METABOLIC CHANGES ASSOCIATED WITH METFORMIN POTENTIATES ANTI‐BCL‐2 INHIBITOR, ABT‐199, AND CDK9 INHIBITOR, BAY‐1143572 AND REDUCES VIABILITY OF LYMPHOMA CELLS

Abstract

Introduction: Metformin exerts anti-tumor effects in part by activating AMP-activated protein kinase (AMPK) with resultant reduction in pro-survival mTOR signaling. Here, we test the hypothesis that metformin induces cell death by altering mitochondrial metabolism in lymphoma cell lines. As Bcl-2 proteins, cyclin-dependent kinases (CDK) and Phosphoinositol-3-kinase (PI3K) also influence mitochondrial physiology and metabolism, we also explored the additive effects of metformin with novel agents including the Bcl-2 inhibitor (ABT-199), the CDK9 inhibitor (BAY-1143572) and the p110δ-selective PI3K inhibitor (CAL-101). Methods: Daudi (Burkitt), SUDHL-4 (GC DLBCL), Jeko-1 (MCL) and KPUM-UH1 (double hit DLBCL) cells were plated in 96-well plates and treated with varying doses of metformin (0–5000 μM). Hoechst 33342 DNA-binding dye or MTS assays were used to measure proliferation and cell viability, respectively. Perturbations in oxidative and glycolytic metabolism were monitored via Seahorse XF Energy Phenotype kits. Lastly, efficacy of combination therapy was evaluated in cells co-treated with metformin and ABT-199 or BAY-1143572 or CAL-101. Results: Daudi and SUDHL-4 cells showed 80% and 50% reduction in viability, respectively, with metformin at levels greater than 1 mM at day 7, while KPUM-UH1 and Jeko-1 cells were unaffected. The Seahorse analyses showed reduction in oxidative phosphorylation, in cells treated with 1mM metformin with a corresponding increase in glycolysis. Co-treating KPUM-UH1 and SUDHL-4 cells with 10 mM metformin resulted in 1.4-fold and 8.8-fold decreases, respectively, in IC50 values of ABT-199 at day 3 (Table 1). Jeko-1 and Daudi cells were resistant to ABT-199. By contrast, 3-fold and 10 fold reduction in IC50 values of BAY-1143572 in Daudi and Jeko-1, respectively, was seen in the presence of 10 mM Metformin, and SUDHL-4 or KPUM-UH1 cells were unaffected. No change in IC50 value for CAL-101 was observed. Conclusions: Metformin induces significant changes in cellular metabolism leading to decreased proliferation and viability in lymphoma cell lines. The combination of metformin and the anti-Bcl-2 agent ABT-199 achieves an additive effect in DLBCL (including double hit) cell lines, whereas the combination with CDK9 inhibitor BAY-1143572 provides more selective additive anti-tumor effect in Burkitt and MCL cell lines. These data support the idea of targeting cancer metabolism with metformin in novel combinatorial therapeutic strategies. Table: IC-50 concentrations for novel agents ±10 mM metformin Keywords: “double-hit” lymphomas; ABT-199; B-cell lymphoma.