Abstract
Malignancies are reliant on glutamine as an energy source and a facilitator of aberrant DNA methylation. We demonstrate preclinical synergy of telaglenastat (CB-839), a selective glutaminase inhibitor, combined with azacytidine (AZA), followed by a single-arm, open-label, phase 1b/2 study in persons with advanced myelodysplastic syndrome (MDS). The dual primary endpoints evaluated clinical activity, safety and tolerability; secondary endpoints evaluated pharmacokinetics, pharmacodynamics, overall survival, event-free survival and duration of response. The dose-escalation study included six participants and the dose-expansion study included 24 participants. Therapy was well tolerated and led to an objective response rate of 70% with (marrow) complete remission in 53% of participants and a median overall survival of 11.6 months, with evidence of myeloid differentiation in responders determined by single-cell RNA sequencing. Glutamine transporter solute carrier family 38 member 1 in MDS stem cells was associated with clinical responses and predictive of worse prognosis in a large MDS cohort. These data demonstrate the safety and efficacy of CB-839 and AZA as a combined metabolic and epigenetic approach in MDS. ClinicalTrials.gov identifier: NCT03047993.
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Data availability
The scRNAseq, scBCRseq and scTCRseq data that support the findings of this study were deposited to the GEO under accession code GSE225352. MS data were deposited to Zenodo (https://doi.org/10.5281/zenodo.7641622)44. The full study protocol is publicly available and provided in the Supplementary Information. All other requests regarding data supporting the findings of this study or related to this paper should be addressed to [email protected]. Source data are provided with this paper.
Code availability
No custom algorithms were used in this study. Open-source software was used to analyze the data. Details of software versions are specified in Methods.
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Acknowledgements
We thank the participants in this trial and their families, the study coordinators and the support staff at the clinical sites and the Calithera team members. We also thank Calithera Biosciences for providing CB-839 for both the preclinical studies and the clinical trial. This work was supported by the National Institutes of Health (NIH) National Cancer Institute (NCI) (R01 CA206210 to M.K., A.V. and S.T.), an NIH/NCI Cancer Center Support Grant (P30 CA016672 to M.K., N.P. and C.D.D.), the MD Anderson Cancer Center Leukemia SPORE (DRP 5 P50 CA100632-12) and Calithera Biosciences. M.R.G. is supported by a Leukemia and Lymphoma Society Scholar award. C.D.D. is supported by the Leukemia and Lymphoma Society Scholar in Clinical Research Award. D.V. is supported by a K12CA279871 NCI Paul Calabresi Award.
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Conceptualization and design, A.V., S.T., M.K., D.V. and C.D.D. Development of methodology, C.D.D., A.V., S.T., M.K., S.D. and D.V. Preclinical experiments and studies, T.C., V.M.K., D.V., A.V., A.S., G.P. and S.C. Acquisition of data (provided animals, acquired and managed participants, provided facilities, etc.), C.D.D., T.D.B., N.B., A.L., K.S., T.C., A.S., D.V., V.A.G., G.P., V.M.K., S. Konoplev, S.G.-M., K.P., S.A., G.L.H., S.C., M.C., S.R.S., J. Busquets, A.S.R., Q.D., M.R.G., S.G., B.A., G.S.C., S.S., M.S., V.T., B.W., U.S., G.D.T., J. Burger, G.B., E.J., N.P., T.K., S. Kornblau, N.G.D., K.N., N.J.S. and G.G.-M. Analysis and interpretation of data (for example, statistical analysis, biostatistics and computational analysis), C.D.D., T.D.B., N.B., A.L., K.S., T.C., X.S., A.S., D.V., S. Konoplev, S.G.-M., K.P., S.A., G.L.H., M.C., S.R.S., J. Busquets, A.S.R., Q.D., M.R.G. and B.A. Writing, review and/or revision of the paper, C.D.D., N.B., A.L., K.S., T.C., X.S., S. Konoplev, D.V., A.S., S.G.-M., K.P., S.A., M.C., S.R.S., J. Busquets, A.S.R., M.G., Q.D. and S.D. Administrative, technical or material support (that is, reporting or organizing data and constructing databases), T.D.B., N.B., A.L., K.S., T.C., X.S., A.S., D.V., V.A.G., V.M.K., K.P., S.A. and S.D. Study supervision, A.V., S.T. and M.K.
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A.V. has received research funding from Prelude, Ryvu, BMS, GSK, Incyte, Medpacto, Curis and Eli Lilly, is a scientific advisor for Stelexis, Aurigene, Acceleron and Celgene, receives honoraria from Stelexis, Aurigene and Janssen and holds equity in Stelexis and Clinstreet. K.S. is a current employee of Gilead Sciences, Inc. All work contributing to the paper was performed while at the University of Texas MD Anderson Cancer Center. N.J.S. has received consulting fees from Pfizer Inc., GSK, NKARTA, Autolus, Adaptive Biotechnologies and Sanofi and research funding from Takeda Oncology, Astellas Pharma Inc., Xencor, GSK, NextCure, Ascentage and Novartis. N.J.S. receives honoraria from Adaptive Biotechnologies, Amgen, Takeda, Pfizer Inc., Astellas Pharma Inc. and Sanofi. C.D.D. is a consultant and/or on the advisory board of Abbvie, AstraZeneca, Astellas, BMS, Genentech, GenMab, GSK, Notable Labs, Rigel, Ryvu, Schrodinger and Servier. S. Konoplev is consultant for Cairo Diagnostics Laboratory. G.S.C. has stock options in privately held company Curis. M.R.G. reports research funding from Sanofi, Kite/Gilead, Abbvie and Allogene; consulting for Abbvie and Allogene; advisory board for Bristol Myers Squibb, Arvinas and Johnson & Johnson; honoraria from BMS, Daiichi Sankyo and DAVA Oncology; and stock ownership of KDAc Therapeutics. S.D. is employed at Calithera. M.K. received research support from Calithera. U.S. has received research funding from GlaxoSmithKline, Bayer Healthcare, Aileron Therapeutics and Novartis; has received compensation for consultancy services and for serving on scientific advisory boards from GlaxoSmithKline, Bayer Healthcare, Celgene, Aileron Therapeutics, Stelexis Therapeutics and Pieris Pharmaceuticals; and has equity ownership in and has served on the board of directors of Stelexis Therapeutics. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 In vitro evaluation of GLS KO on cellular respiration.
(a) Validation of knockout efficiency in stable transfected OCI-AML3 cells, generated using shRNA control, puro-GAC plasmid, puro-KGA plasmid, puro-D3 (both isoforms) plasmid with puromycin incubation (3 days, 1 µg/ml). The expression of GAC and KGA in OCI-AML3 cells was determined by immunoblotting. Data were quantified in reference to loading control (n = 2 independent experiments, in 3 cell lines tested with similar level of knockdown). (b) Representative Seahorse Mito Stress Test assay graphs obtained from OCI-AML3 cells subjected to inducible knockout of KAG, GAC or GAC/KGA; Results were normalized to viable cell count. (n = 3-4 technical replicates, mean±SD, n = 3 independent experiments). (c) Basal, maximal oxygen consumption rate, OCR-linked ATP production, extracellular acidification rate (ECAR) and global metabolic phenotype (OCR/ECAR) were measured and calculated for OCI-AML3 cells subjected to doxycycline inducible knockout of KGA, GAC or GAC/KAG (mean ± SD, n = 3-4 technical replicates, n = 3 independent experiments, ordinary one-way ANOVA); (d) Representative Seahorse Mito Stress Test assay graphs obtained from four AML cell lines: MV4-11, MOLM13, OCI-AML3, and U937 treated with DMSO or telaglenastat (CB-839) at 1µM, for 12 hours; Results were normalized to viable cell count (n = 3–6 technical replicates, mean±SD, n = 3 independent experiments). (e) Basal, maximal oxygen consumption rate, OCR-linked ATP production, extracellular acidification rate (ECAR) and global metabolic phenotype (OCR/ECAR) were measured and calculated for AML cells as in (D) (n = 3–6 technical replicates, mean ± SD, n = 3 independent experiments, ordinary one-way ANOVA). (f) Seahorse Mito Stress Test assay graphs in OCI-AML3 cells treated with DMSO or BTES at 20µM. Results were normalized to viable cell count (n = 4 technical replicates, mean±SD, n = 3 independent experiments). (g) Mass spectrometry measurement of UMP and AMP in OCI-AML3 cells treated with DMSO or CB-839 1µM for 24 hr (n = 1 biological experiment with n = 4 technical replicates, mean±SD, unpaired t-test).
Extended Data Fig. 2 Evaluation of CB-839/AZA drug synergy in AML cell lines in vitro.
Representative graphs demonstrating synergistic effect of CB-839 and azacytidine (AZA) combination in AML cells. Cells were exposed to increasing doses of CB-839 and AZA alone or at constant drugs’ ratios for 5 days. Cell viability was determined by CellTiter-Glo luminescence assay. Combination index (CI) is the average ±SD of CIs at effective doses EC50, EC75, and EC90 calculated using CalcuSyn software. The CI < 1 = synergism; CI > 1 = antagonism (n = 4 technical replicates, mean ± SD).
Extended Data Fig. 3 Patient outcome analysis and biomarker studies.
(a) Overall survival of patients with complex cytogenetics. (b) Overall survival of patients with TP53 mutation. (c) Overall survival of patients harboring mutations in receptor tyrosine kinase gene (RTK). (d) Level of telaglenastat measured in plasma by LC-MS at indicated timepoints as compared between responders: CR and mCR, n = 7, mean ± SD, and patients with HI and NR n = 9, mean ± SD, ns-no significance. (e) Dynamic of changes in telaglenastat level measured in responders and non-responders at day15 of cycle1, day 1 of cycle 2 and at cycle 4 or later, (CR and mCR, n = 7, HI and NR n = 9, mean ± SD, ns-no significance).
Extended Data Fig. 4 scRNA UMAP presentation of selected biomarkers.
scRNAseq was conducted on bone marrow cells from CB839 treated patients. 5 clinical responders and 3 non responders were analyzed, and 11 distinct cell populations identified based on gene expression patterns. Representative UMAP figures show enrichment of CXCL8 (A), S100A9 (B) and S100A8 (C) expression shown between responders and non-responders.
Extended Data Fig. 5 Results for secondary transplantation of MDS PDX models.
(a-b) Percentage of mCD45−hCD45+ (a) and percentage of hCD45 + hCD33+ myeloid (b) cells in the bone marrow aspirate of NSG mice 5 weeks after the transplantation of human AML cells. Mice were randomly and blindly assigned to the vehicle and CB-839 treatment groups (mean ± SD, n = 5, unpaired t-test, ns-no significant). (c) Representative Flow cytometer on serial bone marrows from patients in complete remission (CR) before and after the combined treatment with AZA/Telaglenastat, showing a reduction in leukemic stem cells (LSCs) (CD34+/CD38-/Lin−ve with IL1RAP+).
Extended Data Fig. 6 Validation of SLC38A1 KO in vitro.
(a) Evidence of successful knockdown of SLC38A1 with siRNAs measured by RT-PCR in HEL cells. (mean ± SD, n = 3, unpaired t-test). (b) The intracellular levels of glutamine, glutamate and aspartate decrease in leukemic HEL cells following knockdown of the glutamate transporter SLC38A1 with siRNA. Knockdown of SLC38A1 led to reduced glutamine transport in leukemic HEL cells as measured by LCMS (mean ± SD, n = 4 replicates per condition, unpaired t-test). (c) Evidence of successful knockdown of SLC38A1 with lentiviral shRNA measured by RT-PCR in MOLM13 and THP1 cells (n = 3, mean ± SD, unpaired t-test). SLC38A1 KO GFP+ cells were FACS-sorted and used for growth and viability monitoring (D). (d) Cell growth and viability of MOLM13 and THP1 cells transfected with scrambled or SLC38A1 shRNA lentiviral vectors (n = 3, mean ± SD, unpaired t-test).
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DiNardo, C.D., Verma, D., Baran, N. et al. Glutaminase inhibition in combination with azacytidine in myelodysplastic syndromes: a phase 1b/2 clinical trial and correlative analyses. Nat Cancer 5, 1515–1533 (2024). https://doi.org/10.1038/s43018-024-00811-3
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DOI: https://doi.org/10.1038/s43018-024-00811-3
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