Development of AMPK Inhibitors for the treatment of leukemia

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PROJECT SUMMARY Despite advances in the treatment of acute myeloid leukemia (AML), only 20–30% of patients achieve long-term disease-free survival (DFS) and treatment options for relapsed AML are extremely limited. The recurrence of AML has been attributed to leukemic stem cells (LSCs) and efforts are now focused on targeting this drug resistant population of cells in order to “cure” AML. Our studies measuring energy metabolism in primary human AML specimens, using reactive oxygen species (ROS) as an indicator of metabolic activity, revealed that LSCs preferentially reside in a ROS-low state. Furthermore, high levels of activated 5' AMP-activated protein kinase (AMPK), a central regulator of metabolic pathways, were detected in the LSCs and that knockdown of AMPK resulted in increased ROS levels and concomitant loss of LSCs. Based on these findings, we propose that AMPK inhibition will leverage LSCs out of the ROS-low state decreasing their viability which may be sufficient for LSC elimination or may sensitize them to conventional therapy. There are few potent and selective AMPK inhibitors; however, the multi-kinase inhibitor sunitinib has been reported as a potent inhibitor of AMPK kinase activity. Therefore, the central goal of our research is to develop potent and selective oxindole-based AMPK-targeted agents and examine the effect of AMPK inhibition or degradation in AML models. We have developed an initial series of oxindoles and although we identified potent AMPK inhibitors from this initial series, we believe further AMPK selectivity and inhibitory potency is possible. We will use computational-based modeling to guide the development of inhibitors and evaluate their AMPK inhibitory activity using in vitro kinase assays. Then, inhibition of cellular AMPK will be determined by measuring the phosphorylation of the AMPK substrate acetyl-CoA carboxylase (ACC) by ELISA in MOLM13 and MOLM14 cells, and select inhibitors will be submitted for kinome profiling (Aim 1). AMPK inhibitors that retain an aminoalkyl side-chain extending out of the ATP-binding site will be coupled to a proteolysis targeting chimera (PROTAC) degrader and their ability to degrade cellular AMPK will be evaluated. The oxindole-based AMPK inhibitors or degraders that have a terminal dimethylamino group that interacts with the DFG motif of AMPK will be modified to incorporate a nitroimidazole hypoxia-activated prodrug moiety that are designed to introduce a tier of LSC selectivity (Aim 2). The effect of AMPK inhibitors and PROTAC degraders on cell viability, metabolism and ROS levels as single agents will be determined in MOLM13 and MOLM14 cells and in primary AML cells, LSCs, and normal hematopoietic stem cells (HSCs). Then, the effect of AMPK inhibitors or degraders in combination with venetoclax will be determined in MOLM13 and MOLM14 cells and in primary AML cells. Finally, the effect of our AMPK inhibitors or degraders as single agents and in combination with venetoclax will be evaluated using primary AML specimens transplanted into advanced in vivo tumor xenograft models (Aim 3). The objective of these studies are to develop a range of chemical tools to evaluate the role of AMPK in maintaining LSC viability and the therapeutic potential of targeting AMPK in AML.