AMPK INHIBITION INDUCES APOPTOSIS IN PEDIATRIC B-ALL CELLS WITH MLL GENE REARRANGEMENTS

Background and Aims. Remarkable progress has been made in the past decade in pediatric Acute Lymphoblastic Leukemia (ALL) treatment, reaching cure rates of about 80%, but therapy is not yet effective in all cases. Infants with MLL gene rearrangements form the most striking example of patients who have not benefited from the improved treatment regimens. Consequently, current interest focuses on identifying new specific molecular targets to find new patient-tailored therapies. Thus, to identify aberrantly activated signal transduction pathways in MLL-rearranged patients, we used Reverse Phase Protein Microarrays (RPMA). This innovative technique can be used to quantify a highly multiplexed “portrait”of hundreds of signalling proteins at once from small clinical samples in a very reproducible, precise, sensitive and high-throughput manner. We further investigate RPMA results with in vitro studies testing the effects of a specific kinase inhibitor on apoptosis induction in leukemia cell lines. Methods. We compared with RPMA the signal transduction pathways working state of 8 MLL-rearranged patients vs 41 without known genomic translocations ones. The informed consent was obtained from all patients following the tenets of the Declaration of Helsinki. Phosphorylation status of 92 signalling proteins was analyzed. Based on RPMA results, we tested through proliferation and apoptosis assays the effect of Compound C, an AMPK inhibitor, on selected B-ALL human cell lines: 2 MLL-rearranged (SEM and RS4;11) and 2 non-translocated (MHH-CALL-2 and MHH-CALL-4). We then performed additional experiments to describe Compound C-induced apoptosis. Results. MLL-rearranged patients show an hyperactivated pathway that, through AMPK phosphorylation, leads to BCL-2 activation. Selected cell lines respond very differently to AMPK inhibition. GI50 (Growth Inhibition) at 48h is 0.2 µM for SEM, 3 µM for RS4;11, and 26 µM for non-translocated cell lines. LC50 (Lethal Concentration) at 48h is 7.5 µM for SEM, 8.5 µM for RS4;11 and 38 µM for non-translocated cell lines. Compound C treatment induces activation of Caspase-3, mitochondrial depolarization, ROS production, PARP cleavage, and DNA fragmentation. Conclusions. Our results thus demonstrate that the AMPK pathway is hyperactivated in MLL-rearranged patients, and it appears to directly contribute to the survival of MLL-rearranged cells. This study emphasizes the importance of protein pathway analysis as a route for discovery of functional derangement that may be functional, causative agents of cancer. Our data suggest AMPK as a new molecular target and encourage further studies of AMPK inhibitors as potential new drugs for treatment of MLL-rearranged leukemia patients.