Mitochondrial Fission 1 Regulates GSK3 and AMPK Signaling to Sustain Leukemia Stem Cell Function in Acute Myelogenous Leukemia

Abstract Mitochondrial dynamics describes the ability of mitochondria to switch between fission and fusion-active states to change morphology. Although it has long been thought that mitochondrial dynamics is downstream of various growth/differentiation signals and metabolic cues, recent studies have shown that mitochondrial morphology can also actively regulate cellular metabolism and the cell fate of normal stem cells including hematopoietic stem cells. In the present studies, we examined the role of mitochondrial dynamics in the biology of human leukemia stem cells (LSCs). We observe that LSCs are highly sensitive to the perturbation of mitochondrial morphology and that defined signal transduction pathways are involved in this process. We therefore propose that maintenance of the LSC state is critically linked to mitochondrial dynamics. Our data show that LSC enriched populations derived from primary human acute myeloid leukemia (AML) specimens have hyper-active mitochondrial fission regulators as evidenced by higher levels of mitochondrial fission 1 (FIS1) and activating phosphorylation of dynamin-related protein 1 (DRP1). LSCs also contain an increased number of smaller and globular-shaped mitochondria, indicating that they reside in a fission-active state relative to non-LSCs. Inhibition of mitochondrial fission through genetic knock-down of FIS1 (FIS1-KD) induces mitochondrial fusion and dramatically diminishes both colony-forming ability and serial engraftment potential of primary AML cells, suggesting a fission-active state of mitochondrial morphology is critical for LSC function. To dissect the mechanism by which inhibition of mitochondrial fission impairs the stem and progenitor potential of LSCs, we performed detailed analyses of molecular events following FIS1-KD. We determined that FIS1-KD in AML cells simultaneously induces activating phosphorylation of AMPK and inhibitory phosphorylation of GSK3. Consistent with AMPK activation, FIS1-depleted AML cells had elevated oxidative phosphorylation (OXPHOS) activity, increased cellular ATP, and dramatic changes of various metabolomic intermediates, indicating a clear shift to active global energy metabolism. With regard to GSK3 inhibition, we found that the FIS1-KD-induced gene expression signature strongly phenocopied the gene signature obtained using a pan-GSK3 inhibitor or shRNA-mediated knock down of GSK3. Further, FIS1-KD in AML cells also strongly induced hematopoietic differentiation as evidenced by increased surface expression of hematopoietic lineage markers, global upregulation of hematopoietic differentiation genes, and collapse of the HOXA9 transcriptional program. Importantly, both shRNA-mediated AMPK inhibition and overexpression of a constitutively active GSK3 allele can rescue the FIS1-KD-induced hematopoietic differentiation phenotype, suggesting FIS1-KD-induced differentiation is dependent on both AMPK activation and GSK3 inhibition. To investigate if FIS1-KD-induced hematopoietic differentiation depends on an altered state of mitochondrial morphology, we also studied other regulators of mitochondrial dynamics including fission player DRP1 and fusion players mitofusin 2 (MFN2) and Optic Atrophy 1 (OPA1). We showed that shRNA-mediated KD of fusion players MFN2 and OPA1 can also rescue FIS1-KD induced hematopoietic differentiation, suggesting the balance between the activity of fission and fusion regulators is critical in determining the fate of LSCs. In addition, we showed that shRNA-mediated KD of the fission player DRP1 could also lead to AMPK activation and GSK3 inhibition, in strong agreement with FIS1-KD. Finally, we showed that combined treatment of AMPK activator and GSK3 inhibitors can severely kill AML cells, suggesting a potential therapeutic strategy. Taken together, we propose that mitochondrial dynamics plays a previously unrealized yet important role in sustaining LSCs of AML. Further, inhibition of mitochondrial fission players can activate AMPK signaling, force active energy metabolism, collapse GSK3-mediated transcription programs, and induce hematopoietic differentiation, all of which lead to loss of stem and progenitor potential of AML cells. Thus the current study reveals a novel dependence of LSCs on mitochondrial dynamics, and provides novel insights towards improved therapeutic regimens. Disclosures Pollyea: Celgene: Other: advisory board, Research Funding; Ariad: Other: advisory board; Glycomimetics: Other: DSMB member; Pfizer: Other: advisory board, Research Funding; Alexion: Other: advisory board.