Mitochondrial Carrier Homolog 2 (MTCH2) Regulates the Differentiation of AML Cells By Controlling Pyruvate Entry into the Mitochondria, Nuclear Localization of Pyruvate Dehydrogenase Complex and H3 and H4 Histone Acetylation

Abstract

Mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein that functions as a receptor for pro-apoptotic BID. MTCH2 also regulates metabolism through undefined mechanisms.We identified MTCH2 as essential for AML growth and viability in a CRISPR screen. OCI-AML2 leukemia cells overexpressing CAS9 were transduced with a library of 91,320 sgRNAs in barcoded lentiviral vectors. We focused on the gRNA that targeted the 1050 mitochondrial proteins and identified MTCH2 within the top 1% of hits. We validated this finding using two individual sgRNAs, where knockout of MTCH2 reduced the growth and viability of CAS9-OCI-AML2 cells by >75%. Additionally, knockdown of MTCH2 with shRNA reduced growth and viability of OCI-AML2 (>90%), TEX (>80%), U937 (>65%), and HL60 (>75%) leukemia cells. Knockdown of MTCH2 targeted the leukemia initiating cells as it abrogated engraftment of TEX cells into immune deficient mice. We also infected hematopoietic cells from wild type and MTCH2 flox/flox Vav1-cre mice with the MLL-AF9 oncogene. Knockout of MTCH2 decreased the leukemogenic potential of these cells and increased survival of the mice.In contrast, knockout of MTCH2 has more subtle effects on normal murine hematopoiesis. flox/flox Vav1-cre MTCH2 knockout mice had normal peripheral blood counts, but a ~15% reduction in the abundance of hematopoietic stem cells with a slight increase in the restricted progenitor populations (Maryanovich et al, Nat Comm 2015).Murine hematopoietic cells from MTCH2 -/- mice have increased mitochondrial mass and increased oxidative phosphorylation. Yet, no changes in basal oxygen consumption, mitochondrial ROS, or respiratory chain activity were seen in AML cells after MTCH2 knockdown.We examined changes in gene expression by RNA-sequencing in OCI-AML-2 cells after MTCH2 knockdown. MTCH2 knockdown increased expression of genes involved in cellular differentiation and decreased genes that maintain stemness. Therefore, we analyzed differentiation of AML cells after MTCH2 knockdown and demonstrated increased non-specific esterase staining and increased expression of CD cell surface markers associated with differentiation.In lung cancer mitochondrial stress induces the translocation of pyruvate dehydrogenase (PDH) from the mitochondria to the nucleus, where it increases conversion of pyruvate to Acetyl CoA which, in turn, acetylates histones. Therefore, we examined changes in PDH localization and histone acetylation after MTCH2 knockdown in OCI-AML-2 and TEX cells. MTCH2 knockdown decreased mitochondrial pyruvate, increased nuclear localization of pyruvate dehydrogenase complex (PDH) and increased acetylation of H3 and H4 histones. Over-expression of the PDH E2 subunit with a nuclear localizing signal mimicked MTCH2 knockdown and decreased cell growth, induced differentiation, and increased H3 and H4 histone acetylation in. Likewise, the acetyltransferase inhibitor, C646, abrogated the effects of MTCH2 knockdown on histone acetylation, growth and differentiation. We also demonstrated that knockdown of MTCH2 decreased levels of mitochondrial pyruvate in OCI-AML-2 cells.The entry of pyruvate into the mitochondria is regulated by the Mitochondrial Pyruvate Carrier (MPC). Therefore, we treated AML cells (OCI-AML2, TEX, HL60, and 8227) with the selective MPC inhibitor UK5099. UK5099 mimicked the effects of MTCH2 knockdown and decreased mitochondrial levels of pyruvate, reduced cell growth and viability, increased levels of acetylated histones, increased nuclear levels of PDH and promoted differentiation of AML cells. Treatment of mice with UK5099 (40mg/kg) decreased growth of OCI-AML2 xenografts. Finally, UK5099 induced cell death by Annexin V/PI staining in undifferentiated primary AML cells, but differentiated primary AML cells (by morphology and CD marker staining) and normal hematopoietic cells were more resistant.In summary, inhibiting entry of pyruvate into the mitochondria is a new mechanism to regulate differentiation of AML cells and stem cells by increasing PDH localization to the nucleus and histone acetylation. Thus, MTCH2 or MPC inhibitors may be novel therapeutics agents for AML. DisclosuresSchimmer:Medivir: Research Funding; Takeda Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.