ASTX295 a Novel Potent MDM2 Antagonist Induces More Than One Mechanism of Programmed Cell Death (PCD) in Lymphoid Malignancies

Introduction: Targeting the Mouse double minute 2 (MDM2)-p53 interaction, results in reactivation of wild-type (WT) TP53 and induction of transcriptional targets, causing cell death and cell cycle arrest. However, previous clinical studies with MDM2 inhibitors have shown limited efficacy and on-target thrombocytopenia. ASTX295 is an isoindolinone-based MDM2 antagonist that inhibits MDM2 with an IC50 of <1nM by ELISA. It has a short plasma half-life of <6 hours, resulting in reduced hematological toxicity. Previous studies have shown in vitro activity in acute myeloid leukemia both alone and in combination with decitabine. We sought to assess the activity of ASTX295 in vitro in lymphoid malignancies and to determine mechanisms of cell death.

Methods: Using a panel of 12 TP53^WT DLBCL, MCL, and T-cell lymphoma cell lines and 25 primary MCL patient samples, cells were treated for up to 72 hours with a range of concentrations of ASTX295 (0-10000nM) and viability measured using Cell-Titer Glo (CTG). IC50 values were calculated. Cells were stained with Annexin V and Propidium Iodide and cell death measured by flow cytometry. TP53 mutational status of patient samples was determined by Sanger sequencing. To determine the mechanisms of programmed cell death (PCD), cell lines were pre-treated with both caspase dependent, QVD-OPh, and caspase independent inhibitors of cell death, Ferrostatin-1 and Necrostatin-1 prior to treatment with ASTX295 and electron microscopy imaging performed. Activity of ASTX295 was compared with AMG232 and Idasanutlin.P53 pathway protein modulation in vivo (p53, MDM2 and p21) was assessed by western blot. The effects of ASTX295 in combination with clinically relevant small molecule inhibitors including BH3 mimetics, PI3K inhibitors and CDK4/6 inhibitors were assessed to identify synergistic combinations.

Results: All 12 TP53^WT cell lines were sensitive to ASTX295 treatment (IC50: < 1nM - 100nM). In most cell lines tested, cell death was slow and maximal after 72h exposure. In contrast, Z138 showed evidence of cell death after 24hours. MDM2 inhibition induced the expression of p53 and its transcriptional targets including p21. In primary TP53^WT MCL patient samples 12/25 patient samples were sensitive to ASTX295 (IC50: 4.3nM - 100nM at 72 hours). ASTX295 showed higher levels of cell death at 72 hours than other MDM2i. Screening of cell lines and patient samples also revealed a subset that were less sensitive to ASTX295 despite expressing TP53^WT. ASTX295 in combination with the specific BCL2 inhibitor Venetoclax in the GRANTA519 cell line (CI value 0.2) and KARPAS384 (CI value 0.4) demonstrated strong synergy, despite as monotherapy resulting in <50% fall in viability. Biochemical studies, chemical inhibitors and transmission electron microscopy in TP53^WT DLBCL/MCL cell lines showed ASTX295 induced changes associated with both caspase-dependent and independent cell death pathways. Cell death occurring within 24h of exposure to ASTX295 was associated with features of classical caspase-mediated apoptosis, whereas later time points were not.

Conclusions: ASTX295 shows greater potency than other MDM2i in lymphoid cell lines, demonstrating up to 10 fold greater potency and is now undergoing evaluation in the clinical trial NCT03975387. ASTX295 like other MDM2i showed synergy with BCL2i in DLBCL and MCL models. ASTX295 induced multiple forms of PCD; the precise form of non-caspase dependent PCD remains under investigation.

Walter:NIHR/CRUK, Pfizer, Innovate UK: Honoraria, Research Funding. Sims:Astex Pharmaceuticals: Current Employment.