Targeting MicroRNAs to Eradicate Leukemia Stem Cells

PROJECT SUMMARY Leukemia stem cells (LSCs) are at the apex of the acute myeloid leukemia (AML) cellular hierarchy. The quiescent fraction of LSCs provides a reservoir of self-renewing cells that sustain leukemia growth, prevent clonal exhaustion, and are treatment resistant; thus, eliminating LSCs is the `holy grail' of any anti-leukemia treatment. In previous studies, we showed that miR-126 is necessary to maintain a quiescent subfraction of LSCs that prevent clonal exhaustion. We demonstrated how SPRED1/miR-126 autoregulatory loop in LSCs and in BM endothelial cells (ECs) converge to increase miR-126 levels in LSCs, protect them and support leukemia growth. We showed that high miR-126 levels are due to both LSC autonomous mechanisms, resulting in enhanced endogenous production, and non-autonomous mechanisms, through exogenous miR-126 supply from ECs. To deplete miR-126 in LSCs and ECs, we designed a novel oligodeoxynucleotide anti-miR-126 inhibitor, called miRisten. Our data show that pharmacological miR-126 deprivation by miRisten significantly decreases LSC endogenous production of miR-126 and decreases the exogenous supply of endothelial miR-126. The net result is a significant decrease of miR-126 that damages the homeostasis and activity of LSCs, as demonstrated in serial transplant experiments. In addition, we now have evidence that miR-126 enhances mitochondrial metabolism (i.e., oxidative phosphorylation) and mitochondrial dynamics (i.e., mitochondrial fusion) in LSCs through SPRED1/ERK/p-BCL-2/NRF2 signaling. Accordingly, depletion of miR-126 by miRisten treatment significantly downregulates BCL-2 and disrupts mitochondrial metabolism, leading to increased levels of reactive oxygen species and apoptosis of LSCs. In addition, miRisten disrupts LSC mitochondrial function by upregulating the dynamin related protein 1 (DRP1), inducing mitochondrial fission, decreasing mitochondrial membrane potential, and inducing expression of mitophagy marker proteins. Since mitochondria-centered metabolism is the main metabolic energetic source for LSCs, we propose to dissect how miRisten exploits the mitochondrial metabolic vulnerability as a novel mechanism of action to eliminate LSCs. Furthermore, after conducting Investigational New Drug application (IND)-enabling pharmacokinetic, pharmacodynamic and toxicology studies, we will rapidly translate miRisten from bench to beside with a first-in-human phase 1 clinical trial of miRisten in patients with relapsed/refractory (r/r) AML. The central hypothesis of this proposal is that miRisten targets miR- 126-depended metabolic vulnerability of LSCs and will provide a novel therapeutic approach for LSC elimination in AML. We propose the following Specific Aims (SAs): SA#1: Determine the mechanisms of miRisten-induced mitochondrial metabolic vulnerability in LSCs. SA#2: Conduct pharmacokinetic, pharmacodynamic, efficacy and toxicology studies of miRisten to inform dose and schedule selection for human studies. SA#3: Conduct a first-in-human phase 1 trial of miRisten in patients with r/r AML. This project will translate novel discoveries on miR-126 into the clinic, by conducting preclincal studies that culminate in a first-in-human trial of miRisten.