Beschreibung:
Abstract A subset of AML and stem cells have increased mitochondrial stress and increased expression of mitochondrial proteases that degrade misfolded mitochondrial proteins. Given the recent findings of the interplay between mitochondrial homeostasis and mitochondrial protein import, we hypothesized that AML cells have an increased reliance on mitochondrial protein import as a compensatory mechanism for increased mitochondrial stress. To test this hypothesis, we measured expression levels of key mitochondrial protein import genes in publicly available datasets (GSE30377, GSE42414 and GSE 24759) and demonstrated their up regulation in a subset of AML cells over normal hematopoietic cells. Increased expression occurred across the spectrum of molecular mutations and cytogenetic abnormalities. Moreover, expression levels of mitochondrial protein import genes were enriched in functionally defined AML stem cells over bulk cells. To assess the impact of inhibiting mitochondrial protein import in AML, we knocked down the outer mitochondrial membrane import channel TOM40, the inner membrane import channel Tim23 and the oxidase ALR, that folds proteins through a disulfide relay system in the mitochondrial inner membrane space and is a rate limiting step for the import of a subset of mitochondrial proteins. Knockdown of these targets in OCI-AML2, TEX and U937 leukemia cells with shRNA reduced growth and viability of AML cells. Knockdown of ALR targeted the leukemia initiating cells as it abrogated engraftment of TEX leukemia cells into immune deficient mice (shRNA ALR = 5.481 +/- 0.9 % engraftment vs shRNA control= 29.44 +/- 5.4 % engraftment; p =0.0004) . Mechanistically, knockdown of mitochondrial import genes reduced levels of nuclear (ATP5A, SDHB and NDUFB8), but not mitochondrial (CoxII) encoded proteins of the OXPHOS chain. This in turn led to decreased basal oxygen consumption in leukemic cells. As a chemical approach to investigate the impact of inhibiting mitochondrial protein import in AML and normal cells, we tested the effects of MitoBloCK-6 and related analogues that selectively inhibit ALR in zebrafish, hESCs and yeast models. MitoBloCK-6 and related analogues killed leukemia cell lines (OCI-AML2, TEX, Jurkat and NB4) with an IC50of 5-10 µM. At these concentrations, MitoBloCK-6 decreased levels of nuclear (ATP5A, SDHB and NDUFB8), but not mitochondrial encoded (CoxII) proteins of the OXPHOS chain. Demonstrating the functional importance of changes in mitochondrial metabolism by these compounds, rho zero 143B rhabdomyosarcoma cells that lack mitochondrial DNA and rely solely on glycolysis were resistant to MitoBloCK-6. Finally, we tested the effects of MitoBloCK-6 on primary AML and normal cells. Treatment with MitoBloCK-6 (2 µM) inhibited clonogenic growth of (4 of 5) primary AML > 64% but produced <3% loss of clonogenic growth in normal hematopoietic cells. Finally, in an OCI-AML2 xenograft model, systemic administration of MitoBloCK-6 reduced tumor growth > 50% of control without toxicity. Thus, we have demonstrated that AML cells have a unique reliance on mitochondrial protein import and inhibition of this pathway may be a new therapeutic strategy to selectively target a subset of AML and AML stem cells. Disclosures Schimmer: Novartis: Honoraria.