Targeting the Mitochondrial Metallochaperone Cox17 Reduces DNA Methylation and Promotes AML Differentiation through a Copper Dependent Mechanism
Authors: Singh Rashim Pal , Jeyaraju Danny V. , Voisin Veronique , Xu Changjiang , Barghout Samir H , Khan Dilshad H. , Hurren Rose , Gronda Marcela , Wang Xiaoming , Jitkova Yulia , Sharon David , Liyanage Sanduni U. , Soriano Joelle , Lechman, Eric R. , MacLean Neil , Minden Mark D. , Chan Steven M , Dick John E. , Bader Gary D , Schimmer Aaron 
Published: October 2019

The majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and imported into the mitochondria. A subset of cysteine-rich proteins destined for the mitochondrial intermembrane space are oxidized and folded by the Mitochondrial IMS Assembly (MIA) pathway. We found that genes encoding substrates of the MIA pathway are over-expressed in leukemic stem cells compared to bulk AML cells. Therefore, we assessed the effects of inhibiting the MIA pathway in AML by targeting the FAD linked sulfhydryl oxidase ALR, an integral part of the MIA machinery.

Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, ALR knockdown reduced engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6 killed AML cells (IC50 of 5-10 mM) and preferentially reduced the clonogenic growth of primary AML cells over normal hematopoietic cells. MitoBloCK-6 treatment (80 mg/kg i.p. 5 of 7 days x 2 weeks) of mice engrafted with primary AML cells strongly reduced the leukemic burden without changing mouse body weight, serum chemistries, or organ histology. In contrast, MitoBloCK-6 did not change engraftment of normal cord blood in similar experiments. As evidenced by secondary transplants, MitoBloCK-6 also targeted leukemic stem cells.

As expression levels of ALR substrates are increased in AML stem cells we assessed the effects of ALR inhibition on differentiation in AML. Genetic or chemical inhibition of ALR induced the differentiation of AML cells as evidenced by changes in gene expression, increased differentiation associated CD surface marker expression and increased non-specific esterase.

Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Genetic or chemical inhibition of ALR selectively reduced levels of Cox17 protein. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation and induced AML differentiation.

Cox17 is a copper metallochaperone that promotes respiratory chain complex IV assembly by loading copper into the respiratory complex. COX17 knockdown slightly reduced the complex IV enzymatic activity, but did not change basal oxygen consumption or ROS production. However, COX17 knockdown increased intracellular levels of free copper 16-fold as measured by atomic mass spectrometry. Copper is a known inhibitor of adenosylhomocysteinase, a key enzyme involved in the preservation of S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio in cells. SAM is a global methyl donor and is critical for DNA methylation. Knockdown of COX17 or ALR inhibition with MitoBloCK-6 decreased levels of SAM and reduced DNA methylation in AML cells. Likewise, the enzymatic activity of adenosylhomocysteinase was reduced in OCI-AML2 cells after MitoBloCK-6 treatment. Importantly, co-treatment with the copper chelator, penicillamine, rescued reductions in SAM, DNA methylation, and cell viability after COX17 knockdown or MitoBloCK-6 treatment.

Thus, we have discovered a novel copper-dependent mechanism by which mitochondrial pathways regulate epigenetics and stemness in AML. Moreover, inhibitors of ALR or COX17 may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells.

Disclosures

Schimmer: Otsuka Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medivir AB: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.