Inhibition of PINK1-Dependent Mitochondrial Quality Control Pathways Induces Senescence of Acute Myeloid Leukemia Stem Cells

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Novel therapies that target LSCs have the potential to improve clinical outcomes. An important step towards achieving this goal is identifying the fundamental processes that regulate cell fate decisions in LSCs. Perturbation of these processes may impair LSC activity and form the basis of novel therapies.

Here, we investigated the role of mitochondrial quality control in the regulation of LSCs by inhibiting PTEN-induced kinase 1 (PINK1). PINK1 is a serine/threonine kinase that serves as a critical sensor of mitochondrial damage and is at the apex of multiple quality control pathways that maintain mitochondrial homeostasis. Damage to mitochondria results in activation of PINK1 on the outer mitochondrial membrane, which in turn triggers mitochondrial fission and mitophagy.

We first determined if the overall quality of the mitochondrial pool differs between LSCs and their more differentiated progenies (henceforth termed "non-LSCs") in two human AML cultures known as OCI-AML-8227 and OCI-AML-21. In both cultures, LSCs assayed by xenotransplantation are found only in the CD34 ⁺CD38 ^-fraction and not in the CD34 ⁺CD38 ⁺ and CD34 ^- fractions. We monitored the turnover rate of the mitochondrial pool in the different fractions by expressing MitoTimer, a mitochondria-targeted fluorescent protein that matures from green to red fluorescence over 48 hours. A high ratio of green to red fluorescence is indicative of active mitochondrial protein turnover and high overall mitochondrial quality. In both cell lines, the fluorescence ratio was highest in the CD34 ⁺CD38 ^- fraction compared with the other fractions, suggesting that LSCs maintain a higher quality pool of mitochondria than non-LSCs.

Based on the above findings, we hypothesized that PINK1-dependent mitochondrial quality control mechanisms are involved in the regulation of LSC fate. To test this hypothesis, we silenced the expression of PINK1 in OCI-AML-8227 cells using lentiviral vectors expressing validated shRNAs under a doxycycline-inducible promoter. Depletion of PINK1 shifted the MitoTimer fluorescence from green to red, reduced oxygen consumption rate, and disrupted mitochondrial ultrastructure in all cell fractions, consistent with a reduction in mitochondrial quality. Unexpectedly, PINK1 downregulation resulted in a block in differentiation and cell cycle arrest at G1/G0 phase in CD34 ⁺CD38 ^- cells. Re-expression of PINK1 was unable to reverse the cell cycle arrest, suggestive of a state of cellular senescence. Indeed, we observed other hallmarks of senescence including an increase in p21 ^WAF1 and p16 ^INK4a expression and SA-β-gal activity. To determine the mechanism by which PINK1 depletion causes senescence, we performed RNA sequencing analysis of sorted CD34 ⁺CD38 ^- cells expressing PINK1 shRNAs. This analysis revealed a marked decrease in the expression of MYC target genes, with TERT (Telomerase Reverse Transcriptase) being the most downregulated gene. Consistent with the role of TERT in telomere maintenance and telomere shortening as a trigger of senescence, we found that PINK1 knockdown decreased telomere length in CD34 ⁺CD38 ^- cells, and overexpression of TERT effectively rescued the senescence phenotype. These findings collectively indicate that inhibition of PINK1-dependent mitochondrial quality control pathways induces senescence of LSCs through telomere shortening.

To determine whether these changes translated to a reduction in functional LSC activity, we performed colony forming unit assays and serial xenotransplantation assays in immunodeficient NSG mice. Depletion of PINK1reduced the colony forming capacity and engraftment potential of 3 primary AML samples in primary and secondary recipients. Importantly, PINK1 depletion had minimal impact on the colony forming capacity and engraftment potential of normal CD34 ⁺ hematopoietic stem and progenitor cells (HSPCs) derived from cord blood, suggestive of a therapeutic window in vivo.

In summary, our results demonstrate that mitochondrial quality control pathways regulate cell fate decision in LSCs. Inhibition of PINK1 activity impairs LSC activity by inducing senescence, while sparing normal HSPCs. Our findings provide the basis for exploring PINK1 as a therapeutic target against LSCs in AML.