Inhibition of De Novo Pyrimidine Synthesis Depletes Acute Myleogenous Leukemia Stem Cells (LSCs) Burden and Triggers Apoptosis and Differentiation Associated with Oxidative Stress

ABSTRACT

Background: De novo nucleotide synthesis is necessary to meet the enormous demand for nucleotides, other macromolecules associated with acute myeloid leukemia (AML) progression 1, 2, 34. Hence, we hypothesized that targeting de novo nucleotide synthesis would lead to the depletion of the nucleotide pool, pyrimidine starvation and increase oxidative stress preferentially in leukemic cells compared to their non-malignant counterparts, impacting proliferative and differentiation pathways. Emvododstat (PTC299) is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis that is currently in a clinical trial for the treatment of AML. Objectives: The goals of these studies were to understand the emvododstat-mediated effects on leukemia growth, differentiation and impact on Leukemia Stem Cells(LSCs). Comprehensive analyses of mitochondrial function, metabolic signaling in PI3K/AKT pathways, apoptotic signatures, and DNA damage responses were carried out. The rationale for clinical testing emvododstat was confirmed in an AML-PDX model. Results: Emvododstat treatment in cytarabine-resistant AML cells and primary AML blasts induced apoptosis, differentiation, and reduced proliferation, with corresponding decreased in cell number and increases in annexin V- and CD14-positive cells. Indeed, the inhibition of de novo nucleotide synthesis compromises the dynamic metabolic landscape and mitochondrial function, as indicated by alterations in the oxygen consumption rate (OCR) and mitochondrial ROS/membrane potential and corresponding differentiation, apoptosis, and/or inhibition of proliferation of LSCs. These effects can be reversed by the addition of exogenous uridine and orotate. Further immunoblotting and mass cytometry (CyTOF) analyses demonstrated changes in apoptotic and cell signaling proteins (cleaved PARP, cleaved caspase-3) and DNA damage responses (TP53, γH2AX) and PI3/AKT pathway downregulation in response to emvododstat. Importantly, emvododstat treatment reduced leukemic cell burden in a mouse model of AML PDX ( Complex karyotype, mutation in ASXL1, IDH2, NRAS), decreased levels of leukemia stem cells frequency (1 in 522,460 Vs 1 in 3,623,599 in vehicle vs emvododstat treated mice), and improved survival. The median survival 40 days vs. 30 days, P=0.0002 in primary transplantation and 36 days vs 53.5 days, P=0.005 in secondary transpantation in a PDX mouse model of human AML. This corresponded with a reduction in the bone marrow burden of leukemia and increased expression of differentiation markers in mice treated with emvododstat (Fig. 1). These data demonstrate effect of emvododstat on mitochondrial functions. Conclusion: Inhibition of de novo pyrimidine synthesis triggers differentiation, apoptosis, and depletes LSCs in AML models. Emvododstat is a novel dihydroorotate dehydrogenase inhibitor being tested in a clinical trial for the treatment of myeloid malignancies and COVID-19. Keywords AML, emvododstat, DHODH, apoptosis, differentiation References 1 Thomas, D. & Majeti, R. Biology and relevance of human acute myeloid leukemia stem cells. Blood 129, 1577-1585, doi10.1182/blood-2016-10-696054 (2017). 2 Quek, L. et al. Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage. The Journal of experimental medicine 213, 1513-1535, doi10.1084/jem.20151775 (2016). 3 Villa, E., Ali, E. S., Sahu, U. & Ben-Sahra, I. Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides. Cancers (Basel) 11, doi10.3390/cancers11050688 (2019). 4 DeBerardinis, R. J. & Chandel, N. S. Fundamentals of cancer metabolism. Sci Adv 2, e1600200, doi10.1126/sciadv.1600200 (2016). [Formula presented] Disclosures Weetall PTC therapeutics Current Employment. Sheedy PTC therapeutics Current Employment. Ray PTC therapeutics Current Employment. Andreeff Karyopharm Research Funding;AstraZeneca Research Funding;Oxford Biomedica UK Research Funding;Aptose Consultancy;Daiich -Sankyo Consultancy, Research Funding;Syndax Consultancy;Breast Cancer Research Foundation Research Funding;Reata, Aptose, Eutropics, SentiBio;Chimerix, Oncolyze Current holder of individual stocks in a privately-held company;Novartis, Cancer UK;Leukemia & Lymphoma Society (LLS), German Research Council;NCI-RDCRN (Rare Disease Clin Network), CLL Foundation;Novartis Membership on an entity's Board of Directors or advisory committees;Senti-Bio Consultancy;Medicxi Consultancy;ONO Pharmaceuticals Research Funding;Amgen Research Funding;Glycomimetics Consultancy. Borthakur ArgenX Membership on an entity's Board of Directors or advisory committees;Protagonist Consultancy;Astex Research Funding;University of Texas MD Anderson Cancer Center Current Employment;Ryvu Research Funding;Takeda Membership on an entity's Board of Directors or advisory committees;Novartis Consultancy, Membership on an entity's Board of Directors or advisory committees;GSK Consultancy.