Decitabine Activity Against <i>TP53</i>-Mutated Myeloid Neoplasms Is Enhanced By Combination with a Non-Cytotoxic p53/BAX-Independent Inhibitor of Pyrimidine Synthesis

Introduction. DNA methyltransferase 1 (DNMT1)-degraders decitabine (Dec) or 5-azacytidine (Aza) (hypomethylating agents, HMA) with or without the BCL2-inhibitor venetoclax (Ven), are standard-of-care for TP53-mutated (m) acute myeloid leukemias (AML). Relapse is, however, rapid, motivating study and remedy of HMA-resistance. Methods and Results. (i) HMA-resistant TP53-m (THP1, K562) and TP53-wildtype (WT) AML cells (MOLM13) were selected by culture in clinical Dec and Aza concentrations (1.5 and 5 µM respectively). DNMT1 protein (Western blot, WB) was preserved despite continuous HMA exposures, corroborated by rebounding of DNMT1-protein (immunohistochemistry) in marrow biopsies obtained at resistance vs response to HMA clinical therapy (n=27). (ii) To deplete DNMT1, Dec and Aza, being analogs of deoxycytidine (dC) and cytidine (C) respectively, must compete with these endogenous pyrimidine nucleosides. We found 2 to 7-fold higher dC/C in resistant versus parental AML cell lysates (liquid chromatography tandem mass spectrometry, LCMS/MS). Methods to decrease dC/C and/or their phosphorylated (nucleotide) forms, are thus candidate solutions for HMA-resistance. (iii) Ven inhibits BCL2, which releases BAX that depolarizes mitochondrial membranes, thereby inhibiting nucleotide synthesis at the mitochondrial step. We measured Ven impacts on nucleotide levels in TP53-m vs WT AML cells and found less decrease in TP53-m cells. Showing a reason for this, BAX was ~2-fold lower in TP53-m versus WT AML cells (p&amp;lt;0.001, TCGA, CCLE data)(BAX is a p53-target gene). (iv) We thus considered p53/BAX-independent methods to reduce pyrimidine nucleotides: teriflunomide (Teri), approved to treat multiple sclerosis, directly inhibits de novo pyrimidine synthesis at dihydroorotate dehydrogenase (DHODH). Clinical concentrations of Teri (10-20 µM, clinical Cmax &amp;gt;100 µM) decreased pyrimidine nucleotides in both TP53-m and WT AML cells &amp;gt;5-fold more than Ven (p&amp;lt;0.001). (v) Teri, but not Ven, accordingly renewed DNMT1-depletion and terminated proliferation of p53-null HMA-resistant AML cells (THP1). (vi) Moreover, Teri single-agent, but not Ven, significantly decreased proliferation of parental p53-null AML cells (THP1) without activating apoptosis (annexin/PI) (non-cytotoxic, p53-independent mechanism). However, Teri-resistant AML cells rapidly emerged. (vii) To investigate Teri-resistance, we measured metabolites up- and down-stream of DHODH (LCMS/MS): upstream substrate aspartate was higher, while downstream metabolites orotate and oritidine monophosphate were lower vs controls, indicating ongoing DHODH-inhibition. However, dC-monophosphate (dCMP) levels, expected from deoxycytidine kinase (DCK)-mediated salvage, were similar to controls. Explaining this, DCK protein (WB) was increased. Moreover, DCK mRNA and protein increased within 24 hrs of AML cell exposures to Teri, indicating automatic adaptive response to de novo synthesis inhibition, extending data showing Dec/Aza interactions with pyrimidine-metabolism also trigger metabolic rewiring for homeostasis (adaptive resistance) (Leukemia. 2021;35(4):1023). (viii) We thus evaluated metronomic drug administration schedules in vivo (1X/wk), to allow metabolic shifts to revert between drug exposures, and decrease chances of stabilizing these shifts into adaptive-resistance. Since Teri triggers DCK-mediated salvage, and Dec is taken-up by DCK salvage, we also tested different timings for Teri/Dec combinations. NSG mice were tail-vein inoculated with 1x106 AML cells from a patient with TP53-mutated complex cytogenetics AML. Treatment was initiated after confirmation of &amp;gt;10% hCD45+ cells in marrow: Teri 35 mg/kg oral day-before Dec 0.2 mg/kg subcutaneous 1X/wk was superior to same-day or day-after (n=6/gp, Log-Rank p=0.02, median time-to-distress days: Vehicle 50; Teri 70; Dec 70; Teri same-day or day-after Dec 85; Teri day-before Dec 95). In a separate experiment, Teri/Dec 35/0.2 mg/kg 1X/wk was superior to Ven/Dec 100/0.2 mg/kg 1X/wk (n=7/gp, Log-Rank p=0.001, median time-to-distress days: vehicle 65; Ven/Dec 72; Teri/Dec 95) - repeated with similar results. Conclusion. Mechanism and in vivo proof-of-principle thus support metronomic, timed-combination Teri/HMA, or Teri pro-drug leflunomide/HMA, as candidate solutions for Ven/HMA-resistance, including to treat TP53-mutated disease.