog, and cynomolgus monkey; these PK profiles indicated that the compound displayed low clearance, large volume of distribution, and long terminal half-life

(Supplementary Table 3). In a battery of in vitro studies, IACS- 010759 did not show any concerning effects in binding assays on a panel of 80 receptors and ion channels (Supplementary Table 4), nor did IACS-010759 inhibit the human ERG K+ channel current (IC50 > 30 µM) (Supplementary Fig. 16) or modulate CYP enzyme activity (data not shown). In in vivo toxicity studies, including dose-range finding, acute intravenous dosing, repeated oral dosing

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(both daily and intermittent), and a pivotal good laboratory practice (GLP)-compliant 28-d oral toxicity study with a 28-d recovery period in the Sprague–Dawley rat and cynomolgus monkey, drug-related adverse events included emesis and decreased body weight at lower doses. At the highest doses, decreased core body temperature and death were observed, which are consistent with anticipated effects of excessive OXPHOS inhibition. No-observed-adverse-effect dose levels (NOAELs) were identified in rat and monkey that supported the ability for oral daily doses to yield plasma drug concentrations above the anticipated amount needed to result in on-target drug effects. These data were used to establish the human starting dose per Food and Drug Administration (FDA) guidelines46.
Discussion
Our findings contrast with the century-old tenet, put forth in Otto Warburg’s seminal work, that glycolysis is the dominant metabolic pathway to which tumors become addicted1. In recent years, the field has dispelled the notion that the increased glycolysis observed in tumors is simply a mechanism for providing additional energy to support the high rate of proliferation, and instead has indi- cated that the adaptation serves to provide macromolecule build- ing blocks for biosynthetic processes that are critical for enhanced tumor growth. Through the development and characterization of IACS-010759, we now provide evidence that clinically translatable contexts exist in which tumors are highly dependent on OXPHOS for survival. We further demonstrate that subsets of tumors depend on OXPHOS not only for ATP synthesis, but also to produce the macromolecules necessary for biosynthetic processes, similar to glycolysis. In the most sensitive tumor cells, OXPHOS disruption creates an environment of energy and macromolecule depletion that leads to cell cycle arrest, apoptosis, and, in the case of AML, differentiation, similar to what has been reported with small- molecule inhibitors of mutant IDH47. Taken together, our preclini- cal data support clinical evaluation of IACS-010759 in AML and solid tumors that lack compensatory glycolytic capacity. First-in- human clinical studies with IACS-010759 are ongoing in patients with AML and solid tumors to establish proof of concept, define the maximum tolerated dose, and provide initial verification of the hypotheses generated by this work.
Methods
Methods, including statements of data availability and any asso- ciated accession codes and references, are available at https://doi. org/10.1038/s41591-018-0052-4.
Received: 11 January 2018; Accepted: 27 March 2018;

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Acknowledgements
We thank members of the Center for Co-Clinical Trials, Institute for Applied Cancer Science, the Glioblastoma (GBM) and AML/MDS Moon Shots for intellectual and financial support. We would especially like to express our gratitude and appreciation to
C. Vellano for his help editing and assembling this manuscript; F. F. Lang and J. Gumin for providing GSC models; C. Kingsley and the MDACC Small Animals Imaging Facility; and N. Satani and E. Lin for validation of antibodies and preparation of samples for analysis, D. Bigner for D423 cells and D. N. Louis for Gli56 cells. P.Mo. was supported by The Agilent Technologies Thought Leader Award. R.A.D., A.-N.A.A., R.S., and J.Hi. were supported by The Medical Research Council (MC_U105663141 and MC_UU_00015/2 to J.Hi.). M.K., S.T., A.L., P.Ma., H.M., and Q.Z. were supported by CPRIT grant

RP140218. F.L.M. was supported by the CPRIT RP140612 and National Institutes of Health (NIH) Brain SPORE P50CA127001, and A.C.S. was supported by the Research Scholar Grant RSG1514501CDD. G.F.D. was supported by the American Association for Cancer Research (AACR) 14-90-25 and by the Sheikh Ahmed Bin Zayed Al Nahyan Center for Pancreatic Cancer Grant. This work was supported in part by the Leukemia & Lymphoma Society through its Therapy Acceleration Program (TAP) and by the MD Anderson Moon Shots program.
Author contributions
The studies were designed with input from J.R.Ma., J.R.Mo., Y.S., M.Pr., M.B., J.Ha., C.B., P.Mo., J.Hi., M.K., P.J., M.E.D.F., C.T., T.P.H., G.F.D., and F.M. In vitro experiments were
performed by J.R.Ma, J.R.Mo., V.G., L.Ha., Y.T., Y.S., M.Pr., S.G., M.M., T.K., M.B., P.Mo.,
J.B., G.G., M.G.D., J.Ha., Y.J., T.L., H.M., P.Ma., M.Pe., R.S., T.S., M.S., V.K.H., C.C.C., and
Q.Z. IACS-010759 was developed and conceived by M.E.D.F., T.M., C.C., B.C., G.L., Z.K., A.P., J.T., and P.J. In vivo studies were performed by Y-H. L., N.F., J.Ga., J.Gr., and R.M. Immunohistochemistry was performed by J. Ac., E.C., S.K., and J.R-C. Computational chemistry was performed by J.Hi. and J.B.C. Pharmacokinetic analysis was performed by S.H., Q.X., and Y.J. FACS data analysis was performed by J.R.Mo. and S.G. Glioma stem cell work was performed by Y.S., T.S., J-W.D., V.K.H, J.F.d.G., and C.C.C. Bioinformatic analysis was performed by C.B. Clinical positioning in AML was designed by M.K., N.D., J.R.Ma, M.E.D., and P.J. Metabolomic data for glycolysis deficient was generated by Y.S., C.B., and J.As. and for leukemia by J.R.Mo., S.T., A.L., and P.Mo. Stable-isotope labeling study design, analysis and interpretation was performed by P. Mo. Mouse complex I assays were designed and performed by J.Hi., A.-N.A.A., and R.S. Normal bone marrow samples were provided by S.C. and G.A-A. Writing and preparation of the manuscript and figures were performed by J.R.Ma., J.R.Mo., T.P.H., R.A.D., A.D., Y.S., M.E.D.F., M.K., P. Mo., and P.J.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/ s41591-018-0052-4.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to J.R.M.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Methods
Cell culture. Unless otherwise specified, all cell lines (H460, 293 T, H292, RCC4,
RCC4 +VHL, Gli56, D423, and NB-1) were cultured in DMEM (Gibco) with 10% FBS (Sigma). AML cell lines (KG1, THP1, MOLM13, K562, MV4-11, OCI-AML3,
U937, Kasumi, and HL60) were cultured in RPMI (Gibco) with 5–20% FBS. D423 cells were provided by D. Bigner (Duncan et al.48, Oncotarget 2010). Gli56 cells were provided by D. N. Louis (Mueller et al.49, Oncogene 2005). NB-1 cells were obtained from the Japanese Cancer Research Resources Bank (JCRB) Cell Bank. All GSCs were kindly provided by F. Lang at MD Anderson Cancer Center (MDACC). GSCs were cultured in serum-free DMEM/F12 from American Type Culture Collection (ATCC) supplemented with 20 ng/ml basic fibroblast growth factor (bFGF) (Sigma), 20 ng/ml epidermal growth factor (EGF) (Sigma) and
1 × B27 supplement (Invitrogen). Normal diploid cells were purchased from ATCC and cultured following ATCC’s guidelines. All other cell lines are from ATCC. RCC4 and RCC4VHL were obtained from European Collection of Authenticated Cell Cultures (ECACC). Cell lines were maintained at an internal core facility that routinely used short tandem repeat (STR) fingerprinting to verify cell line identity and performed mycoplasma testing to confirm they were negative.
Compound synthesis. Step 1: synthesis of ethyl 3-(4(trifluoromethoxy)phenyl)-1,2,4- oxadiazole-5-carboxylate. To a solution of (E)-N′-hydroxy-4-(trifluoromethoxy) benzimidamide (Sigma-Aldrich, 60.0 g, 272 mmol) and pyridine (32.3 g, 408 mmol) in CHCl3 (400 ml) at 0 °C was slowly added ethyl 2-chloro-2-oxoacetate (44.6 g, 327 mmol). The mixture was stirred at reflux for 3 h, cooled to RT and diluted
with H2O (400 ml). The mixture was extracted with dichloromethane (DCM, 400 ml × 2), the combined organic layers were washed with aqueous HCl (1 M,
300 ml × 2) and H2O (400 ml), dried over Na2SO4, filtered, evaporated to give ethyl 3-(4(trifluoromethoxy)phenyl)-1,2,4-oxadiazole-5-carboxylate as a light yellow solid (77.7 g, 94%), which was used for next step without further purification.
Proton nuclear magnetic resonance (1H NMR) (300 MHz, CDCl3): δ 8.22 (d,
J = 11.2 Hz, 2 H), 7.36 (d, J = 11.2 Hz, 2 H), 4.60 (q, J = 9.6 Hz, 2 H), 1.51 (t,
J = 9.6 Hz, 3 H). 13C NMR (126 MHz, DMSO-d6): δ 167.5, 167.1, 153.5, 150.7, 129.5,
124.4, 121.7, 119.9 (q, J = 257 Hz), 63.3, 13.7. 19F NMR (471 MHz, DMSO-d6): δ
–56.7. High-resolution mass spectrometry, electrospray ionization (HRMS, ESI+) m/z: [M + H]+ calculated for C12H10F3N2O4, 303.0587; found, 303.0584.
Step 2: synthesis of 3-(4-(trifluoromethoxy)phenyl)-1,2,4-oxadiazole -
5-carbohydrazide. To a solution of ethyl 3-(4-(trifluoromethoxy)phenyl)-1,2,4- oxadiazole-5-carboxylate (80.0 g, 265 mmol) in EtOH (800 ml) was added NH2NH2. H2O (85%, 76.0 ml, 1,325 mmol). The reaction mixture was stirred at RT for 16 h.
The desired compound precipitated from the reaction mixture and was filtered
and washed with EtOH (200 ml) to produce 3-(4-(trifluoromethoxy)phenyl)-1,2,4- oxadiazole-5-carbohydrazide (70.2 g, 92%) as a light yellow solid, which was used for the next step without further purification. 1H NMR (600 MHz, Methanol-d4): δ 8.24 (d, J = 8.8 Hz, 2 H), 7.47 (d, J = 8.6 Hz, 2 H). 13C NMR (126 MHz, DMSO-d6):
δ 166.9, 129.4, 124.7, 121.7, 119.9 (q, J = 258 Hz). 19F NMR (471 MHz, DMSO-d6):
δ –56.6. HRMS (ESI+) m/z: [M + H]+ calculated for C10H8F3N4O3, 289.0543; found, 289.0538.

Step 3: synthesis of 5-(5-methyl-1H-1,2,4-triazol-3-yl)-3-(4-(trifluoromethoxy) phenyl)-1,2,4-oxadiazole. NaOH (9.4 g, 234 mmol) was added to a mixture
of 3-(4-(trifluoromethoxy)phenyl)-1,2,4-oxadiazole-5-carbohydrazide (45.0 g, 156 mmol) and acetimidamide hydrochloride (22.2 g, 234 mmol) in
tetrahydrofuran (THF) (500 ml). The mixture was refluxed for 3 d and then cooled to RT, concentrated under reduced pressure and diluted with H2O (500 ml). The resulting suspension was stirred at RT for 30 min and then filtered to produce a crude solid product, which was treated with ethyl acetate (EtOAc) (400 ml). The resulting suspension was stirred at RT for 30 min and then filtered to produce
5-(5-methyl-1H-1,2,4-triazol-3-yl)-3-(4-(trifluoromethoxy)phenyl)-1,2,4- oxadiazole as a white solid (30.3 g, 62%). 1H NMR (600 MHz, DMSO-d6): δ 14.65 (s, 1 H), 8.22 (d, J = 8.7 Hz, 2 H), 7.61 (d, J = 8.6 Hz, 2 H), 2.51 (s, 3 H). 13C NMR
(126 MHz, DMSO-d6): δ 169.4, 167.2, 165.8, 155.3, 150.5, 129.4, 125.0, 121.7, 119.9 (q, J = 258 Hz), 11.5.19F NMR (471 MHz, DMSO-d6): δ –56.6. HRMS (ESI+) m/z:
[M + H]+ calculated for C12H9F3N5O2, 312.0703; found, 312.0699.
Step 4: synthesis of 5-(1-(3-bromobenzyl)-5-methyl-1H-1,2,4-triazol-3-yl)-3-(4- (trifluoromethoxy)phenyl)-1,2,4-oxadiazole. 1-bromo-3-(bromomethyl)benzene (48.2g,
192.8 mmol) was added to a suspension of 5-(5-methyl-1H-1,2,4-triazol-3-yl)-3- (4-(trifluoromethoxy)phenyl)-1,2,4-oxadiazole (60.0 g, 192.8 mmol) and potassium carbonate (66.6g, 482.0 mmol) in DMF (200ml). The mixture was stirred at RT for
16 h and then diluted with water (500ml) and extracted with EtOAc (3 ×500 ml). The combined organic layers were washed with H2O (300 ml) and concentrated under reduced pressure to produce the crude product, which was purified using a silica gel chromatography column (eluent: 4:1 to 3:2, petroleum ether (PE):EtOAc) to produce 5-(1-(3-bromobenzyl)-5-methyl-1H-1,2,4-triazol-3-yl)-3-(4-(trifluoromethoxy) phenyl)-1,2,4-oxadiazol as a white solid (53.6g, 57.9%). 1H NMR (600 MHz, CDCl3): δ 8.28 (d, J =8.8 Hz, 2 H), 7.48 (d, J =7.9 Hz, 1 H), 7.41 (s, 1 H), 7.34 (d, J =8.8 Hz, 2 H),
7.26 (d, J =7.6 Hz, 1 H), 7.17 (d, J =7.9 Hz, 1 H), 5.43 (s, 2 H), 2.55 (s, 3 H). 13C NMR
(126 MHz, DMSO-d6): δ 168.9, 167.2, 155.4, 150.5, 148.2, 137.8, 131.0, 131.0, 130.5,

129.4, 126.9, 124.9, 121.9, 121.6, 119.9 (q, J=258 Hz), 51.1, 11.6. 19F NMR (471 MHz, DMSO-d6): δ –56.6. HRMS (ESI+) m/z: [M + H]+ calculated for C19H14BrF3N5O2, 480.0277, 482.0257; found 480.0270, 480.0247.

Step 5: synthesis of IACS-010759 5-(5-methyl-1-(3-(4-(methylsulfonyl)piperidin-1-yl) benzyl)-1H-1,2,4-triazol-3-yl)-3-(4-(trifluoromethoxy)phenyl)-1,2,4-oxadiazole.
2-dicyclohexylphosphino-2’,6’-di-i-propoxy-1,1’-biphenyl (580 mg, 1.25 mmol) and tris(dibenzylideneacetone)dipalladium (760 mg, 0.83 mmol) were added to a mixture of 5-(1-(3-bromobenzyl)-5-methyl-1H-1,2,4-triazol-3-yl)-3-(4-
(trifluoromethoxy)phenyl)-1,2,4-oxadiazole (2.00 g, 4.16 mmol), 4-(methylsulfonyl)
piperidine (1.02 mg, 6.24 mmol), and t-BuONa (800 mg, 8.33 mmol) in toluene (80 ml), and the reaction mixture was degassed with argon for 3 min and then was heated to 140 °C for 18 h under an argon atmosphere. The mixture was then
cooled to RT, diluted with EtOAc (100 ml), filtered through a pad of celite, washed with EtOAc (100 ml), and concentrated under reduced pressure. The residue was purified using a silica gel chromatography column (PE:EtOAc = 1:1 with pure EtOAc) to produce a crude product, which was treated with EtOAc and Et2O (vol/ vol = 1:9, 30 ml). The resulting suspension was stirred at RT for 30 min and then filtered to produce 5-(5-methyl-1-(3-(4-(methylsulfonyl)piperidin-1-yl)benzyl)- 1H-1,2,4-triazol-3-yl)-3-(4-(trifluoromethoxy)phenyl)-1,2,4-oxadiazole as a white solid (905 mg, 39%). 1H NMR (600 MHz, DMSO-d6): δ 8.22 (d, J = 8.8 Hz, 2 H),
7.61 (d, J = 8.2 Hz, 2 H), 7.21 (t, J = 7.9 Hz, 1 H), 6.97 (bs, 1 H), 6.94 (dd, J = 8.3,
2.4 Hz, 1 H), 6.64 (d, J = 7.5 Hz, 1 H), 5.48 (s, 2 H), 3.86 (bd, J = 13.4 Hz, 2 H), 3.28
(m, 1 H), 2.94 (s, 3 H), 2.76 (m, 2 H), 2.57 (s, 3 H), 2.06 (bd, J = 13.4 Hz, 2 H), 1.68
(ddd, J = 16.5, 12.5, 4.1 Hz, 2 H). 13C NMR (126 MHz, DMSO-d6): δ 169.0, 167.2,
155.1, 150.7, 150.5, 147.9, 136.0, 129.6, 129.4, 124.9, 121.7, 119.9 (q, J = 258 Hz),
117.8, 115.5, 115.2, 58.6, 52.3, 47.2, 37.4, 23.7, 11.7. 19F NMR (471 MHz, DMSO-d6): δ –56.6. HRMS (ESI+) m/z: [M + H]+ calculated for C25H26F3N6O4S, 563.1683; found, 563.1675.
Oxygen consumption. AML cells were suspended normal growth medium at a concentration of 1 × 107 cells/ml, and 100 µl of cells was added to Seahorse 96-well plates precoated with Cell-Tak. Plates were centrifuged at 1,000 r.p.m., and medium was replaced with prewarmed (37 °C) 125 µl Seahorse medium (Seahorse XF medium with 2 mM glutamax, 10 mM glucose, 2 mM pyruvate) containing IACS- 010759 or rotenone (41 nM). For AML cell lines, 3 × 105 total cells were seeded per well in Seahorse XF medium with 2 mM glutamine, 10 mM glucose, and 2 mM pyruvate. The mitostress test was performed with 1 µM oligomycin, 0.4–1 µM FCCP, and 1 µM antimycin. For oxygen consumption in permeabilized tumor cells (Fig. 1d), H460 cells were suspended in Seahorse XF assay medium and plated
at a concentration of 15 × 103 cells/well of Seahorse 96-well plates precoated with Cell-Tak. Plates were centrifuged at 1,000 r.p.m., and medium was replaced with prewarmed (37 °C) 100 µl of 10 mM pyruvate and 2 mM malate substrate + 4 µM FCCP (uncoupled) in the 1 × Mitochondria Assay Solution (MAS) medium.
Immediately before IACS-010759 injection, plasma membrane permeabilizer (PMP) was added to a final concentration of 1 nM, and ADP was added to a final concentration of 4 nM. Final drug and substrate concentrations were 10, 100, or 1,000 nM for IACS-010759; 100 nM for rotenone; 10 mM for succinate; 4 µM for antimycin. A detailed protocol is provided by Agilent at https://www.agilent.com/ cs/library/usermanuals/public/insert-xf-pmp-reagent-web.pdf.
Seahorse analyses for AML cell lines and the patient-derived xenograft (PDX) model were performed according to Seahorse Biosciences protocol for the
mitostress test. Briefly, 300,000 cells per well were seeded in 175 µl of Seahorse XF medium supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate. FCCP concentration was optimized for each cell line model (1.6–0.2 μM), and data were normalized to cell number.
Seahorse analyses for basal OCAR and ECAR were performed using reagents from Seahorse Bioscience, as previously reported50.
Ectopic expression of NDI-1. HEK293T cells were transfected with pCMV-dR8.9 packaging DNA vector, pMD2.G VSV-G-expressing envelope vector, and pLenti6.3 NDI-1, pLenti6.3/V5 NDI-1, or pLenti6.3/V5 GFP plasmid DNA. 2 × 105 H460 cells were transduced in a 6-well plate with 2 ml of viral supernatant supplemented with polybrene to a final concentration of 8 µg/ml. After infection, transduced cells were selected through growth in 7 µg/ml blasticidin. The concentration of IACS- 010759 used in the graph in Fig. 1e was 14 nM.
Isolated mouse complex I assay. Complex I was isolated from mouse heart mitochondria using an adaptation of the method of Sharpley and colleagues51. The concentration of IACS-010759 in Fig. 1f was 60 nM. The NADH:decylubiquinone assay is described in Sharpley et al.52, and the APAD+ and H2O2 assays are described in Birrell et al.52.

Generation of clonal cell lines resistant to IACS-010759. H292 cells (1 × 106 cells/ plate) were seeded in 15-cm dishes in galactose growth medium and treated with
1 nM IACS-010759 (IC65) for 3 weeks, followed by exposure to 8 nM IACS-010759 (IC95) until resistant clones emerged. Twenty-six resistant clones were isolated from four independent experiments and were seeded at 5 × 103 cells/well in 96- well plates in 100 µl galactose growth medium. After cells became fully attached,

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IACS-010759 or rotenone was added to a final concentration of 370 nM to 18 pM for 3 d. Plates were scanned in the IncuCyte live-cell analysis system before
analysis via Hoechst and PI. Subsequently, both Hoechst and PI using an Operetta high-content imaging system. RNA-seq was conducted on the parental line and 12 resistant clones, uncovering a single nonsynonymous, heteroplasmic (35–50%), recurrent mutation in the mitochondrial-encoded gene MT-ND1 in 9 of the 12 resistant clones that conferred the L55F (T3469C) amino acid change. Paired-end
reads were initially aligned to transcript sequences of complex I genes with Bowtie 2 (ref.53), and the aligned fragments were probabilistically assigned to transcripts using eXpress54. Variants from the reference genome were called using the ‘mpileup’ command in SAMtools. MutPred55 analysis of the L55F variant classifies the alteration as potentially pathogenic (MutPred score = 0.8); this alteration is found at a very low frequency in mtDNA sequences in Genebank (1:30,589 based on full- length mitochondrial genomes deposited in Genebank before 28 October 2015), suggesting it is unlikely to be a polymorphism. The mutation was confirmed in four of the resistant clones by cloning the MT-ND1 gene sequence (ZERO blunt PCR; Invitrogen) and analyzing purified plasmid DNA via Sanger sequencing using the following primers: Forward: 5′-GTAAAACGACGGCCAGT-3′ and Reverse: 5′-AACAGCTATGACCATG-3′.
Metabolomics for glycolysis-deficient models. NB-1, D423, Gli56, A1207, SW1088, and U87 were plated and treated with DMSO or 100 nM IACS-010759 in a 10-cm plate. Two days after, the cells were briefly washed in cold PBS, scrapped in 80% methanol, and spun down. The supernatant was dried in a GeneVac HT4 using the low-temperature program (SP Scientific). The dried samples were
resuspended and subjected to a liquid chromatography and mass spectrometry (LC–MS) analysis that covers over 200 metabolites by J. M. Asara’s group at Beth Israel Deaconess Medical Center, as previously described56. Analysis of metabolite peak area integrated total ion chromatogram values was carried out using the statistical computing language R. Metabolites with missing data in any sample were excluded, and the remaining values were quantile normalized. Statistical comparisons of groups was performed with limma, and all P values were reportedcorrected for multiple-hypothesis testing using the Benjamini– Hochberg method.

LC–MS for targeted metabolomics and stable-isotope tracing in AML cell lines. For targeted metabolomics, dried cellular extracts from OCI-AML3 cultures were reconstituted with 1:1 acetonitrile:water (40 µl), and aliquots (5 µl) were analyzed on an Agilent 1290 ultra-high performance liquid chromatography (UHPLC) system coupled with an Agilent 6550 quadrupole-time of flight (Q-TOF) mass spectrometer operating in negative ion mode. Details about LC–MS analysis conditions are reported in Appendix I. Acquired Q-TOF raw data were processed using Agilent MassHunter Profinder 8.0 software, and target metabolites were identified using the built-in batch-targeted feature extraction algorithm, which used an in-house accurate mass-retention time (AMRT) database library, including 126 endogenous metabolites. Identified compound signal intensities were extracted and subjected to statistical analysis in Agilent Mass Profiler Professional (MPP).
Sample metabolite raw abundancies were log2 transformed, normalized by their correspondent sample protein content or viable cell count, and centered to their median signal intensities. The unpaired Welch’s t-test was used to determine statistically significant variations across sample groups (for detailed methodology, refer to Supplementary Methods).
For stable-isotope tracing experiments, samples for LC–MS analysis were prepared as described above and analyzed on an Agilent 6550 Q-TOF. The raw data were analyzed in MassHunter Profinder 8.0 by running the batch isotopologue extraction algorithm against an accurate mass retention time (AMRT) in-house compound library including the compounds of interest. The resulting isotopologue abundancies were corrected for their isotopic natural abundance, extracted as detailed CVS files, and tested for significant variations using a Welch’s t-test.
Details about cell growth and LC–MS analysis are reported in Appendix I (for detailed methodology, refer to Appendix I).

Primary AML cells and normal bone marrow. Peripheral blood samples from patients with AML were collected during routine diagnostic procedures after informed consent was obtained in accordance with the regulations and protocols (LAB 01-473) approved by the MDACC Investigational Review Board (IRB) in accordance with IRB regulations of The University of Texas MD Anderson Cancer Center and the Declaration of Helsinki. AML samples were analyzed under the IRB-approved laboratory protocol PA13-1025. Briefly, mononuclear cells were separated by Ficoll–Hypaque density gradient centrifugation and incubated in RBC lysis buffer (ammonium chloride solution) to remove red blood cells. Primary leukemia samples were maintained in StemEZ Serum-Free Medium. Viability and induction of apoptosis were assessed simultaneously using flow cytometry. 1 to 3 million cells were grown in triplicate in 24-well plates and exposed to DMSO or IACS-010759. Cells were harvested after 3, 4, or 5 d of exposure to agent and were resuspended in binding buffer containing Annexin V. Apoptotic cells were detected by Annexin V flow cytometry after gating on CD45+ leukemic cells. Viable cells were detected by flow cytometry with anti–human CD45-FITC antibody (BD Pharmingen) staining after exclusion of nonviable cells determined with DAPI staining (Sigma-Aldrich) and apoptotic cells. Flow cytometry was performed

on Gallios Flow Cytometer, and data were analyzed using Kaluza Flow Analysis software (Beckman Coulter). For normal bone marrow, the same procedure was followed.
Gene expression signature. To determine whether specific gene pathways were altered by treatment with IACS-010759, REACTOME pathway analysis was performed on the genes that were significantly upregulated (132 genes) or
downregulated (132 genes) in > 2 cell lines following 24-h treatment with 100 nM IACS-010759. The top four gene pathways upregulated in response to IACS-010759 were all related to amino acid biosynthesis (Supplementary Fig. 13a), consistent with the finding that metabolite levels are altered in response to IACS-010759 described above. The fifth upregulated pathway included genes regulated by ATF4, which is a transcription factor that upregulates genes that that deal with cellular stress in response to decreased amino acids and energy intermediates. The top
five downregulated pathways all involve genes involved in cell cycle progression (Supplementary Fig. 13b).
Genes whose expression was modulated by treatment with IACS-010759 in > 3 cell lines were selected for validation in vivo with a custom NanoString
codeset. Samples from an acute pharmacokinetics and pharmacodynamics (PK/ PD) study conducted in a primary patient-derived xenograft mouse model of AML were harvested in triplicate after 48 h of treatment with 1, 2.5, or 7.5 mg/ kg IACS-010759 and in duplicate for vehicle treatment. Analysis of NanoString data was carried out using the statistical computing language R. Normalization
factors were calculated on the basis of the internal positive controls and a selected set of housekeeping genes (CLTC, TBP, ALAS1, ACTB, and RPL19) according to the nCounter Expression Data Analysis Guide. For each sample, to control for variability in hybridization across samples, the geometric mean of the internal controls was calculated and then used to normalize across samples. To control for variability associated with the amount of input mRNA, the geometric mean of the housekeeping genes was calculated and then used to normalize samples for the amount of input mRNA. Data were normalized to vehicle samples, and genes were ranked by the magnitude of alteration at the highest dose (Supplementary Fig. 8c).
A subset of genes were dose-dependently downregulated upon treatment, with a more than twofold decrease in expression at the highest dose of IACS-010759
(Supplementary Fig. 13c–e). Variability in mRNA signal for each gene was minimal across the three mice within a dosing group.
The top-scoring gene was IL8, and it was equally inhibited at all three dose levels (Supplementary Figs. 13d,e). In contrast, other genes showed a dose- dependent inhibition over this range (Supplementary Figs. 13d,e). A ‘summary score’ was constructed on the basis of the average fold change of the 12 genes
for which the highest dose-dependent downregulation was observed in response to IACS-010759 (RRM2, HMGN2, DHCR24, PCNA, KIF11, TK1, KLGAP25,
KIF20A, CDCA5, CCNB1, and FEN1) and plotted against plasma levels for
the PK/PD analysis shown in Supplementary Fig. 13f. This analysis shows a dose-dependent decrease in expression of this group of genes in response to IACS-010759 treatment.
Reporting Summary. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Data availability. All data supporting this study are available from the corresponding author upon reasonable request.

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