Pathway Analysis Provides Mechanistic Insights into Navtemadlin (KRT-232) Activity and Synergy with Decitabine in Acute Myeloid Leukemia Preclinical Models

Background: Murine double minute 2 (MDM2) is the primary negative regulator of the tumor suppressor protein, p53. Navtemadlin (KRT-232), a potent, selective, orally available MDM2 inhibitor, restores p53 activity to drive apoptosis of cancer cells in TP53 WT malignancies through the intrinsic (mitochondrial) pathway. Navtemadlin is being evaluated in a phase 3 trial in relapsed or refractory myelofibrosis, and in phase 1b/2 trials in various hematologic malignancies and solid tumors. Because p53 is infrequently mutated and MDM2 is often overexpressed in acute myeloid leukemia (AML), navtemadlin represents a rational therapeutic candidate (Prokocimer et al. Blood. 2017). Synergy between navtemadlin and the hypomethylating agent decitabine has been demonstrated in preclinical AML models (Canon et al. AACR. 2016), although the mechanisms mediating this effect remain unclear. The present study aims to identify mechanisms of navtemadlin-mediated apoptosis within AML cell lines, timing of pathway activation, and effects of combination treatment with navtemadlin + decitabine. Methods: MOLM13 or MV-4-11 AML cell lines were treated with a vehicle control, 0.5-1.0 µM navtemadlin, 1.0 µM decitabine, or with a combination of navtemadlin + decitabine. Cells were pretreated with decitabine for 42-48 hours followed by navtemadlin + fresh decitabine for 0, 5, 10, or 24 hours, and were then harvested for RNA-Seq and immunoblotting analyses of apoptotic pathway proteins. Gene set enrichment analysis (GSEA) and signaling pathway impact analysis (SPIA) were performed on RNA-Seq data to determine the most differentially regulated pathways (using KEGG curated signaling pathways) between controls, single-agent navtemadlin or decitabine, or combination treatment. Protein expression analyses were conducted to validate RNA-Seq data. Results: GSEA of MOLM13 and MV-4-11 cells at 10 hours showed that, relative to controls, several signaling pathways were differentially regulated to a statistically significant level (P≤0.05) with navtemadlin alone, including activation of apoptosis and p53 signaling pathways, and suppression of DNA replication and cell cycle pathways, in line with previous reports (Figure 1a). In MOLM13 cells treated with single-agent decitabine, lipid and proteoglycan-related signaling pathways were activated, whereas several metabolic pathways were suppressed. With combination treatment in both cell lines, unique activated pathways relative to controls included immun