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Characterization of the miR-185-PAK6-mediated survival and cell cycle controls in chronic myeloid leukemia

University of British Columbia

Overcoming drug resistance and targeting leukemic stem cells (LSCs) remain major challenges for curative treatment of human leukemia, including chronic myeloid leukemia (CML). Thus, there is a need to identify novel biomarkers and therapeutic strategies to target LSCs. Increasing evidence indicates that LSCs are susceptible to disturbances in cellular metabolism and cell cycle regulation. Previously, through global transcriptome profiling, our lab identified a key microRNA (miRNA), miR-185 as a predictive biomarker which had significantly reduced expression levels in CD34⁺ (LSC/progenitor) treatment-naïve CML cells. Through RNA-seq analysis, PAK6, a serine/threonine-protein kinase, was identified as a target gene of miR-185; it is upregulated in CD34⁺ TKI-nonresponder cells vs. TKI-responders, correlating with reduced miR-185 expression. Gene set enrichment analysis (GSEA) in the same CD34⁺ patient cells where miR-185 and PAK6 were identified as being differentially expressed revealed a significant gene set enrichment of oxidative phosphorylation (OXPHOS) and reactive oxygen species (ROS) in CD34⁺ CML cells compared to healthy CD34⁺ cells. These levels were also significantly higher in TKI-nonresponder cells than in TKI-responders. Thus, the miR-185-PAK6 axis may contribute to the perturbation of specific metabolic pathways in TKI-nonresponder LSC/progenitor cells and confer therapy-resistance to these cells. Indeed, a pre-clinically validated pan-PAK inhibitor (PF-3758309) alone, or in combination with a TKI, greatly reduced mitochondrial quantity (MitoTracker) and ROS production (CellROX) in TKI-nonresponder cells, an effect that was not seen in the same cells treated with a TKI. Notably, PF-3758309 also significantly reduced the growth of TKI-resistant cell lines and CD34⁺ TKI-nonresponder cells and increased their apoptosis; these effects were greatly enhanced by TKIs. These results were further confirmed in TKI-resistant cells using a lentiviral shRNA knockdown system that specifically targets PAK6. Interestingly, cell cycle analysis demonstrated an accumulation of cells in G2/M phase with increased senescence associated (SA) β-galactosidase staining in TKI-resistant cells following PAK6 knockdown. These observations were accompanied by increases in cell cycle and senescence protein markers p21 and p27. Taken together, these findings indicate that dual targeting of miR-185-PAK6-mediated survival, cell cycle and metabolic pathways, along with BCR-ABL, selectively eradicates therapy-resistant LSC/progenitors, providing a valuable therapeutic strategy for improved treatment and care.

Text

2021-04-01

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/77702

Interdisciplinary Oncology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/