SIRT1 Mediates Enhanced Mitochondrial Oxidative Phosphorylation in Chronic Myelogenous Leukemia Stem Cells
Authors: Abraham Ajay , Qiu Shaowei , Chacko Balu K , Li Hui , Paterson Andrew J , He Jianbo , Agarwal Puneet , Shah Mansi , Welner Robert S. , Darley-Usmar Victor M , Bhatia Ravi 
Published: October 2019

Despite the success of BCR-ABL tyrosine kinase inhibitors (TKIs) in inducing remission and prolonging survival of CML patients, cures remain elusive because of persistence of primitive leukemia stem cells (LSC). We have reported that SIRT1, a NAD+ dependent deacetylase, is overexpressed in CML LSC and contributes to their maintenance and TKI resistance, related at least in part to increased p53 acetylation and transcriptional activity (Li et.al; Cancer Cell 2012). However, these studies using RNAi and pharmacological inhibitors were limited by possible off-target effects and limited duration of exposure. Therefore, to definitively delineate the role of SIRT1 in CML development, we developed a conditional SIRT1 knockout mouse model by crossing SIRT1 floxed mice (SIRT1fl/fl) with the Mx1-Cre strain to delete SIRT1 in hematopoietic stem cells (HSC), and crossed with transgenic BCR-ABL mice (BA Mx1-Cre SIRT1fl/fl). SIRT1 deletion in normal HSC did not inhibit normal hematopoiesis. In contrast, SIRT1 deletion in LSC profoundly inhibited the development of CML, and SIRT1-deleted CML mice did not develop evidence of morbidity and demonstrated significantly lower leukocytosis, neutrophilia and splenomegaly, and markedly improved survival, compared to controls (Fig-1). CML development in the SCL-tTA/BCR-ABL mouse model is associated with reduction in BM stem cells, and increase in stem/progenitor cell populations in the spleen. Splenic stem and progenitor populations were significantly decreased in SIRT1-deleted mice, whereas BM LTHSC were increased, indicating reversal of alterations associated with CML. SIRT1 deletion was associated with a significant decrease in LTHSC in G0 phase and increase in LTHSC in G1 phase of cell cycle. SIRT1-deleted LTHSCs generated significantly lower WBC counts, donor cell and donor LTHSC engraftment following transplantation into wild-type recipients compared to controls, indicating reduced in vivo repopulating ability. Treatment with the BCR-ABL TKI Nilotinib (50mg/kg/day) or vehicle for 4 weeks resulted in additional suppression of CML hematopoiesis in SIRT1-deleted mice. Gene expression analysis identified significant inhibition of mitochondria and oxidative phosphorylation related genes in SIRT1-deleted CML stem cells compared to controls. We performed extracellular flux analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF analyzer to evaluate mitochondrial respiration and glycolysis in c-Kit+ stem/progenitor cells from SIRT1-deleted and control CML and normal mice. CML LSC demonstrated significantly increased ATP-linked respiration, maximal mitochondrial respiration and mitochondrial reserve capacity compared to normal HSC. SIRT1-deletion resulted in significant reduction in basal and maximal mitochondrial respiration, and in mitochondrial reserve capacity, in CML LSC (Fig-2). In contrast, SIRT1-deletion did not result in significant change in mitochondrial respiration in normal HSC, suggesting that effects of SIRT1 deletion on mitochondrial respiration were specific to CML cells. The SIRT1 substrate PGC-1α is a master regulator of mitochondrial biogenesis and activity. Treatment of CML mice with the PGC-1α inhibitor SR-18292 significantly reduced basal, ATP-linked and maximal mitochondrial respiration. Consistent with results obtained with murine LSC, SIRT1 inhibition with TV39OH resulted in significant reduction in mitochondrial respiration in CML CD34+ cells compared to vehicle controls. Treatment of CML mice with Nilotinib did not significantly alter mitochondrial respiration in LSC. Similarly TKI treatment did not affect mitochondrial respiration in CML CD34+ cells compared to vehicle controls. In conclusion, our studies definitively demonstrate that SIRT1 is required for leukemia development, and reveal its critical role in increased mitochondrial respiration in CML LSC. Increased mitochondrial metabolism is kinase-independent, and is mediated by PGC-1a. SIRT1 inhibition results in enhanced inhibition of TKI-treated LSC. Our studies demonstrate an essential role for SIRT1 in altered mitochondrial metabolism in CML LSC, and support further exploration of SIRT1 pathway inhibition for LSC targeting.

Disclosures

No relevant conflicts of interest to declare.