Synergistic Cytotoxicity in Acute Myeloid Leukemia By Combined Treatment with Metformin and 6-Benzylthioinosine

Background: Cytogenetic and molecular aberrations have improved risk stratification of acute myeloid leukemia (AML) patients, however survival continues to be low (40-50%) and current therapy primarily includes high dose cytotoxic chemotherapy with or without allogeneic stem cell transplant. In AML, metabolomic analysis has identified key differences in glycolysis, gluconeogenesis and tri-carboxylic acid (TCA) pathways between normal and AML cells. Monotherapy with mitochondrial inhibitors like Metformin have met with little success since cancer cells use glycolytic escape mechanisms to overcome the decreased mitochondrial oxidative phosphorylation. Here we demonstrate a novel therapeutic approach for overcoming this route of Metformin resistance.

Methods: Metformin, a well-established anti-diabetic drug, is a strong inhibitor of mitochondrial respiration and is also considered an inhibitor of mTOR signaling acting via AMPK activation. Despite its therapeutic potential, cytotoxicity of AML cells with Metformin alone at achievable plasma levels (10-20 mM) is modest due in part to increased glycolytic ATP production as a bypass mechanism. This has limited the effectiveness of Metformin in AML and led us to investigate mechanisms for reversal of this resistance mechanism to improve the pre-clinical efficacy. 6-Benzylthioinosine (6-BT) is a small molecule that we have shown depletes ATP and can cause differentiation in myeloid leukemia cells (at 10µM concentration) with minimal toxicity to normal cells (LD50 >100µM) (Wald et al. Cancer Res, 2008). To improve on cytotoxicity seen with Metformin, we hypothesized that the combination of 6-BT and Metformin would enhance cytotoxicity in AML. The combination of Metformin (10mM) and 6-BT (10 µM) were tested alone or combined. We compared the effects of the combination treatment on mTOR signaling and caspase-3 activation. We also measured changes in reactive oxygen species (ROS), intracellular ATP, extracellular glucose levels and GLUT-1 expression. Glycolytic flux was measured by Extracellular Acidification Rate (ECAR) and oxidative phosphorylation was measured by Oxygen Consumption Rate (OCR) using Seahorse XF Analyzer. All experiments were performed in triplicate and Student’s two tailed t-test was used to calculate p-values (less than 0.05 were considered to be statistically significant).

Results: Metformin and 6-BT alone caused inhibition of cell growth (< 3-fold) and modest cytotoxicity (10-40%, SD ± 6.3%) in all AML cell lines tested (MV4-11, MOLM-14, OCI-AML3, Nomo-1 and THP-1). However the combination of 6-BT with Metformin resulted in marked growth inhibition (30-50 fold) and cytotoxicity (90-100%, SD ± 8.3%) in monocytic AML cell lines (MV4-11 and MOLM-14) within 48 hours of exposure, which was partially dependent on caspase-3 induction. Apoptosis induction was also detected by Annexin V assay. Cytotoxic effects were independent of mTOR inhibition since AZD8055 showed no synergy with the 6-BT or the combination. 6-BT has structural similarity with 6-Mercaptopurine (6-MP), however the combination of Metformin and 6-MP also showed no synergy. 6-BT combined with Metformin caused significant (50-70%) reduction in intracellular ATP levels in the monocytic AML lines without significant extracellular glucose depletion suggestive of glycolytic inhibition. As expected, Metformin significantly suppressed oxidative phosphorylation as evident by near complete inhibition of OCR (<0 pmols/min) and subsequent reduction of ROS to levels <40% of normal. 6-BT alone showed significant suppression of glycolysis with reduction in ECAR but more strikingly reduced by 50% the glycolytic flux normally induced by Metformin and was accompanied by a 50% reduction in the mRNA levels for the glucose transporter GLUT-1.

Conclusions: The anti-tumor and metabolic effects of Metformin have been limited by the metabolic reprogramming within cells that result in low cytotoxicity. Here we show that the novel combination of 6-BT and Metformin targets this metabolic bypass mechanism resulting in increased cell death. This is the first demonstration that 6-BT, a drug that is selectively taken up and phosphorylated by AML cells, can exert cytotoxicity involving down modulation of glycolysis. We propose that the combination of 6-BT and Metformin represents a new therapeutic strategy that warrants further testing in primary patient samples.