P413: THE POTENTIAL USE OF AN ORAL HYPOMETHYLATING AGENT, OR-2100, AS A COMBINATION THERAPY FOR MYELOID MALIGNANCIES

Abstract

Background: The combination therapy of hypomethylating agents (HMAs), azacytidine (AZA) and decitabine (DAC), with venetoclax (VEN), a selective BCL-2 inhibitor, have revolutionized the outcome of elderly patients with acute myeloid leukemia (AML), which have demonstrated the potential effect of HMAs as a combination therapy with molecular targeting agents for myeloid malignancies. However, there are a few studies on the combined effect of HMAs with tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and the mechanism by which HMAs enhance the efficacy of molecular targeting agents remain poorly understood. We have developed OR-2100 (OR21), an oral HMA, a 5’-O-trialkylsilylated DAC. OR21 is as effective as DAC, but less myelosuppressive, in xenograft mouse models of solid and hematological malignancies (Hattori et al. Clin Epigenetics 2019, Watanabe et al. Blood 2020, Ureshino et al. Mol Cancer Ther 2021, Kamachi et al. Cancer Letters 2022). We investigated the efficacy and mechanism of OR21 in combination with VEN or TKIs for myeloid malignancies, AML, and CML. Aims: To investigate the efficacy and mechanism of the combination therapy of OR21 for myeloid malignancies. Methods: We investigated the efficacy and mechanism of OR21 in combination with VEN against AML and with TKIs against CML, in vitro and in vivo. Results: In AML, OR21 plus VEN synergistically inhibited cell growth, increased apoptosis in AML cell lines as similar to AZA and DAC, and significantly prolonged survival in a xenograft mouse model using HL60 cell line (p<0.05). Gene expression analysis using AML cell lines (HL60, KG1a) showed that OR21 plus VEN significantly downregulated VAMP7 which regulates mitophagy/autophagy to maintain mitochondrial homeostasis compared to VEN monotherapy. Consistently, OR21 plus VEN enhanced apoptosis by increasing ROS accumulation and attenuated mitophagy/autophagy response triggered by decreased mitochondrial membrane potential. The addition of mitophagy inducers, such as rapamycin and p62-mediated mitophagy inducer, to OR21 plus VEN decreased ROS accumulation and apoptosis, indicating OR21 enhanced ROS accumulation via attenuating VEN-induced mitophagy/autophagy pathway. In CML, OR21 plus TKIs (imatinib, dasatinib, ponatinib) synergistically inhibited cell growth, increased apoptosis in CML cell lines as similar to AZA and DAC, and significantly suppressed the colony-forming capacity of bone marrow CD34+ stem/progenitor cells in CML patients. OR21 plus imatinib significantly suppressed tumor growth compared to imatinib monotherapy in a xenograft mouse model using K562 cell line (p<0.05). In phosphoproteomic, transcriptome and methylome analysis (K562, KBM5), OR21 augmented imatinib-induced apoptosis by dephosphorylating of STAT and Src family proteins by upregulating several tumor suppressor genes, including PTPN6, through demethylation of their promoter CpG regions. Image:Summary/Conclusion: OR21 enhanced the anti-tumor effect in combination with the molecular agents for myeloid malignancies with different mechanisms: OR21 increased ROS accumulation in combination with VEN, and dephosphorylated BCR-ABL1 downstream protein kinases by demethylating effect in combination with TKIs. With its advantage as an oral HMA, OR21 is expected to have future clinical trials as a combination therapy for myeloid malignancies.