The Roles And Mechanisms Of PAK1 Regulating ERK1/2 Signaling Pathway In Bone Marrow Stromal Cell-induced Drug Resistance In Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia(AML)is a common hematological malignancy characterized by the abnormal clonal proliferation of myeloid precursor cells,cell differentiation disorder,and obstructed apoptosis.Although the clinical standard"3+7" chemotherapy regimens,including anthracyclines and cytarabine,have made some progress during the past decades,the prognosis of AML patients remains poor.Leukemia drug resistance and recurrence represent major challenges in the treatment of AML.Therefore,exploring the relevant mechanisms underlying leukemia drug resistance and recurrence is crucial and will provide a basis for the combined targeted therapy of AML.The bone marrow microenvironment is composed of soluble factors and supporting tissues,such as bone marrow stromal cells(BMSCs)and the extracellular matrix(ECM),which provide a favorable environment for AML cells;BMSCs play an important role in disease recurrence and drug resistance.Studies have shown that under BMSC co-culture conditions,the expression levels of genes such as those encoding galectin-3,cysteine-rich 61,and autophagy-related E1 ligase 7 are significantly increased in AML cells.However,the mechanism underlying BMSC-mediated AML resistance has not been fully elucidated.The p21-activated kinase(PAK)is a member of the serine/threonine family of kinases and is divided into two groups based on the structural homology and regulatory functions:group I(PAK1-3)and group ?(PAK4-6).Upstream signals activate PAKs through binding partners and promote the phosphorylation of effector substrates,thereby mediating related signal transduction pathways,including cytoskeleton remodeling,cell motility,apoptosis,cell cycle,DNA damage response,gene expression,as well as transformation and invasion.The PAK family is overexpressed in a variety of tumors.PAK1 and PAK2 are overexpressed in breast and liver cancer and promote the occurrence and development of tumors;PAK3 is overexpressed in neuroendocrine tumors whereas PAK4 promotes the proliferation of multiple myeloma cells.Recent studies have shown that PAK1 has a critical effect on a variety of oncogenic signaling pathways and has emerged as a potential therapeutic target for tumors.It has been reported that PAK1 inhibitors and BCR-ABL1 tyrosine kinase inhibitors exert a synergistic effect on chronic myelogenous leukemia cells.Studies have reported that the combined inhibition of PAK1 and receptor tyrosine is a valuable treatment strategy for childhood acute lymphoblastic leukemia.However,the role of PAK 1 in BMSC-mediated AML resistance has not been reported.In this study,we used publicly available data sets to investigate the expression of PAK1 in AML patients and determined whether it is correlated with clinical and molecular characteristics as well as clinical prognosis.Overall,our research,aimed at elucidating the role of PAK1 in BMSC-mediated AML drug resistance,might provide novel insights for potential combined targeted therapies for AML.PART ONECorrelation of PAK1 expression with clinical features and prognosis in acute myeloid leukemiaObjective1.To use public databases,clinical specimens,and cell lines to screen out PAK members that play an important role in AML2.To analyze the expression level of PAK1 in AML and the relationship between PAK1 expression and patient survival,risk stratification,as well as gene mutation relevanceMethods1.Downloading and integrating The Cancer Genome Atlas(TCGA)information and subsequent analysis of the expression of PAK family members in AML samples and their impact on patient survival1.1 We used GEPIA(http://gepia.cancer-pku.cn/)to analyze the RNA-seq data as well as the clinical data from 173 AML patients from TCGA database.Next,we compared the PAK1-6 gene mRNA levels between AML patients and healthy controls.1.2 According to the median expression levels of PAK 1-6,AML patients were divided into the low and high expression groups,and the difference in survival of AML patients between the groups was analyzed.2.Collection,extraction,and testing of bone marrow specimensTo detect the expression level of PAK1 in AML patients,we collected bone marrow specimens from 41 AML patients and 8 healthy controls.Among the AML patients,20 were newly diagnosed whereas 12 were in complete remission and 9 were relapsed/refractory AML.The lymphocyte separation solution was used for density gradient centrifugation to separate bone marrow mononuclear cells.Real-time PCR was used to detect the expression of PAK1 at the mRNA level in bone marrow mononuclear cells of AML patients and normal controls.3.AML cell line culture and detection of PAK1 expressionThe human leukemia cell line HL60 was cultured in IMDM with 10%FBS and 1%penicillin/streptomycin.The human leukemia cell lines THP1,K562,Kasumi-1,U937,Jurkat,and KG1 were cultured in RPMI 1640 culture medium.They were cultivated in a complete medium with 10%FBS and 1%penicillin/streptomycin.The cells were collected and subjected to total protein extraction followed by western blotting to detect the PAK1 and p-PAK1 protein expression levels.4.Correlation analysis of PAK1 expression level and clinical characteristics of AML patientsPAK1 expression and clinical data from 173 AML patients(TCGA database)were downloaded,screened,and integrated.According to the PAK1 median expression,AML patients were divided into high and low expression groups.4.1 The correlations between PAK1 expression and patient gender,age,peripheral blood blast count,bone marrow blast count,peripheral blood white blood cell count,hemoglobin amount,and platelet count were analyzed.4.2 According to the FAB classification criteria,AML patients were divided into different morphological subtypes,and the correlation between PAK1 expression and different FAB subtypes was analyzed.4.3 The correlation between PAK1 expression level and cytogenetic abnormalities in AML patients was analyzed.5.Relationship between PAK1 expression levels and the risk stratification of AML patientsAccording to the AML patient information provided in TCGA database as well as the National Comprehensive Cancer Network(NCCN)guidelines,the prognostic risk of AML patients was stratified,and AML patients were divided into a good prognosis group,a medium prognosis group,and a poor prognosis group.The relationship between PAK1 expression and the different risk stratification of AML patients was analyzed.6.Influence of PAK1 expression level on prognosis in AML patients with gene mutationsTCGA database information was used to analyze the significance of PAK1 expression in the prognosis of AML patients with FLT3,IDH1,RAS,and NPMC mutations as well as other molecular genetic abnormalities.We divided AML patients into PAK1 high and low expression groups.On this basis,we combined different gene mutation states,including FLT3,IDH1,RAS and NPMC,and divided AML patients into four groups.Subsequently,we analyzed the survival rate of AML patients in the different groups.7.Statistical methodsWe used SPSS V20.0 for statistical analysis.Paired or unpaired Student t-tests were used to assess statistical differences between the two groups,and one-way analysis of variance was used to determine significant differences between multiple groups.The Mann-Whitney U test was used for data with unequal variances.The Pearson chi-square test was used to compare the differences in clinical characteristics between groups.Survival time was presented in a Kaplan-Meier survival chart.Spearman correlation test was used to test the correlation between different variables.P-values<0.05 were considered statistically significant.Results1.PAK expression levels in the bone marrow of AML patients and healthy controlsWe evaluated the expression levels of PAK family members in AML patients'data from TCGA database.The expression levels of PAK1 and PAK6 in AML patients were higher than those in normal controls,whereas the expression of other PAK family members was not significantly different between AML patients and normal controls.2.Influence of PAK expression levels on the survival of AML patientsThe overall survival time of AML patients in the PAK1 high-expression group was significantly shorter than that in the PAK1 low-expression group.The expression levels of other PAK family members were not significantly correlated with the overall survival of AML patients.This suggests that PAK1 may play an important role in the occurrence and development of AML.3.PAK1 expression level in bone marrow mononuclear cells of AML patients and normal controlsReal-time PCR results showed that compared to that in the healthy control group,the expression level of PAK 1 in AML bone marrow mononuclear cells was significantly increased.Additionally,the expression level of PAK 1 in newly diagnosed AML patients was significantly higher than that in the complete remission group,and the expression of PAK1 in refractory/relapsed AML patients was also significantly higher than that in the complete remission group.These findings suggest that PAK1 may be closely related to the onset and drug resistance of AML.4.Expression level of PAK1 in AML cell linesSeven leukemia cell lines were cultured,of which THP1,Kasumi-1,U937,HL60,and KG1 were AML cell lines.K562 was a CML cell line,and Jurkat was a T-ALL cell line.Western blotting results showed that PAK1 and p-PAK1 were highly expressed in most AML cell lines.5.Relationship between PAK1 expression and the clinical characteristics of AML patients5.1 Following integration and analysis of the AML patients' data from TCGA database,high PAK1 expression was found to be associated with the patient's advanced age,lower bone marrow blast cell count,lower hemoglobin amount,and higher platelet count.5.2 The dataset used in this study consisted of 16 cases of M0 type,42 cases of M1 type,39 cases of M2 type,16 cases of M3 type,35 cases of M4 type,18 cases of M5 type,2 cases of M6 type,3 cases of M7 type,and 2 cases of M7 type.The PAK1 expression in patients with M4 and M5 subtypes was relatively high,whereas the PAK1 expression in patients with M3 subtypes was relatively low;the difference between them was statistically significant.5.3 In patients with high PAK1 expression,complex karyotypes were more common,whereas t(8;21)and t(15;17)cytogenetic abnormalities were less common.5.4 COX multivariate analysis showed that similar to age and cytogenetic risk status,PAK1 expression level may be one of the prognostic factors of AML.6.Relationship between PAK1 expression level and the risk stratification of AML patientsThe correlation between PAK1 expression level and different prognostic risk strata of AML patients was analyzed.Among the 173 cases,32 were in the good prognosis group,103 were in the medium prognosis group,36 were in the poor prognosis group,and 2 could not be grouped.PAK1 expression levels in the good prognosis group were significantly reduced compared to those in the medium prognosis group and the poor prognosis group.7.Effect of PAK1 expression level on AML prognosis in patients with gene mutations In AML patients without FLT3,IDH1,NPMC,or RAS mutations,high PAK1 expression was shown to be associated with a poor prognosis.For AML patients with FLT3 mutation,the prognosis of patients with high expression of PAK1 was worse than that of patients with low expression of PAK1,and patients with both FLT3 mutation and high expression of PAK1 experienced the shortest survival time.Conclusion1.Among the members of the PAK family,only PAK1 is highly expressed in AML patients and is closely related to poor prognosis.2.The expression level of PAK1 in newly diagnosed,relapsed and refractory AML patients is significantly higher than that in patients with complete remission.3.High expression of PAK1 is closely related to a higher cytogenetic risk and more complex karyotype.PART TWOStudy on the biological function and mechanism of action of PAK1 in acute myeloid leukemiaObjectiveTo clarify the effect of the PAK1 gene on AML cell proliferation,apoptosis,as well as drug sensitivity and explore the downstream regulatory mechanism of PAK1.Methods1.Downregulation of PAK1 expression via lentivirus infection in AML cell lines In order to study the biological function of PAK1 in AML,we selected two AML cell lines(THP1 and Kasumi-1).The PAK1 gene in THP1 and Kasumi-1 cells was stably down-regulated by lentiviral transfection technology carrying shRNA.1.1 The AML cell lines were transfected with the lentivirus containing PAK1 shRNA,and fluorescence-activated cell sorting was performed using the GFP label as a sorting marker.The sorted cells continued to be expanded and cultured to obtain a cell line with stable downregulation of PAK1 expression.1.2 The transfection efficiency of the PAK1 downregulation group(shPAK1)and control group(shCtrl)cells was evaluated using fluorescence microscopy and flow fluorescence analysis.1.3 Real-time RT-PCR and western blotting were used to detect the downregulation efficiency of PAK1 in THP1 and Kasumi-1 cells.2.Detection of AML cell proliferation following shPAK1-induced downregulation of PAK1Using lentiviral transfection and screening,we constructed a cell line with stable downregulation of PAK1 and used a CCK-8 detection kit to detect changes in the proliferation ability of AML cells in the PAK1 downregulation group and the control group at different time periods.3.Detection of AML cell apoptosis following lentivirus-induced PAK1 downregulationFollowing lentiviral transfection of the AML cells,Annexin V/PI or Annexin V-FITC/7-ADD double staining was used to detect apoptosis in the PAK1 down-regulated and control cell groups using flow cytometry.4.Drug sensitivity of AML cells following PAK1 downregulation4.1 Following lentiviral transfection with PAK1 shRNA,THP1 cells were treated with different concentrations of Ara-C(2 ?M,4?M)or IDA(10 ?g/L,20 ?g/L)for 24 h.Subsequently,Annexin V-FITC/7-ADD double staining and flow cytometry were used to evaluate the apoptotic rate in the PAK1 downregulation group(shPAK1)and control group(shCtrl).4.2 After down-regulating the expression of PAK1 in Kasumi-1 cells using a lentivirus,the cells were treated with different concentrations of Ara-C(4 ?M,8 ?M)or IDA(20 ?g/L,40 ?g/L)for 24 h,and Annexin V-FITC/7-ADD double staining and flow cytometry were used to evaluate the apoptosis rate in the PAK1 downregulation.group(ShPAK1)and control group(shCtrl).5.Inhibition of PAK1 expression using small molecule inhibitorsWe used IPA-3,a selective non-ATP competitive PAK1 small molecule inhibitor,to inhibit PAK1 expression.First,THP1 and Kasumi-1 cells were treated with different concentrations of IPA-3(0 ?M,10 ?M,and 20 ?M)for 12 and 24 hours,respectively.Western blotting was used to detect the expression of p-PAK1 and PAK1 in AML cells and examine the effect of IPA-3 on PAK1 expression in AML cells.6.Effect of small molecule inhibitors on the proliferation and apoptosis of AML cells6.1 THP1 cells were treated with different concentrations of IPA-3(0 ?M,10 ?M,20 ?M)for 24 hours and the proliferation ability of THP1 cells was detected using CCK-8.6.2 THP1 and Kasumi-1 cells were treated with 20 ?M and 30 ?M IPA-3,respectively,for 24 hours and the apoptotic rate was investigated using the Annexin V/PI kit and flow cytometry.7.Effect of small molecule inhibitors on drug sensitivity of AML cells7.1 THP1 cells were treated with IPA-3(10 ?M)and Ara-C(1 ?M)or IDA(10?g/L)for 24 hours,and cell proliferation was measured using the CCK-8 method.7.2 THP1 cells were treated with IPA-3(10 ?M)and Ara-C(1 ?M)or IDA(10?g/L)for 24 hours.The Annexin V/PI kits and flow cytometry were used to detect the apoptotic effects of IPA-3 combined with chemotherapy drugs.8.Study of PAK1 regulating the ERK signaling pathway8.1 First,we used the GEPIA and TCGA databases to analyze the correlation between PAK1 mRNA levels and ERK1/ERK2 signaling in AML patients.8.2 Next,we used lentiviral transfection(shPAK1)or IPA-3 to inhibit the expression of PAK1 in THP1 and Kasumi-1 cells.Subsequently,we used western blotting to detect the changes in ERK 1/2 and p-ERK protein expression.9.Statistical analysisWe used SPSS V20.0 software for data analysis.The experimental data are presented as mean ± standard deviation.The data were obtained from at least three independent experiments.A paired or unpaired Student's t-test was used to assess the statistical difference between two groups.The differences between three groups or more were analyzed via analysis of variance or a non-parametric test.P-values<0.05 were considered statistically significant.Results.1.Establishment of an AML cell line exhibiting stable PAK1 downregulationWe used the lentivirus containing PAK1 shRNA to infect THP1 and Kasumi-1 cells and then amplified the GFP-positive cells using flow cytometry.Fluorescence microscopy and flow fluorescence analysis revealed that the PAK1 downregulation group(shPAK1)and the control group(shCtrl)cells were transfected.The dyeing efficiency exceeded 95%.2.PAK1 expression in AML cells decreases following lentivirus transfectionReal-time RT-PCR and western blot results showed that the mRNA as well as protein expression levels of PAK1 in THP1 and Kasumi-1 cells following transfection were significantly lower than those in the negative control group.This indicates that the lentivirus carrying PAK1 shRNA can significantly down-regulate the expression of PAK1 in THP1 and Kasumi-1 cells.3.Effect of PAK1 downregulation on the proliferation of THP1 and Kasumi-1 cellsThe CCK-8 assay showed that PAK1 downregulation(shPAK1)led to a decreased proliferation ability in d2-d4 THP1 cells compared to that in the negative control group(shCtrl).A similar inhibition of proliferation was observed in Kasumi-1 cells on Day 4,suggesting that PAK1 downregulation can reduce the proliferation ability of AML cells.4.Effect of PAK1 downregulation on the apoptosis of THP1 and Kasumi-1 cellsThe apoptotic rate of THP1 and Kasumi-1 cells in the PAK1 downregulation group was significantly higher than that in the negative control group,suggesting that PAK1 downregulation can promote AML cell apoptosis.5.Effect of PAK1 downregulation on chemotherapeutic drug sensitivity in THP1 and Kasumi-1 cellsDifferent concentrations of the chemotherapeutic drugs Ara-C and IDA were used to treat AML cells in the PAK1 downregulation group and the control group,respectively.Results showed that downregulation of PAK1 could significantly increase Ara-C-and IDA-induced AML cell apoptosis.This finding suggests that PAK1 downregulation can increase the sensitivity of AML cells to chemotherapeutic drugs.6.Effect of IPA-3 on PAK1 expression in AML cellsWestern blotting results showed that the PAK1 small molecule inhibitor IPA-3 could significantly reduce the expression of p-PAK1 and PAK1 in a dose-dependent manner.7.Effect of IPA-3 on the proliferation and apoptosis of AML cells7.1 IPA-3 could significantly inhibit the proliferation of THP1 cells in a dose-dependent manner.7.2 Following the analysis of the apoptotic assay,we observed that the apoptotic rate of TPH1 and Kasumi-1 cells in the IPA-3 treatment group was increased compared to that in the control group.This suggests that IPA-3 inhibition of PAK-1 can promote AML cell apoptosis.8.Effect of IPA-3 on drug sensitivity in AML cells8.1 The CCK-8 assay showed that the combination of IPA-3 and Ara-C or IDA led to significantly increased inhibition rates of THP1 cells compared to those observed in the group treated with Ara-C or IDA alone.8.2 Following treatment with IPA-3 combined with Ara-C or IDA,THP1 and Kasumi-1 cells exhibited a significantly increased apoptotic rate compared to those treated with Ara-C or IDA alone.9.PAK1 regulates the ERK signaling pathway9.1 Results of the database analysis showed that the mRNA expression levels of ERK1 and ERK2 were positively correlated with the expression of PAK1 in AML patients.9.2 In THP1 and Kasumi-1 cells,lentivirus or IPA-3 was used to inhibit the expression of PAK1.Western blotting results showed that the decrease in PAK1 expression was correlated with a decrease in p-ERK1/2 levels,thus indicating that PAK1 can regulate the ERK signal pathway.Conclusion1.Down-regulating the expression of PAK1 can significantly inhibit the proliferation of AML cells and promote cell apoptosis.2.Down-regulating the expression of PAK1 can enhance the sensitivity of AML cells to Ara-C/IDA traditional chemotherapeutic drugs.3.PAK1 may play a role in AML onset and development by regulating the ERK1/2 signaling pathway.PART THREERole and mechanism of action of PAK1 in bone marrow stromal cell-mediated drug resistance in acute myeloid leukemiaObjectiveTo clarify the role of PAK1 in BMSC-mediated drug resistance of AML cells and the downstream signaling pathway of BMSC-mediated AML chemotherapy resistance.Methods1.Co-culture model1.1 Direct contact co-culture model1)We seeded human bone marrow stromal cells HS-5 onto cell culture plates overnight so as to ensure cell adherence to the wall.Next,we aspirated the culture solution and added fresh complete medium.2)We collected primary AML cells,THP1 9 Kasumi-1 or KG1 cells in the logarithmic growth phase,adjusted the cell density,and added them to the above-mentioned adherent HS-5 cell culture(the ratio of AML cells:stromal cells was1:1)for 24 hours.1.2 Transwell indirect co-culture model1)We spread the HS-5 cells on the cell culture plate overnight so as to ensure cell adherence to the wall.Next,we aspirated the culture solution and added fresh complete medium.Lastly,we placed the Transwell chamber on the well plate.2)AML cells in the logarithmic growth phase were added to the Transwell chamber and cultured(AML cell:stromal cell ratio was 1:1)for 24 hours.2.Cellular immunofluorescence detectionThe THP1 cells and HS-5 cells were cultured in a direct co-culture or indirect co-culture model for 24 hours.THP1 cells were collected for cell immunofluorescence detection,and the fluorescence expression of PAK1 as well as p-PAK1 was examined and imaged using a fluorescence microscope.3.Western blot detectionPrimary AML cells,THP1,Kasumi-1,or KG1 were cultured separately or in a co-culture model with HS-5 cells for 24 hours.AML cells were collected,and the total protein was extracted.Western blotting was used to detect PAK1 and p-PAK1 protein expression levels in AML cells.4.Apoptosis detection4.1 Primary AML cells,THP1 or Kasumi-1 cells were cultured alone or co-cultured with HS-5 cells overnight,following which the AML cells were treated with 2 ?M Ara-C for 24 hours.The AnnexinV-FITC/7-AAD apoptosis kit and flow cytometry were used to evaluate the apoptotic rate.4.2 After down-regulating PAK1 expression in THP1 and Kasumi-1 cells using the shRNA lentivirus,AML cells and HS-5 cells were co-cultured directly or indirectly.Next,the AML cells were treated with 2 ?M Ara-C for 24 hours.Flow cytometry was used to evaluate the apoptotic rate.4.3 After THP1 cells were directly or indirectly co-cultured with HS-5 cells,IPA-3(20 pM)and Ara-C(2 ?M)were added for 24 hours.The apoptotic rate was detected using flow cytometry.5.Downstream mechanism detectionFirst,THP1 and Kasumi-1 cells were transfected with lentiviral shPAK1.AML cells were co-cultured for 24 hours alone or in direct or indirect contact with HS-5 cells and then treated with Ara-C 20 ?M for 24 hours.Western blotting was used to detect the expression of PAK1,p-PAK1,ERK1/2,p-ERK1/2,and apoptotic product PARP as well as cleaved-PARP.GAPDH was used as an internal control.6.Statistical methodsSPSS V20.0 was used for statistical analysis.The experimental data were presented as mean ± standard deviation.The data were obtained from at least three independent experiments.Paired or unpaired Student's t-tests were used to assess the differences between the two groups,and one-way analysis of variance was used to determine the differences between multiple groups.P-values<0.05 were considered statistically significant.Results1.PAK1 protein expression levels in AML cells co-cultured with BMSCsPAK1 and p-PAK1 protein expression levels in AML cells co-cultured with HS-5 cell were significantly increased compared to those in the AML cell culture alone.Furthermore,the increase in PAK1 and p-PAK1 expression was the most obvious in the direct co-culture model.2.Immunofluorescence detection of PAK1 expression level in AML cells co-cultured with BMSCsCellular immunofluorescence detection showed that following co-culture of AML cells and BMSCs,the fluorescent expression of PAK and p-PAK1 increased.In addition,the fluorescent expression of PAK1 and p-PAK1 was higher in the direct co-culture model than in the indirect Transwell co-culture model.3.Apoptosis of AML cells induced by chemotherapeutic drugs is reduced after BMSC co-cultureThe Ara-C-induced AML cell apoptotic rate was significantly reduced in the two co-culture models compared to that in the AML cell culture alone.Furthermore,the apoptotic rate of AML cells in the direct co-culture model was lower than that in the indirect Transwell co-culture model.This effect was opposite to the effects associated with PAK1 expression.Thus,we hypothesize that PAK1 plays an important role in AML cell protection mediated by BMSCs.4.PAK1 downregulation can weaken the protective effect of BMSC on AML cellsFollowing PAK1 downregulation using the lentiviral shPAK1,THP1 cells and Kasumi-1 cells were co-cultured with HS-5 in a direct or indirect manner.Next,AML cells were treated with Ara-C for 24 hours.Using flow cytometry,we observed that the apoptotic rate of AML cells in the co-culture model was significantly higher than that in the control group after PAK1 was down-regulated.5.Inhibition of PAK1 expression by IPA-3 can weaken the protective effect of BMSC on AML cellsAfter AML cells and HS-5 cells were co-cultured directly or indirectly,they were treated with Ara-C alone or in combination with IPA-3(24 hours).We observed that the apoptotic rate of AML cells in the IPA-3 combined with Ara-C treatment group was increased compared to that in the.Ara-C-alone treatment group.This indicates that inhibiting PAK1 can weaken the protective effect of BMSCs on AML cells and increase the sensitivity of AML cells to chemotherapeutic drugs.6.Down-regulating PAK1 can inhibit BMSC-mediated activation of the ERK signaling pathwayWestern blotting results showed that the expression level of p-ERK1/2 was significantly increased in AML cells co-cultured with HS-5 cells when compared to that in AML cells cultured alone.This phenomenon is consistent with our findings associated with the role of PAK1 and p-PAK1 expression.Furthermore,PAK1 downregulation in AML cells led to a decrease in p-ERK1/2 expression in co-cultured AML cells.In AML cells cultured alone or in a co-culture,PAK1 downregulation increased the expression of the apoptotic marker cleaved-PARP.This suggests that PAK1 downregulation can inhibit the activation of the BMSC-mediated ERK signaling pathway.Conclusion1.PAK1 plays a role in BMSC-mediated drug resistance of AML cells.2.Inhibition of PAK1 can weaken the protective effect of BMSC on AML cells and increase the sensitivity of AML cells to chemotherapeutic drugs.3.BMSC-mediated AML chemotherapy resistance may be related to activation of the ERK1/2-cleaved-PARP signaling pathway by PAK1.