Inhibiting the Mitochondrial Sulfhydryl Oxidase Alr Reduces Cox17 and Alters Mitochondrial Cristae Structure Leading to the Differentiation of AML and Stem Cells

The vast majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and then imported into the mitochondria. A subset of these imported proteins are folded into their mature and functional forms in the mitochondrial inter-membrane space (IMS) 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. We knocked down the mitochondrial sulfhydryl oxidase ALR, a key regulator of the MIA pathway. Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, knockdown of ALR reduced the engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6, mimicked the effects of ALR knockdown and killed AML cells with an IC₅₀ of 5-10 μM. MitoBloCK-6 preferentially reduced the clonogenic growth of primary AML cells (n=4/5) over normal hematopoietic cells (n=4). However, only 3/10 bulk AML cells were sensitive to MitoBloCK-6 induced cell death by Annexin V/PI staining.

Next, we evaluated the efficacy and toxicity of ALR inhibition in vivo . We injected primary AML cells or normal cord blood into the femurs of mice and then treated mice with MitoBloCK-6 (80 mg/kg i.p. 5 of 7 days x 2 weeks). MitoBloCK-6 strongly reduced the engraftment of primary AML samples but did not affect engraftment of cord blood. In secondary transplants, MitoBloCK-6 also targeted leukemic stem cells. No change in mouse body weight, serum chemistries, or organ histology was seen.

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 increased CD surface marker expression and increased non-specific esterase. In addition, ALR inhibition was preferentially cytotoxic towards undifferentiated cells and stem cells over differentiated bulk AML cells.

Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Inhibition of ALR selectively reduced levels of Cox17 protein and altered mitochondrial cristae structure. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation, induced differentiation of AML cells, and altered mitochondrial cristae structure, without changing respiratory chain activity or oxygen consumption. Of note, cristae remodelling independent of respiratory chain function has been recently implicated in cellular differentiation and in yeast, Cox17 regulates the cristae organizing machinery.

Thus, we have identified novel mechanisms by which mitochondrial pathways regulate the fate and differentiation of AML cells and stem cells Moreover, inhibition of ALR may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells.