Leukemia stem cell regulation and resistance

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PROJECT SUMMARY/ABSTRACT Chronic myelogenous leukemia (CML) results from hematopoietic stem cell (HSC) transformation by the BCR- ABL tyrosine kinase. Tyrosine kinase inhibitors (TKI) are effective in inducing remission and prolonging survival in CML patients, but fail to eliminate primitive leukemia stem cells (LSC) that can regenerate disease. Most patients need ongoing TKI treatment to maintain remission, and remain at risk of toxicity, financial hardship and non-adherence. The long-term goal of our research is to improve understanding of mechanisms of LSC resistance to treatment, to support development of effective and safe strategies for LSC targeting, and enhance possibilities of treatment-free remissions in CML patients. Mitochondrial metabolism plays a critical regulatory role in normal HSC function. CML LSC demonstrate increased mitochondrial oxidative phosphorylation (OXPHOS) compared to low OXPHOS in normal HSC. However, mitochondria also play important roles in metabolic processes besides OXPHOS, including fatty acid, glutamine and glucose oxidation, and generation of biosynthetic intermediates. The rationale for our studies is that specific mitochondrial metabolic alterations that contribute to altered LSC growth and TKI resistance are not known. Our preliminary studies show initial inhibition of OXPHOS in CML LSC after TKI treatment, but subsequent restoration of OXPHOS, and increased fatty acid oxidation (FAO), with continued treatment. A SIRT1, P53 and MYC regulatory network plays an important role in LSC propagation. We show that SIRT1 and its target PGC-1α play an important role in increased OXPHOS in CML LSC. PPARa, a PGC-1α-coactivated transcription factor and a key regulator of FAO, shows increased expression in CML LSC after TKI treatment, and contributes to increased OXPHOS, proliferation and survival. We will explore the hypothesis that increased FAO following BCR-ABL kinase inhibition, together with maintenance of high levels of OXPHOS, glycolysis and glutaminolysis, contributes to TKI resistance in CML LSC, and that metabolic regulatory mechanisms represent potential targets for elimination of TKI-treated CML LSC. In Specific Aim 1 we will use a combination of gene expression, extracellular flux, metabolite profiling and in vitro and in vivo metabolic labeling to study effects of TKI treatment on mitochondrial metabolism in CML LSC, examine the role of SIRT1, PGC1a and PPARa in metabolic alterations, and study interactions of MYC and p53 regulatory networks with mitochondrial metabolism. In Specific Aim 2 we will investigate the role of increased OXPHOS and FAO in promoting TKI resistance in CML LSC. Bone marrow microenvironment niches play a critical role in maintaining quiescent, TKI-resistant LSC populations. However, the role of the microenvironment in metabolic regulation of LSC growth is not known, and will be evaluated here . These studies are significant since they are expected to identify mechanisms of metabolic regulation underlying TKI resistance in CML LSC, establish connections between metabolism and other regulatory mechanisms in CML LSC, and identify new targets for therapy. The concepts developed here will have broad implications for other malignancies.