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International Journal of Molecular Sciences 26 (13): 6533 (2025)
BC Cancer Research Centre. Terry Fox Laboratory
Leukemia/Bone Marrow Transplant Program of British Columbia
Wu, Andrew
Chen, Min
Phoa, Athena
Yang, Zesong
Forrest, Donna L.
Jiang, Xiaoyan
Chronic myeloid leukemia (CML) is a leading example of a malignancy where a molecular targeted therapy revolutionized treatment but has rarely led to cures. Overcoming tyrosine kinase inhibitor (TKI) drug resistance remains a challenge in the treatment of CML. We have recently identified miR-185 as a predictive biomarker where reduced expression in CD34⁺ treatment-naïve CML cells was associated with TKI resistance. We have also identified PAK6 as a target gene of miR-185 that was upregulated in CD34⁺ TKI-nonresponder cells. However, its role in regulating TKI resistance remains largely unknown. In this study, we specifically targeted PAK6 in imatinib (IM)-resistant cells and CD34⁺ stem/progenitor cells from IM-nonresponders using a lentiviral-mediated PAK6 knockdown strategy. Interestingly, the genetic and pharmacological suppression of PAK6 significantly reduced proliferation and increased apoptosis in TKI-resistant cells. Cell survivability was further diminished when IM was combined with PAK6 knockdown. Importantly, PAK6 inhibition in TKI-resistant cells induced cell cycle arrest in the G2-M phase and cellular senescence, accompanied by increased levels of DNA damage-associated senescence markers. Mechanically, we identified a PAK6-mediated MDM2-p21 axis that regulates cell cycle arrest and senescence. Thus, PAK6 plays a critical role in determining alternative cell fates in leukemic cells, and targeting PAK6 may offer a therapeutic strategy to selectively eradicate TKI-resistant cells.
Academic Editor: Alessandro PoggiReceived: 6 June 2025Revised: 28 June 2025Accepted: 4 July 2025Published: 7 July 2025Citation: Wu, A.; Chen, M.; Phoa, A.;Yang, Z.; Forrest, D.L.; Jiang, X.Identification of a PAK6-MediatedMDM2/p21 Axis That ModulatesSurvival and Cell Cycle Control ofDrug-Resistant Stem/Progenitor Cellsin Chronic Myeloid Leukemia. Int. J.Mol. Sci. 2025, 26, 6533. https://doi.org/10.3390/ijms26136533Copyright: © 2025 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).ArticleIdentification of a PAK6-Mediated MDM2/p21 Axis ThatModulates Survival and Cell Cycle Control of Drug-ResistantStem/Progenitor Cells in Chronic Myeloid LeukemiaAndrew Wu 1,2, Min Chen 1, Athena Phoa 1, Zesong Yang 1,3, Donna L. Forrest 2,4 and Xiaoyan Jiang 1,2,5,*1 Collings Stevens Chronic Leukemia Research Laboratory, Terry Fox Laboratory, British Columbia CancerResearch Institute, Vancouver, BC V5Z 1L3, Canada; mchen@bccrc.ca (M.C.)2 Department of Medicine, University of British Columbia, Vancouver, BC V6T 1Z4, Canada3 Department of Hematology, The First Affiliated Hospital of Chongqing Medical University,Chongqing 400000, China4 Leukemia/Bone Marrow Transplant Program of British Columbia, Vancouver, BC V5Z 1M9, Canada5 Department of Medical Genetics, University of British Columbia; Vancouver, BC V6T 1Z4, Canada* Correspondence: xjiang@bccrc.caAbstractChronic myeloid leukemia (CML) is a leading example of a malignancy where a moleculartargeted therapy revolutionized treatment but has rarely led to cures. Overcoming tyrosinekinase inhibitor (TKI) drug resistance remains a challenge in the treatment of CML. We haverecently identified miR-185 as a predictive biomarker where reduced expression in CD34+treatment-naïve CML cells was associated with TKI resistance. We have also identifiedPAK6 as a target gene of miR-185 that was upregulated in CD34+ TKI-nonresponder cells.However, its role in regulating TKI resistance remains largely unknown. In this study,we specifically targeted PAK6 in imatinib (IM)-resistant cells and CD34+ stem/progenitorcells from IM-nonresponders using a lentiviral-mediated PAK6 knockdown strategy. In-terestingly, the genetic and pharmacological suppression of PAK6 significantly reducedproliferation and increased apoptosis in TKI-resistant cells. Cell survivability was furtherdiminished when IM was combined with PAK6 knockdown. Importantly, PAK6 inhibitionin TKI-resistant cells induced cell cycle arrest in the G2-M phase and cellular senescence,accompanied by increased levels of DNA damage-associated senescence markers. Me-chanically, we identified a PAK6-mediated MDM2-p21 axis that regulates cell cycle arrestand senescence. Thus, PAK6 plays a critical role in determining alternative cell fates inleukemic cells, and targeting PAK6 may offer a therapeutic strategy to selectively eradicateTKI-resistant cells.Keywords: CML; leukemic stem cells; TKI therapy; TKI resistance; cell cycle; Imatinib;PAK6; small molecule inhibitors; senescence; DNA damage1. IntroductionChronic myeloid leukemia (CML) is a myeloproliferative disorder characterized bythe t(9;22)(q34;q11) chromosomal translocation, which results in the constitutively activeBCR::ABL1 fusion oncogene [1]. Although CML is successfully managed by tyrosinekinase inhibitors (TKIs) for the most part, the disease remains elusive to curative therapy,as patients who respond to therapy often receive continuous treatment or risk relapsedue to the persistence of leukemic stem cells (LSCs) [2–4]. In addition, several drugresistance mechanisms also contribute to a barrier to TKI response, leading to the studyInt. J. Mol. Sci. 2025, 26, 6533 https://doi.org/10.3390/ijms26136533Int. J. Mol. Sci. 2025, 26, 6533 2 of 19and development of combination strategies that simultaneously target BCR::ABL1 andthese alternative oncogenic pathways [5–8]. We have recently identified p21-activatedkinase 6 (PAK6) as a target gene of miR-185, a microRNA suppressed in TKI-resistantstem/progenitor cells, which is also a predictive biomarker for TKI response in CMLpatients treated with imatinib (IM) or nilotinib [9–12]. However, its precise role in regulatingTKI resistance remains largely unknown.The p21-activated kinases (PAKs) are a family of serine/threonine kinases that regulateessential biological processes such as cytoskeletal dynamics, cell survival, and prolifera-tion [11,13–15]. The six PAK family members are divided into two groups based on theirstructure and activation mechanisms. Group I PAKs (PAK1-3) have an autoinhibitorydomain (AID) that suppresses their kinase activity until the binding of Cdc42/Rac GTPasesinduces a conformational change [13,15]. In contrast, Group II PAKs (PAK4-6) lack a well-defined AID and are constitutively phosphorylated but are regulated by pseudosubstrateregions that bind the kinase domain [13,15]. Dysregulation of PAKs is frequently observedin human cancers [11,14].PAK6 has been known to play a prevalent role in promoting several solid tumors,including prostate, gastric, and colorectal cancers [14,16]. Although some group I PAKs,such as PAK1 and PAK2, have been identified as therapeutic targets in hematologic malig-nancies, little is understood about the role of PAK6, especially in the context of CML [17–19].Interestingly, PAK6 was more highly expressed in CD34+ CML cells compared to healthybone marrow samples and in TKI-nonresponder patients compared to responders [12].PAK6 was also elevated in the more primitive CD34+CD38− compartment relative to thedifferentiated CD34− compartment [12].In this study, we investigated the role of PAK6 in mediating TKI resistance andcell survival using the pan-PAK inhibitor PF-3758309, as well as a lentiviral-mediatedPAK6 knockdown strategy, in IM-resistant cell lines and CD34+ TKI-nonresponder cells.We find that pharmacological and genetic suppression of PAK6 reduces survivabilityand proliferation, particularly when combined with IM treatment. We also demonstratethat PAK6 knockdown can induce DNA damage-associated G2-M cell cycle arrest andsenescence by modulating the expression of MDM2, p21, and other senescence proteinmarkers. Thus, our findings suggest that PAK6 may be an attractive therapeutic targetdue to its role in mediating the DNA damage response and governing multiple cell fates,including senescence and apoptosis, in the context of CML.2. Results2.1. Lentiviral-Mediated PAK6 Knockdown Sensitizes TKI-Resistant CML Cells to IMWe have previously shown that PAK6 mRNA transcript levels are highly expressedin CD34+ CML cells, including IM-nonresponder patients, compared to IM-responder pa-tients [12]. We validated this observation in CD34+ cells from an additional 23 CML patientsamples and 11 normal bone marrow (NBM) samples by qRT-PCR (Table S1) as well as byanalyzing the GSE14671 microarray dataset of CD34+ BM samples from 24 IM-respondersand 12 IM-nonresponders. Indeed, PAK6 gene expression was significantly higher in CMLcompared to NBM (p = 0.0042) and in the microarray set of IM-nonresponders compared toIM-responders (p = 0.0264, Figure 1A) [20].Int. J. Mol. Sci. 2025, 26, 6533 3 of 19K562 IMRCD34+ CML CellsK562 IMRA BIMRK562 K562 IMRCID CD34+ CML Cells PAK6 Transcript Levels in CD34+ CellsFigure 1. Knockdown of PAK6 inhibits cell survival and proliferation. (A) qRT-PCR analysisof PAK6 transcript levels in CD34+ stem/progenitor cells from 23 CML samples compared to 11normal bone marrow (NBM) samples (left) and analysis of the GSE14671 microarray dataset of CD34+BM samples from 24 IM-responders and 12 IM-nonresponders (right). (B) Western blot analysis ofknockdown of PAK6 with two shRNA constructs in K562 and IMR cells and CD34+ primary CMLcells, quantifying protein expression levels relative to β-Actin as indicated. (C) Viability and apoptosisassays were conducted in PAK6 knockdown cells with or without IM. (D) qRT-PCR analysis of PAK6transcript levels in CD34+ nonresponder cells with PAK6 knockdown (n = 3). Viability, apoptosis,and CFC assays were conducted in these PAK6 knockdown cells with or without IM.To explore the specific role of PAK6 in mediating TKI resistance in CML, we gen-erated a lentiviral-mediated shRNA knockdown system with two constructs targetingdifferent sequences of PAK6 and demonstrated that both specifically suppressed PAK6gene expression at both the RNA and protein levels (up to 80%, Figure 1A and FigureInt. J. Mol. Sci. 2025, 26, 6533 4 of 19S1A). Interestingly, PAK6 knockdown alone significantly decreased viability and increasedapoptosis more than IM in K562- and K562-resistant (IMR) cell lines in vitro (p < 0.045,Figure 1C). The addition of IM combined with the PAK6 knockdown had an increasedadditive effect (p < 0.0214). These results support our findings from a previous study usingthe pan-PAK inhibitor PF-3758309 alone or in combination with TKIs to suppress CML cellsurvivability [12].We then knocked down PAK6 in CD34+ stem/progenitor cells from IM-nonresponderpatient samples (n = 3) to determine if we could inhibit survivability to a similar degree.Although PAK6 mRNA transcript and protein expression were reduced by 50% in thesecells, we found that PAK6 knockdown could indeed reduce cell viability and induceapoptosis (p < 0.0095, Figure 1B,D). Additionally, combining IM with PAK6 knockdown inthese cells could significantly enhance TKI-mediated killing compared to either treatmentalone (p < 0.001). Colony-forming cell (CFC) assays also showed that combining IM andPAK6 knockdown inhibited CFC output over single treatment conditions (p = 0.0094,Figure 1D). The results of these survival assays, especially in CD34+ stem/progenitorcells, suggest that PAK6 knockdown re-sensitizes resistant CML cells to TKI-mediatedkilling effects.2.2. Lentiviral-Mediated PAK6 Knockdown Induces G2/M Cell Cycle Arrest in IM-Resistant CellsSince we demonstrated that PAK6 suppression can inhibit CML cell proliferationand survival, we wanted to investigate additional oncogenic mechanisms governed byPAK6 in CML. To uncover the biological processes involved downstream of PAK6, we firstcompiled gene sets of known and predicted PAK6 substrates, as well as common proteininteractors, from the Harmonizome 3.0 dataset repository and ran gene set enrichmentanalyses (GSEAs) using the Enrichr platform [21]. For both the PAK6 substrate and commonprotein interactor gene sets, we noticed the G2-M checkpoint biological processes to besignificantly enriched (Figure 2A,B, Table S3). However, since the GSEA was performedusing gene sets largely curated through mass spectrometry, synthetic peptides, and in vitrokinase experiments in non-leukemic cells, we then conducted cell cycle assays on K562 andIMR cells following PAK6 knockdown to investigate changes in cell cycle dynamics in thecontext of CML. In support of the enrichment analysis, we found that PAK6 knockdownreduced the number of cells in the G0/G1 (p = 0.0058) and S (p = 0.0104) compartmentsand significantly induced accumulation of cells in the G2-M compartment in K562 cellstransduced with the two different PAK6 knockdown constructs as compared to the control(up to 35% vs. 20%, p = 0.0407, Figure 3). Interestingly, this arrest of cells in the G2-Mcompartment was greater in IMR cells, showing that IMR cells may experience moresensitivity to PAK6 knockdown compared to K562 cells (up to 44%, p = 0.0038, Figure 3).This finding suggests that PAK6 plays a role in facilitating leukemia growth and progressionby dysregulating G2-M checkpoint controls.Int. J. Mol. Sci. 2025, 26, 6533 5 of 19DABFigure 2. Bioinformatics analysis of PAK6 substrates and common protein interactors. (A,B) Geneset enrichment analyses (GSEAs) of PAK6 substrates and common protein interactors identifiedfrom the Harmonizome 3.0 dataset repository revealed significant enrichment of biological processesrelated to the G2-M checkpoint. * = p value (left). Green line indicate enrichment profile and graylines indicate raking metric scores (right).shPAK6 2shPAK6 1shPAK6 2K562IMRP = 0.0407P = 0.0038P = 0.0247P = <0.0001P = < 0.0001Figure 3. Lentiviral-mediated PAK6 knockdown induces G2/M cell cycle arrest in IM-resistantcells. Flow cytometry analysis of cell cycle distribution in K562 and IM-resistant cells (IMR) followingknockdown of PAK6 with two shRNA constructs. PAK6 knockdown reduced the number of cellsin the G0/G1 and S phases and induced the accumulation of cells in the G2-M phases compared tothe control.2.3. Identification of an MDM2-P21 Axis Mediated by PAK6 in IM-Resistant CellsGiven that PAK6 knockdown could induce G2-M cell cycle arrest, we explored themolecular pathways mediated by PAK6 to dysregulate the cell cycle by searching PAK6substrates identified from in vitro kinase studies curated by the PhosphoSitePlus platform,Int. J. Mol. Sci. 2025, 26, 6533 6 of 19one of which was MDM2, a key negative regulator of p53 and cell cycle checkpoint blockerp21 (Figure S1B) [22]. Interestingly, we demonstrated that MDM2 mRNA transcript levelswere highly elevated in CD34+ stem/progenitor cells from 23 CML samples as compared to11 NBM samples by qRT-PCR analysis (p = 0.0396, Figure 4A). This is further supported byshowing increased MDM2 expression in IM-nonresponders as compared to IM-respondersby analyzing MDM2 expression in the GSE14671 microarray dataset (Figure 4A) [20]. Therewere no noticeable differences in gene expression of the other PAK6 substrates betweenthese two groups (Figure S1C). We then conducted univariate and correlation analyses onleukemic cell line and PAK inhibitor (PF-3758309) drug activity data from the Cancer Ther-apeutics Research Portal Version 2 (CTRPv2) database using the CellMinerCDB analysistool [23]. As expected, there was a positive correlation (r = 0.45, p = 0.036) between MDM2expression and PF-3758309 area-under-curve (AUC) values, indicating that cells with higherexpression of MDM2 were more susceptible to the PAK inhibitor (Figure 4B). This wassupported by positive correlations with p21 and p53 (Figure 4B). Indeed, we demonstrateda time-dependent reduction in MDM2 total protein levels following inhibition of PAK6phosphorylation by PF-3758309 in K562 and IMR cells (Figure 4C). Interestingly, we alsofound corresponding increases in p21 protein expression that appear to be induced in ap53-independent manner, as the K562 cell line is known to be p53-null (Figure 4C). In ourPAK6 knockdown models, we observed similar protein dynamics where MDM2 is reducedand p21 is significantly increased in response to PAK6 knockdown (Figure 4D). We alsoobserved that treating PAK6 knockdown cells with IM could further inhibit MDM2 proteinlevels. Notably, IM treatment in PAK6 knockdown cells could reverse the induction of p21(Figure 4D). Altogether, it appears that the G2-M cell cycle arrest we observed followingPAK6 knockdown in CML cells may result from the induction of p21 protein expressionmediated by MDM2.R NR3.03.23.43.63.8Log2-Fold IntensityMDM2 CD34+ CML 0.1553A BC DATMATR𝛃-Actin1 1.23 0.40 0.17 1 0.64 0.35 0.251 1.25 0.93 0.21 1 0.68 0.80 0.37EMDM2 Transcript Levels in CD34+ CellsK562 IMR K562 IMR K562 IMR K562 IMRMDM2 p21 p27 MMP-3FFigure 4. Cont.Int. J. Mol. Sci. 2025, 26, 6533 7 of 19R NR3.03.23.43.63.8Log2-Fold IntensityMDM2 CD34+ CML 0.1553A BC DATMATR𝛃-Actin1 1.23 0.40 0.17 1 0.64 0.35 0.251 1.25 0.93 0.21 1 0.68 0.80 0.37EMDM2 Transcript Levels in CD34+ CellsK562 IMR K562 IMR K562 IMR K562 IMRMDM2 p21 p27 MMP-3FFigure 4. Identification of a PAK6-mediated MDM2-P21 axis in CML cells. (A) qRT-PCR analysis ofMDM2 transcript levels in CD34+ cells from 23 CML samples compared to 11 NBM samples (left) andanalysis of the GSE14671 microarray dataset from 24 IM-responders and 12 IM-nonresponders (right).(B) Correlation analysis using the Cancer Therapeutics Research Portal (CTRPv2) database betweenMDM2, p21, and p53 expression and their sensitivities to the PAK inhibitor PF-3758309, as measuredby area-under-curve (AUC) values. (C) Western blot analysis of PAK6 phosphorylation, MDM2, andp21 protein expression in K562 and IM-resistant cells (IMR) treated with PF-3758309 (PF) at 16, 24,and 48 h. Quantification of protein expression levels relative to β-Actin as indicated. (D–E) Westernblot analysis of protein expression and phosphorylation of senescence-associated biomarkers andDNA damage markers, following PAK6 knockdown in K562 and IMR cells, quantifying proteinexpression levels relative to β-Actin as indicated. (F) qRT-PCR analysis of transcript levels ofsenescence-associated biomarkers in PAK6 knockdown cells.2.4. Pharmacological and Genetic PAK6 Suppression Induces Senescence in IM-Resistant CellsIt has been well established that p21 is not just a key regulator of cell cycle checkpointsbut also a marker for senescence. Cellular senescence is traditionally defined as a state ofpermanent cell cycle arrest and is considered an alternative cell fate to apoptosis. WithPAK6 knockdown already shown to cause G2-M cell cycle arrest, we investigated if thisarrest was related to senescence. However, there is a consensus that senescence cannot bedetermined by a single marker alone and that several senescence-associated phenotypesmust be observed for confirmation. One such phenotype is an increase in cell size andmorphology. Interestingly, quantification of phase-contrast images and GIEMSA stainingof K562 and IMR cells following PAK6 knockdown or treatment with a PAK inhibitorrevealed a two-fold increase in cell size compared to control cells transduced with acontrol vector (p < 0.0001, Figure 5A–C). This enlargement of cells was similar to thatinduced by doxorubicin, a senescence-inducing compound serving as a positive control(Figure 5B). Senescent cells are also known to accumulate lysosomal β-galactosidase, whichcan be detected by senescence-associated β-galactosidase (SABG) staining. K562 andIMR cells with PAK6 knockdown placed on Poly-L-Lysine slides and stained for SABGshowed a more intense blue color resulting from the cleavage of X-Gal due to increased β-galactosidase expression (Figure 5D). To validate these results, these PAK6 knockdown cellswere analyzed with a more sensitive C12FDG-mediated SABG assay by FACS. As expected,the total cell number did not increase in K562 (p < 0.0003) and IMR (p < 0.0002) PAK6knockdown cells compared to controls over 48 h of treatment with C12FDG (Figure 6A).FACS analysis revealed a three-fold increase in C12FDG fluorescence following PAK6knockdown in both K562 (p < 0.0487) and IMR cells (p < 0.0367, Figure 6A). This result wasfurther supported by observation of increased senescence cells by treatment of the PAKinhibitor PF-3758309 in both K562 and IMR cells (p < 0.0165, Figure 6B).Int. J. Mol. Sci. 2025, 26, 6533 8 of 19C DK562IMRCTRL shPAK6 PF-375830920µMK562 IMRA B20µMFigure 5. Inhibition of PAK6 increases cell size in CML cells. (A,B) Representative phase-contrastimages and quantification of cell size in K562 and IM-resistant cells (IMR) following PAK6 knockdown,including senescence-inducing compound doxorubicin (Dox), used as a positive control. (C) GIEMSAstaining of K562 and IMR cells following PAK6 knockdown or treatment with PF-3758309 (10 nM).(D) Representative images of senescence-associated β-galactosidase (SABG) staining of K562 andIMR cells following PAK6 knockdown.IMRK562A BCTRL PF-3758309CTRL PF-3758309C12FDGC12FDGCML 1CTRL PF-3758309 DoxCML 2CTRL PF-3758309 DoxCCtrl PF-PAKi0.00.51.01.5Fold Change MFIP = 0.0090DCTRL PF-3758309C12FDGCD34+ CML Cells PF-3758309DOXPF-3758309DOXCTRLCTRL20µM0. 9Figure 6. Cont.Int. J. Mol. Sci. 2025, 26, 6533 9 of 19IMRK562A BCTRL PF-3758309CTRL PF-3758309C12FDGC12FDGCML 1CTRL PF-3758309 DoxCML 2CTRL PF-3758309 DoxCCtrl PF-PAKi0.00.51.01.5Fold Change MFIP = 0.0090DCTRL PF-3758309C12FDGCD34+ CML Cells PF-3758309DOXPF-3758309DOXCTRLCTRL20µM0. 9Figure 6. PAK6 knockdown induces senescence-associated phenotypes in CML cells. (A) Totalcell numbers generated in K562 and IM-resistant cells (IMR) following PAK6 knockdown, includinga Dox control, after 48 h of treatment with C12FDG. C12FDG-mediated SABG assays were thenperformed on these cells and analyzed by FACS histograms, with corresponding quantificationof mean fluorescence intensity MFI (bar plots). (B) Fold changes of differences in K562 and IMRcells treated with the PAK inhibitor PF-3758309 (10 nM), with corresponding quantification of MFIby C12FDG-mediated SABG assays. (C,D) Representative images of SABG staining and C12FDG-mediated SABG assays conducted in CD34+ nonresponder primary cells treated with PF-3758309(10 nM). Intense staining of blue color in cells indicates increased β-galactosidase expression.Interestingly, we also found that senescence could be induced by modulation of theMDM2-p21 axis following PAK6 suppression in CD34+ IM-nonresponder cells. On its own,we have already demonstrated that PF-3758309 increased apoptosis in TKI-insensitive CMLcells. Here, we observed that treatment of PF-3758309 in CD34+ IM-nonresponder cellscould induce senescence, as evidenced by increased chromogenic and FACS-mediatedSABG staining (p = 0.0090, Figure 6C,D).2.5. Suppression of PAK6 Induces Activation of Senescence-Associated Protein Markers inIM-Resistant CellsWe then investigated molecular changes in several senescence-associated proteinbiomarkers in response to PAK6 knockdown at both RNA and protein levels. Aside fromp21 induction, as described earlier, p27, MMP3, and the DNA damage marker γH2Ax weresignificantly increased (Figure 4D,F). To further explore the effect of PAK6 aberration onDNA damage, we also probed for and found increased phosphorylation of ATM and ATR,which are key players and additional markers of DNA Damage Response (DDR), in cellswith PAK6 knockdown alone and in combination with IM (Figure 4E). We also noticed anassociated decrease in total ATM and ATR levels in these conditions. Notably, some ofthese protein phosphorylation and expression changes were more obvious in IM-resistantcells than in the control cells. Additional IM treatment did not enhance senescence andDDR-associated protein expression beyond that achieved with PAK6 knockdown alone.Importantly, we observed changes in senescence-associated protein markers withinthe MDM2-p21 axis following pharmacologic and genetic PAK6 inhibition in CD34+ IM-nonresponder cells (n = 3, Figure 7A). In response to PF-3758309 treatment, we observeda significant decrease in MDM2 protein levels and increases in p53, p21, and γH2Ax(Figure 7A). Interestingly, in both cell line and primary cell models of PAK6 inhibition,treatment of IM in PAK6 knockdown cells decreased the expression of p21 induced byPAK6 knockdown. In primary cells where p53 was also induced following PF-3758309treatment, the addition of IM also negated p53 elevation (Figure 7A). These observationsInt. J. Mol. Sci. 2025, 26, 6533 10 of 19were further supported by lentiviral-mediated PAK6 knockdown in CD34+ CML cells,which led to a decrease in MDM2 as well as associated increases in p21, p27, and γH2Ax,which was reflective of what we observed in our cell line knockdown models (Figure 7B).Altogether, this suggests a possible dynamic interplay between senescence and apoptosiscell fates (Figure 7C).CCD34+ CML CellsBMDM2p53-Actinp21p-PAK6𝛾H2AXp27ACtrl IM PF IM+PF1 0.68 0.39 0.241 0.63 0.88 0.591 0.84 1.37 0.961 1.67 1.54 1.391 2.60 1.83 1.561 0.96 1.02 1.10Ctrl IM PF IM+PF1 0.87 0.88 0.461 0.98 0.44 0.601 1.27 1.06 1.241 1.59 2.42 1.151 1.31 2.90 2.751 2.77 2.19 1.46CTRL IM PF PF+IM1 0.30 0.45 0.261 0.52 0.64 0.691 0.51 2.55 1.111 0.25 13.6 4.781 0.70 5.25 4.47CD34+ CML Cellsp53DNA DamageFigure 7. PAK6 inhibition elevates senescence-associated and DNA damage response biomarkersin CD34+ CML cells. (A) Western blot analysis of protein expression of senescence-associatedbiomarkers, including p21, p27, MMP3, and the DNA damage marker γH2Ax, following IM (5µM) orPF-3758309 (10 nM), alone or in combination in CD34+ CML cells (n = 3). (B) Western blot analysis ofthese proteins in CD34+ CML cells with lentiviral-mediated PAK6 knockdown (n = 2). Quantificationof protein expression levels relative to β-Actin as indicated. (C) Model of the role of PAK6 in cellcycle progression, senescence, and DNA damage response in CML. PAK6 promotes leukemia growthby regulating the MDM2-p21 axis, facilitating cell cycle progression, and suppressing senescence.PAK6 knockdown disrupts this axis, leading to G2-M cell cycle arrest, induction of senescence, andactivation of the DNA damage response.Int. J. Mol. Sci. 2025, 26, 6533 11 of 193. DiscussionThe development of TKIs has significantly improved the treatment of CML. However,TKI therapy is not curative, and patients often require lifelong treatment due to potentialrelapse from drug resistance and activation of BCR::ABL1-independent pathways. Werecently identified PAK6 as a target gene of miR-185 in CD34+ TKI-nonresponder cellsand demonstrated that the use of a pan-PAK inhibitor, PF-3758309, reduced cell growth inTKI-nonresponder CD34+ stem/progenitor cells [12,24]. However, molecular mechanismsof PAK6-mediated drug resistance are largely unknown; in particular, PF-3758309 couldreduce mitochondrial staining and ROS production in TKI-nonresponder CD34+ CML cells,indicating metabolic effects that require further investigation [12].To demonstrate that the observed effects by PF-3758309 were specifically due toPAK6 inhibition, we performed shRNA-mediated PAK6 knockdown. Indeed, in ourIM-resistant cell line model and CD34+ nonresponder patient cells, PAK6 knockdownor pharmacological suppression reduced cell viability, increased apoptosis, and inhibitedcolony formation in TKI-resistant cells. We then found through gene set enrichment analysisthat PAK6 substrates and common protein interactors were heavily involved in the G2-Mcheckpoint, and we demonstrated that PAK6 knockdown in IM-resistant cells inducedG2-M cell cycle arrest [25–27]. To uncover the mechanisms behind PAK6 and cell cyclearrest, we evaluated MDM2 from a list of known PAK6 substrates from PhosphoSitePlusdue to its crucial role as a negative regulator of p53 and the cell cycle checkpoint inhibitorp21 [22,28,29]. Others have previously shown that PAK6 phosphorylates MDM2 at theSer186 and Thr158 sites, activating MDM2 to ubiquitinate the androgen receptor and inhibittumor growth in prostate cancer cells [30]. In contrast to our previous study, where weestablished PAK6 as an oncogene targeted by the miR-185 tumor suppressor, this studysuggests that PAK6 may have tumor-suppressive functions in prostate cancer.In CML, it has been shown that MDM2 is more highly expressed in quiescent CD34+CML progenitor cells compared to their proliferating counterparts and that MDM2 plays avital role in mediating TKI resistance in CML. Inhibition of MDM2 has been demonstratedto sensitize TKI-resistant CD34+CD38- proliferating and quiescent cells to nilotinib [31].In our patient samples, we also found that MDM2 transcript expression was higher inCML patients than in those with NBM. Microarray data from the GSE14671 dataset showedincreased expression in IM-nonresponder samples compared to IM-responders [20]. How-ever, we noticed in both our own and the GSE14671 studies that the expression of MDM2in CML patient cells appeared to be quite heterogeneous. In addition, univariate andcorrelation analyses of leukemic cell line data and PF-3758309 activity, conducted throughCellMinderCDB, revealed that cells with higher expression of MDM2 were more sensitiveto PF-3758309 [23,32]. In this study, we demonstrate for the first time that PAK6 suppres-sion can indeed modulate the MDM2-p21 axis in CML cells. At the basal level, MDM2is strongly expressed in both K562 and IMR cells, whereas p21 is minimally expressed.However, PF-3758309 and PAK6 knockdown induced a corresponding decrease in MDM2total protein expression and a significant increase in p21 protein expression. Canonically,MDM2 is understood to be an E3 ubiquitin ligase that negatively regulates p53 by taggingit for ubiquitination [28,29,33]. As p21 is downstream of p53, reduction in p53 thereforeupregulates p21 activity. Interestingly, in K562 and IMR cells, the upregulation of p21following PAK6 is independent of p53, as these cells are known to have mutational inactiva-tion of the p53 gene and therefore do not express the p53 protein [34]. The existing literaturehas suggested that MDM2 may directly inhibit p21 independent of p53 and ubiquitinationthrough proteasome-mediated degradation in PC3 prostate cancer cells [35]. While thismay also be a possible mechanism of p21 regulation in K562 and IMR cells, further workis required for confirmation. Indeed, it is critical to determine if a physical interactionInt. J. Mol. Sci. 2025, 26, 6533 12 of 19between PAK6 and MDM2 or p21 is involved in understanding the molecular mechanismsby which this pathway mediates protein phosphorylation and protein expression of keyproteins, as well as their biological outcomes, through the knockdown or overexpression ofthese proteins or their mutants. In particular, it would be interesting to determine if PAK6can directly interact with a specific domain or phosphorylate several sites of MDM2 or p21in drug-resistant cells, as well as mediate protein ubiquitination and degradation.Furthermore, CD34+ stem/progenitor cells from primary patient samples, unlikeK562 cells, can be p53 positive. Previous work on human diploid fibroblasts has shownthat the degree of p53 expression can influence whether a cell undergoes senescence orapoptosis [36]. In our hands, CD34+ CML cells exhibited increased p53 levels followingPAK6 inhibition by PF-3758309, which subsequently led to an increase in p21. SincePAK6 inhibition can induce a corresponding decrease in MDM2 protein expression and asignificant increase in p21 protein expression in the presence of p53 in CD34+ CML patientcells, a similar observation was made in our cell line model system without expression ofp53, suggesting that the activity of the MDM2-p21 axis is mainly independent of p53. Itpossibly induces ubiquitination through proteasome-mediated protein degradation in thesecells. However, the MDM2-p53 interaction is a crucial regulatory mechanism in cancercells; this particular interaction may also contribute to the biological and molecular changesobserved in CD34+ CML cells under PAK6 inhibition. Indeed, further work is requiredto elucidate how the presence or absence of p53 affects PAK6 knockdown-associatedphenotypes in the context of CML [31]. P21 upregulation in response to PAK6 knockdownalso contradicts previous findings in prostate cancer cells, where PAK6 knockdown inducedexpression of cyclin D1, a proliferation marker negatively regulated by p21, enhancingtumor growth in vitro and in vivo [30]. This suggests that PAK6-mediated regulation ofMDM2 may exert both oncogenic and tumor-suppressive effects depending on the cancercontext. Nevertheless, our findings support our previous work characterizing PAK6 as anoncogene that facilitates cell cycle progression and survival in CML.PAK6 knockdown also induced senescence-associated phenotypes like enlarged cellsize, increased SABG staining, and elevation of senescence protein markers, including p21.PAK6 knockdown showed increased expression of p27, γH2AX, and MMP3. Like p21, p27is also a tumor suppressor and is implicated in G2-M cell cycle arrest [37]. Interestingly,the induction of p21, p27, and γH2AX also suggests that G2-M arrest and senescence inPAK6 knockdown cells may be part of the DNA Damage Response (DDR) [38–40]. Ithas been demonstrated that p21 is involved in G2 arrest of the cell cycle in p53-deficientDLD1 colon cancer cells following DNA damage [41]. Additionally, p27-deficient micehave been observed to have impaired G2-M arrest in response to DNA damage [37].Recently, it has been revealed that PAK6 enhances gastric cancer chemoresistance bymodulating the DDR [16]. Specifically, PAK6 activates the ATR cascade, which includesCHK1, and promotes homologous recombination [16]. Interestingly, we found that PAK6knockdown cells had increased phosphorylated levels of ATM and ATR, indicating thatPAK6 knockdown possibly induces DNA damage and triggers the DDR. Surprisingly,IM treatment on PAK6 knockdown cells reversed p21 protein expression induction byPAK6 alone. This could be attributed to the dynamic relationship between senescent andapoptotic cell fates simultaneously induced by PAK6 knockdown. While traditionallyviewed as distinct processes, emerging evidence suggests that senescent cells can transitionto apoptosis under prolonged stress conditions such as persistent DDR signals [36,42–44].Thus, it may also be fruitful to study the combination of senolytic drugs with PAK6inhibition and TKI treatment for a more robust targeted therapeutic strategy [45].Int. J. Mol. Sci. 2025, 26, 6533 13 of 194. Materials and Methods4.1. Cell LinesThe K562 human CML cell line originated from a 53-year-old female CML blast crisispatient with no BCR::ABL1 kinase mutations. IM-resistant K562 (IMR) cells, a generous giftfrom Dr. A. Turhan (University of Poitiers, Poitiers, France), were generated by sequentiallytreating K562 cells with increasing doses of IM and selecting resistant clones that did notharbor BCR::ABL1 kinase mutations.4.2. Primary Patient SamplesHeparin-anticoagulated peripheral blood (PB) or bone marrow (BM) cells were ob-tained from 23 CML patients (Table S1) and 11 healthy adult donors from the HematologyCell Bank of BC. Patient samples in this study were retrospectively characterized as eitherIM-responders or IM-nonresponders. IM-responder characterization was defined by theEuropean Leukemia Net Treatment guidelines and required patients to have achieved atleast a 3-log reduction in BCR::ABL1 transcript levels after 12 months of treatment (MMR)and/or show no BCR::ABL1+ status after cytogenetic analysis after 6 months of therapy(CCyR) [46–48]. Those who did not meet the criteria were considered IM-nonresponders.The mononuclear cells were isolated from the samples using Ficoll-Paque density gradientseparation, and CD34+ stem/progenitor cells were enriched using the EasySep™ HumanCD34 Positive Selection Kit (STEMCELL Technologies, Vancouver, BC, Canada). Verifi-cation of stem/progenitor cell purity was performed by staining the cells for the CD34surface marker with an anti-human CD34 antibody conjugated to allophycocyanin (APC)(BD Biosciences, Mississauga, ON, Canada) and then assessing at least 85% of cells withCD34+ expression by FACS analysis.4.3. Small-Molecule InhibitorsIM (Novartis, Montreal, QC, Canada) and PF-3758309 (Pfizer, Kirkland, QC, Canada)were reconstituted in 10 mM water (IM) or dimethyl sulfoxide (PF-3758309) and stored at−20 ◦C.4.4. Lentiviral Transfection of PAK6 shRNA ConstructsTwo PAK6-coding-domain-targeting shRNA sequences were cloned into a pLKO.1-puro vector under a U6 promoter and obtained as liquid cultures from Millipore-Sigma, Oakville, ON, Canada. The first shRNA, shPAK6 1, contained the sense strandCATCCAGAAGTTGTCAGTCAT and the antisense strand ATGACTGACAACTTCTG-GATG, whereas for shPAK6 2, the sense strand was CCCAAAGAAGGCAAGTTTGTG,and the antisense strand was CACAAACTTGCCTTCTTTGGGT (Figure S1A). Followingthe manufacturer’s protocol, the plasmid DNA containing these shRNA sequences wasisolated using the PureLink™ HiPure Plasmid Filter Maxiprep Kit (ThermoFisher, Burnaby,BC, Canada). The purified DNA was stored at −20 ◦C until use.HEK 293T cells were plated at a density of 5.5 × 106 cells per 10 cm tissue cultureplate in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and1% glutamine, incubated at 37 ◦C with 5% CO2 [6,12]. After 24 h, the culture medium wasreplaced with fresh DMEM 4 h before transfection. For each construct, two solutions wereprepared. Solution A included construct DNA, ∆R, packaging constructs, and the envelopeconstruct VSVG in Opti-MEM. Solution B contained PEI in Opti-MEM. Solution B wasadded dropwise to Solution A and incubated at room temperature for 20 min. Subsequently,the DNA-PEI mixture was added to each plate of 293T cells and incubated for 48 h at 37 ◦Cwith 5% CO2. The culture medium containing the virus was then collected and filteredthrough a 0.45 µM syringe filter to remove cellular debris. The filtered virus-containingInt. J. Mol. Sci. 2025, 26, 6533 14 of 19medium was subjected to ultracentrifugation at 2.5 × 103 rpm for 90 min at 4 ◦C. Thesupernatant was discarded, and the virus pellet was resuspended in Iscove’s medium with5% DNase with light agitation for 1 h at room temperature. The virus was then stored at−80 ◦C until needed.4.5. Lentiviral-Mediated PAK6 Knockdown in CML CellsK562 and K562-resistant (IMR) cells (3× 105) were seeded in 24-well plates with 400 µLof RPMI-1640 medium and then transferred to the Biosafety Level 3 facility for transduction.Protamine sulfate was added to each well, along with the scrambled vector control or PAK6knockdown vector. The cells were incubated at 37 ◦C with 5% CO2 for 16 h, followed bythree washes with PBS, then recovered in complete RPMI-1640 medium for at least 24 h. Forcells transduced with the PAK6-coding-domain-targeting shRNA, puromycin was addedto a final concentration of 2.5 µg/mL. The medium was refreshed every 48 h for at least5 days to ensure optimal knockdown efficiency. In primary samples, 2 × 105 CD34+ CMLcells were pre-stimulated in 300 µL of serum-free medium containing growth factors in24-well plates for 16 h and then similarly transduced with PAK6-coding-domain-targetingshRNA lentiviral particles for 6 h (Supplementary Figure S1A). The cells were then washedand allowed to recover for 48 h before puromycin selection (Supplementary Figure S1A).4.6. Trypan Blue Viability AssayCell lines were seeded at a density of 1 × 105 cells/mL in 12-well plates containingcomplete RPMI-1640 medium. PF-3758309 was administered either alone or in combinationwith IM for 48 h. Following treatment, cell viability was assessed using a Neubauerhemocytometer after adding trypan blue and PBS. Cells transduced with shRNA wereallowed to recover in complete RPMI-1640 medium for at least 48 h before seeding. ForIC50 measurements, cells were treated with PF-3758309 at increasing concentrations (0 nM,1 nM, 10 nM, 100 nM, 1000 nM). Viability data were plotted, and the IC50 curve and valueswere calculated using the Dose-Response—Inhibition Non-Linear Fit function in GraphPadPRISM 8 version.4.7. PI-Annexin V Apoptosis AssayCells that remained after the viability assay at 48 h post-treatment were stained withAnnexin V APC and Propidium Iodide (PI) using the Annexin V Apoptosis Detection KitAPC (eBioscience, San Diego, CA, USA) to assess apoptosis according to the manufac-turer’s instructions. The prepared cells were kept on ice and analyzed by flow cytometrywith FACSCalibur (BD Bioscience, Mississauga, ON, Canada) and FlowJo® v10 software.Apoptotic populations were identified based on Annexin V+ staining, distinguishing earlyapoptosis (Annexin V+/PI−) and late apoptosis (Annexin V+/PI+) populations.4.8. CFC AssayFor colony-forming cell (CFC) assays, 3000 CD34+ CML cells were mixed in MethoCultH4230 (STEMCELL Technologies, Vancouver, BC, Canada) containing growth factor cock-tails (IL-6, IL-3, GM-CSF, G-CSF). The MethoCult mixture was aliquoted into polystyreneround-bottom tubes using a syringe with a blunt-end needle, with or without IM. Themixture was vortexed and plated onto culture plates, followed by incubation at 37 ◦C with5% CO2. After 14 days, colonies were counted and expressed as CFC output per input cellfor specific colony types (GEMM, BFU-E, and CFU-GM).4.9. Cell Cycle AssayCells were washed three times with PBS and resuspended to a concentration ofapproximately 3 × 106 cells/mL. Ice-cold 100% ethanol was added to fix the cells, whichInt. J. Mol. Sci. 2025, 26, 6533 15 of 19were left overnight at 4 ◦C. The ethanol was then removed, and the cells were washed withPBS before being resuspended in a solution containing propidium iodide (PI), RNase, andTriton X-100. The cells were stained for 30 min in the dark, washed, and resuspended inice-cold PBS prior to analysis by flow cytometry using the FACSCalibur (BD Bioscience,Mississauga, ON, Canada) and FlowJo® v10 software.4.10. Senescence-Associated β-Galactosidase StainingSenescence-associated β-Galactosidase chromogenic and FACS staining were con-ducted following standard protocols [49]. For chromogenic staining, cells were adheredto Poly-L-Lysine-coated slides for 20 min at 37 ◦C. The cells were then fixed and stainedfor β-Galactosidase using the Senescence-Associated β-Galactosidase Staining Kit (CellSignaling, Danvers, MA, USA) per the manufacturer’s instructions. Slides were examined,and images were captured using a Nikon C1 confocal microscope with a 40× objectivelens. For FACS detection, cells were seeded into 6-well plates, with 80 nM doxorubicin-treated cells as a positive control. After 48 h, a portion of the cells was used to determinethe total cell number using a hemocytometer. The remaining cells were pre-treated with100 nM bafilomycin A1 for 1 h at 37 ◦C, 5% CO2. Cells were then incubated with 33 µM5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C12FDG) for 30 min at 37 ◦C,5% CO2, washed with PBS at room temperature, and resuspended in 400 µL of ice-coldPBS. The cell suspensions were immediately analyzed using a FACSCalibur flow cytometerand FlowJo® v10 software.4.11. Wright-Giemsa StainingCells were placed on slides using a cytospin and stained with the Hematology StainPack Modified Wright’s Stain (VWR) using the Hematek® Slider Stainer instrument, fol-lowing the manufacturer’s instructions. The slides were then examined, and images werecaptured using a Nikon C1 confocal microscope with a 20× objective lens.4.12. RNA ExtractionRNA was extracted using TRIzol (Life Technologies, Toronto, ON, Canada) followingthe manufacturer’s standard protocol. The RNA pellet was resuspended in UltraPureDNase/RNase-free water (Life Technologies, Toronto, ON, Canada) and quantified forconcentration and purity using a NanoDrop spectrophotometer (ThermoFisher Scientific,Burnaby, BC, Canada). The RNA was then stored at −20 ◦C until further use.4.13. Quantitative Real-Time PCRA total of 500 ng of RNA was reverse transcribed into cDNA using the STEMscriptcDNA Synthesis Kit with Random Primers (StemCell Technologies, Vancouver, BC, Canada)according to the manufacturer’s instructions. The cDNA was then combined with oligoprimers for PAK6, MDM2, p21, p27, MMP3, and B2M, along with RNase-free water and 2XSYBR Green PCR Master Mix (Life Technologies, Toronto, ON, Canada) in a 96-well platefor qRT-PCR analysis using the 7500 Real-Time PCR System (Applied Biosystems, FosterCity, CA, USA). Primers used for qRT-PCR are listed in Table S2.4.14. Cell Lysis and Protein QuantificationCell pellets were washed twice with phosphate-buffered saline (PBS) (Life Technolo-gies, Toronto, ON, Canada), flash frozen, and lysed with a homemade lysis buffer consistingof 90% phosphorylation solubilizing buffer (PSB), 10% NP-40, 0.1% sodium dodecyl sulfate(SDS), 0.5% 200 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5% protease inhibitorcocktail for 20 min at 4 ◦C. The lysed pellets were then centrifuged at 14,000 rpm for 20 minat 4 ◦C to separate the cell debris. The supernatant was collected, and protein concentrationInt. J. Mol. Sci. 2025, 26, 6533 16 of 19was measured using the Bradford protein assay with absorbance read at 570 nm on aspectrophotometer, using the lysis buffer as a control.4.15. Western Blot AnalysisProtein lysates were mixed with 2× Laemmli loading buffer and ddH2O, then heatedat 90 ◦C for 10 min for denaturation. Samples were run on 10% SDS-PAGE gels andtransferred to PVDF membranes for 1.5 h on ice. Membranes were blocked with 5% skimmilk for 30 min, then incubated overnight at 4 ◦C with primary antibodies in TBST (5% BSAfor phospho-proteins, skim milk for total proteins). After washing with TBST, secondaryantibodies were added for 1 h at room temperature. The membranes were washed, treatedwith ECL reagent, and imaged using the ChemiDoc MP system (Bio-Rad, Mississauga, ON).Primary antibodies included PAK6 (1:1000, Bio-Rad), β-Actin (1:500), MDM2 (1:200, SantaCruz, Dallas, TX, USA), p21, γH2AX, p27, MMP3 (1:2000, Cell Signaling, Danvers, MA,USA), 4G10 (1:5000), pPAK6 (1:500), ATM and ATR (1:1000, Cell Signaling, Danvers, MA,USA), and pATR and pATM (1:500, Cell Signaling, Danvers, MA, USA). Protein expressionwas quantified using FIJI ImageJ2 software, normalizing to β-Actin.4.16. Bioinformatic AnalysisCancer cell lines and drug activity were profiled using CellMinerDB (version 1.2)and the Cancer Therapeutics Research Portal Version 2 (CTRPv2) database to performunivariate analysis and generate correlation plots between drug sensitivity and gene ex-pression [23,32]. Gene set enrichment analysis (GSEA) was conducted with the Enrichrtool, cross-referencing PAK6 substrate gene sets from the Kinase Library Serine ThreonineKinome Atlas, PhosphositePlus, and Pathway Commons Protein-Protein against the Molec-ular Signatures Database (MSigDB, 2024.1) [25–27,50]. The GSE14671 microarray datasetfrom the Gene Expression Omnibus (GEO) was analyzed to compare gene expressionin CD34+ cells between 12 IM-nonresponders and 24 IM-responders [20]. Samples werecollected at diagnosis, with response status determined 12 months post-IM therapy [20].The dataset used the Affymetrix Human Genome U133 Plus 2.0 Array system, and geneexpression was determined via the median of robust multi-array average (RMA) geneprobe values.4.17. Statistical AnalysisResults are presented as the mean ± standard error of the mean from at least threeindependent experiments. Group differences were assessed using the 2-tailed Student’st-test or one-way ANOVA for multiple comparisons.5. ConclusionsOverall, our findings indicate that PAK6 knockdown induces G2-M cell cycle arrest inCML cells, suggesting that PAK6 facilitates leukemia growth by dysregulating cell cyclecheckpoints. Additionally, PAK6 knockdown mediates the MDM2-p21, contributing tocell cycle arrest and senescence. The observed increase in phosphorylated ATM/ATR andsenescence markers like p21, p27, MMP3, and γH2AX further supports the role of PAK6in regulating DDR-associated senescence. Thus, our study provides promising evidencethat targeting PAK6, either through pharmacological inhibitors or genetic suppression,could offer a new therapeutic strategy in combination with TKIs or other new drugs fordeveloping effective therapies for clinical applications.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26136533/s1.Int. J. Mol. Sci. 2025, 26, 6533 17 of 19Author Contributions: A.W. performed most experiments and data analyses; M.C., A.P. and Z.Y.helped with some primary cell culture, Western blot, and imaging experiments; D.L.F. providedthe clinical data and insightful discussions; A.W. and X.J. wrote the manuscript, and all authorscommented on it. All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the Canadian Cancer Society (#706288), the Canadian Institutesof Health Research (#427201), and the Collings Stevens Chronic Leukemia Research Fund (#1LERG904,X.J.). A.W. received an MITACS Accelerate Fellowship, and A.P. received a Michael Smith HealthResearch BC Postdoctoral Trainee Award.Institutional Review Board Statement: This study was conducted in accordance with the Declarationof Helsinki and approved by the Institutional Ethics Committee of the University of British Columbia—BC Cancer Research Ethics Board (H24-02839) and approved on 15 October 2024.Informed Consent Statement: Patient samples were retrospectively included in our study withprior fully informed patient consent from all subjects involved in the study and approval fromthe Institutional Ethics Committee at the University of British Columbia—BC Cancer ResearchEthics Board.Data Availability Statement: Data are contained within the article and Supplementary Materials.Acknowledgments: The authors thank the Stem Cell Assay Laboratory staff for processing patientsamples, Josephine Leung for excellent technical assistance, members of the Leukemia/Bone MarrowTransplant Program of British Columbia and the Hematology Cell Bank of British Columbia forpatient samples, the Terry Fox Laboratory FACS Facility for cell sorting, STEMCELL Technologiesfor culture reagents, and A. Turhan (University of Poitiers, Poitiers, France) for an IM-resistant K562cell line.Conflicts of Interest: The authors declare no conflicts of interest.References1. Sawyers, C.L. Chronic Myeloid Leukemia. N. Engl. J. Med. 1999, 340, 1330–1340. [CrossRef] [PubMed]2. Graham, S.M.; Jørgensen, H.G.; Allan, E.; Pearson, C.; Alcorn, M.J.; Richmond, L.; Holyoake, T.L. Primitive, Quiescent,Philadelphia-Positive Stem Cells from Patients with Chronic Myeloid Leukemia Are Insensitive to STI571 In Vitro. Blood2002, 99, 319–325. [CrossRef] [PubMed]3. Houshmand, M.; Simonetti, G.; Circosta, P.; Gaidano, V.; Cignetti, A.; Martinelli, G.; Saglio, G.; Gale, R.P. Chronic MyeloidLeukemia Stem Cells. Leukemia 2019, 33, 1543–1556. [CrossRef] [PubMed]4. Holyoake, T.; Jiang, X.; Eaves, C.; Eaves, A. Isolation of a Highly Quiescent Subpopulation of Primitive Leukemic Cells in ChronicMyeloid Leukemia. Blood 1999, 94, 2056–2064. [CrossRef]5. 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Article
eng
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Vancouver : University of British Columbia Library
Multidisciplinary Digital Publishing Institute
CC BY 4.0
Faculty
Researcher
CML
leukemic stem cells
TKI therapy
TKI resistance
cell cycle
Imatinib
PAK6
small molecule inhibitors
senescence
DNA damage
Identification of a PAK6-Mediated MDM2/p21 Axis That Modulates Survival and Cell Cycle Control of Drug-Resistant Stem/Progenitor Cells in Chronic Myeloid Leukemia
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