Navtemadlin, a Novel MDM2 Inhibitor, Potentiated Venetoclax-Induced Antitumor Efficacy in TP53 Wild-Type Acute Myeloid Leukemia (AML)

Introduction: The tumor suppressor p53 ( TP53) maintains genomic integrity, and nearly 50% of cancers are characterized by a loss or mutation in the TP53 gene. Murine double minute 2 (MDM2) negatively regulates p53 expression and promotes oncogenesis in TP53 wild-type ( TP53^WT) tumors. Navtemadlin (nvtm) is a potent, selective, orally available MDM2 inhibitor that overcomes MDM2 dysregulation by restoring p53 activity and inducing apoptosis in TP53^WT tumors.

AML is a heterogeneous hematological malignancy resulting from the transformation of stem cells and is characterized by poor therapy response. Thus, combination therapies are required to avoid tumor escape mechanisms, such as alterations in apoptotic machinery and metabolism. Venetoclax (ven), a BCL-2 inhibitor, is a promising therapy in AML. Overexpression of anti-apoptotic members of the BCL-2 family, including myeloid cell leukemia-1 (MCL-1) limits ven response, thus requiring MCL-1 inhibitors to further improve sensitivity. A proof-of-concept study with idasanutlin (idasa), a first-generation MDM2 inhibitor and ven demonstrated manageable safety and encouraging efficacy in relapsed/refractory AML patients (Daver et al. Blood 2023). Here, we present a preclinical evaluation of nvtm alone and in combination with ven in the setting of TP53^WT AML, with a focus on metabolic pathways, pro-survival signaling, and bone marrow (BM) niche.

Methods: AML-derived cell line, MOLM-13 ( TP53^WT, FLT3-ITD) was cultured alone, with HS5 AML stromal cells or with cytokines. Cultures were treated with DMSO, nvtm (0.1-1.5 μM), or ven (0.5 μM) for short-term (4-8 hrs) or long-term (48-72 hrs) and analyzed for changes in cell cycle progression, viability, clonogenicity potential, and metabolism (glycolysis and mitochondrial oxidative phosphorylation by analyzing extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) respectively). Primary AML BM mononuclear cells (MNCs) were cultured in cytokine support media.

Results: In MOLM-13 cells, short-termnvtm (0.5 µM) treatment rapidly restored p53 expression and phosphorylation, with greater efficacy than idasa. Long-term treatment significantly induced apoptosis in a dose-dependent manner from 0.25 μM (p<0.01) with 80% cytotoxicity at 0.75 μM (p<0.001). Cell cycle analysis at 48 hr showed that nvtm significantly arrested cells in the sub-G0-G1 phase. Clonogenicity potential was significantly lowered in nvtm-treated cells. Metabolic alterations in the Mito Stress and Glycostress tests showed nvtm significantly lowered EACR, OCR, and mitochondrial membrane potential, as compared to idasa.

We next evaluated the effects of nvtm in ven-treated MOLM-13 cells. Combination treatment significantly lowered the metabolism, cell viability, and clonogenicity potential of MOLM-13 cells, compared to nvtm or ven treatment. Immunoblotting analysis revealed a reduction in key anti-apoptosis proteins (BCL-2, MCL-1, PARP). Strikingly, nvtm downregulated ven-induced MCL-1 expression in a dose-dependent manner. Culturing MOLM-13 cells in HS5-conditioned media, transwells overlying HS5 cells, or with HS5 cells increased MOLM-13 cell resistance to nvtm. However, in HS5 co-cultures, a combination of nvtm and ven strongly re-sensitized MOLM-13 cells for apoptosis. Nvtm also significantly blocked the cytoprotective effects of AML-relevant cytokines (IL-6, SCF, FLT3 ligand, TPO, and GM-SCF). In cultures of primary AML BM-MNCs (n=11), nvtm alone at clinically relevant concentrations reduced viability (14-40%), and a combination nvtm and ven significantly enhanced AML cytotoxicity (30-52%).

Conclusions: Nvtm, a potent second-generation MDM2 inhibitor, rapidly restored p53 function, induced apoptosis in p53 ^WT AML MOLM-13 by impeding cell cycle, viability, clonogenicity potential, and reducing both glycolysis and oxidative phosphorylation. Nvtm combined with ven significantly enhanced these effects. Importantly, this combination blocked stromal cell-mediated (contact and soluble factors) cytoprotection. These encouraging results provide a mechanistic basis for the evaluation of this combination in vivo and as a therapeutic strategy to overcome documented ven resistance mechanisms in TP53^WT AML patients.