Inhibition of PAK6-Mediated Survival and Cell Cycle Controls Selectively Targets Therapy-Resistant CML Stem/Progenitor Cells
Authors: Wu Andrew , Chen Min , Yen Ryan , Jiang Xiaoyan 
Published: February 2021

The resistance of chronic myeloid leukemia (CML) leukemic stem cells (LSC) to ABL tyrosine kinase inhibitor (TKI) monotherapy remains a challenge in curing CML. We have recently identified miR-185 as a useful biomarker to predict therapy response in treatment-naïve CML stem/progenitor cells. We also demonstrated that restored miR-185 expression in LSCs impaired survival, sensitizing them to TKIs in vitro and in preclinical patient-derived xenotransplantation models, indicating that miR-185 is a critical regulator mediating TKI response/resistance of CML stem/progenitor cells. PAK6, a serine/threonine-protein kinase, was uncovered as a target gene of miR-185 by RNA-seq and was found to be upregulated in CD34+ TKI-nonresponder cells vs. TKI-responders, but its biological functions in CML are largely unknown. To investigate the biological effects of inhibiting PAK6 activity in TKI-resistant cells, we tested a pre-clinically validated pan-PAK inhibitor (PF-3758309) in vitro. PF-3758309 significantly reduced the growth of IM-resistant cell lines, including K562-resistant cells, BV173 blast cells (IC50 25-70 nM) and CD34+ TKI-nonresponder cells, as assessed by viability and CFC assays, and increased their apoptosis; these effects were significantly enhanced by TKIs (~2-fold, P&lt;0.05). PF-3758309 alone, or in combination with a TKI, did not have obvious inhibitory effects on CD34+ normal bone marrow. These results were further confirmed in IM-resistant cells using a lentiviral knockdown system that specifically inhibits PAK6. Interestingly, PF-3758309 alone, or in combination with a TKI, greatly reduced mitochondrial activity in CD34+ TKI-nonresponder cells, as shown in functional assessments with MitoTracker, a dye that accumulates in active mitochondria, the site of OXPHOS; this was not seen with TKI alone (P&lt;0.002). Similarly, CellROX analysis confirmed a reduction in ROS levels upon PF-3758309 treatment, or a combination of PF-3758309 with TKI, in these cells. In addtion, MDM2, a critical negative regulator of the p53 tumor suppressor, was identified as one of substrates of PAK6, by PhosphoSitePlus analysis. Its expression was found to be correlated with PF-3758309 treatment in CML cells based on CellMinerDB univariate analyses using gene-small-molecule association data from the CTRPv2 database. Indeed, Western blot analysis showed that PAK6 knockdown in K562 and IM-resistant cells led to a reduction in MDM2 protein levels. Furthermore, MDM2 downregulation by PAK6 inhibition corresponded to an increase in p21 levels, suggesting a mechanism of MDM2-mediated p21 regulation independent of p53, as these cells are p53-null. Most interestingly, PAK6 knockdown in IM-resistant cells leads to G2/M phase accumulation and increased senescence levels (2-fold, P&lt;0.05), detected by senescence-associated β-galactosidase staining. PAK6 knockdown induced senescence was further supported by observations of enlarged cell size (p&lt;0.05) and increased granulation, as well as changes in senescence-associated protein markers, including p21, p27, MMP-3 and the DNA damage marker pH2Ax, by Western blot analysis. Hence, our findings indicate that dual targeting of miR-185-PAK6-mediated survival, cell cycle and metabolic pathways, along with BCR-ABL, selectively eradicates drug-resistant CML stem/progenitors. Specifically, PAK6 plays roles in MDM2/p21-mediated apoptosis, senescence and cell cycle controls, offering a valuable therapeutic strategy for improved treatment and care.

Disclosures
No relevant conflicts of interest to declare.