Study Of Resistance Mechanism Of IM In CML And The Predictive Value Of Sokal And ELTS Score Systems On Efficacy And Prognosis In CML Patients

Background:Chronic myeloid leukemia(CML)is a malignant disease caused by malignant gene cloning and evolution of bone marrow hematopoietic stem cells,and its clinical course can be divided into chronic phase(CP),accelerated phase(AP)and blastic crisis(BP).The emergence of tyrosine kinase inhibitors(TKIs)has led to molecular remission in most patients with CML.Imatinib(IM),as the first line drug for CML treatment,is generally regarded as the safest of the TKIs.However,the resistance of IM become the main obstacle to CML-targeted therapy.Our early research found that miR-23a was significantly differentially expressed in CML cell lines and their drug-resistant IM strains,suggesting that miR-23a may play an important role in disease progression and chemoresistance of CML.CRYAB is a principal member of the small molecule heat shock protein family.Recent studies showed that CRYAB might play an important role in tumor genesis and progression.CRYAB has been found to be highly expressed in various human cancers and significantly associated with the unfavorable prognosis of these tumors.Whether miR-23a is involved in the occurrence and development of chemoresistance of CML cells to IM through CRYAB has not been reported.Moreover,the development of new drugs that focus on drug-resistant CML cells is crucial for the development of more effective treatments.As a selective histone deacetylase inhibitor(HDACi)developed in China,chidamide has a good application prospect in the treatment of reversing the resistance of different anticancer drugs.Nevertheless,the potential mechanism has not been well elucidated.Based on the available data,we hypothesize that chidamide may induce apoptosis and increase the sensitivity of CML to IM and that it exerts its function by regulating the expression of miR-23a.Objective:The aim of this study was to investigate the expression of miR-23a in CML patients with and drug resistant cell lines,analyze the relationship between miR-23a and CML clinical progression;and ditermine the roles of miR-23a in IM resistance;We further elucidated the underlying molecular genetic basis of miR-23a in CML chemoresistance,and revealed the mechanism of miR-23a in IM chemoresistance,which is of great importance for the enhancement of CML treatment efficacy.In addition,we studied the expression of miR-23a under the effect of chidamide and its effect on the IM resistance of CML cells,therefore to provide a new target and therapeutic idea for reversing IM resistance of CML cells.Materials and methods:1.Quantitative real-time PCR(qRT-PCR)assay was performed to investigate the expression of miR-23a in bone marrow of patients and cell lines of CML.The relationship between miR-23a expression and chemoresistance of IM in CML was analyzed.2.The mechanisms of miR-23a in CML chemoresistance.After transfected with the synthetic miR-23a mimic or inhibitor,the expression of miR-23a was measured by qRT-PCR,Then,CML cells were treated with IM.The inhibition of cell growth was analyzed by CCK8 assay,and the apoptosis assay was performed using the AnnexinV/FITC and propidiumiodide(PI).3.Screening and identification of the crucial gene in miR-23a downstream,involved in IM resistance.3.1.Gene chip analysis:The key downstream genes of miR-23a involved in imatinib resistance of CML cells were screened by microarray images analysis and standard enrichment computation analysis.The expression of candidate genes was verified by qRT-PCR and Western Blot methods.3.2.The expression of CRYAB in CML patients and cell lines was detected by qRT-PCR.3.3.The expression of target gene CRYAB was down regulated by small interfering RNA(siCRYAB),and then,the drug sensitivity of CML cells to imatinib was detected by CCK8 assay.The apoptosis assay was performed using the AnnexinV/FITC and PI.4.Effects of chidamide on the expression of miR-23a in CML cell lines and its intervention on the biological behavior of CML drug-resistant cell line.4.1.After treated by chidamide with serial dilutions,the expression of miR-23a was detected by qRT-PCR.4.2.K562/G01 cells were treated with IM,after the intervention of chidamide.The inhibition of cell growth was analyzed by CCK8 assay.The apoptosis assay was performed using the AnnexinV/FITC and PI.4.3.After down-regulation of miR-23a expression,the effect ofchidamide on imatinib resistance of K562/G01 cells was detected.Results:1.K562/G01 drug resistance detection:CCK8 assay proved that K562/G01 cells were imatinib-resistant cell lines.2.A signficant downregulation of miR-23a were observed in CML drug-resistant cells K562/G01 compared with that in K562 cells.3.The expression of miR-23a among the clinical samples:The expression of miR-23a in TF group was significantly lower than that in OR group.The expression level of miR-23a was significantly lower in BP BM(bone marrow)samples than in samples obtained from the same patients at the time of CP.4.miR-23a modulates the chemoresistance of CML cells to IM.4.1.The results of CCK8 assay showed overexpression of miR-23a increased the sensitivity of K562/G01 cells to IM,compared with NC conditions.The results of apoptosis assay showed overexpression of miR-23a markedly increased the apoptosis of K562/G01 cells induced by IM.4.2.The results of CCK8 assay showed inhibition of miR-23a decreased the sensitivity of K562 cells to IM.The results of apoptosis assay showed inhibition of miR-23a markedly decreased the apoptosis of K562 cells induced by IM.5.Identification of the crucial gene in miR-23a downstream involved in IM resistance.5.1.Using gene chip for bioinformatics analysis,combined with qRT-PCR and Western Blot results,CRYAB was initially selected as the target gene regulated by miR-23a involved in IM-resistance.5.2.Expression of CRYAB in CML patients and CML cell lines:we observed that the expression level of CRYAB in TF group was higher than that in the OR group.CRYAB expression level in K562/G01 cells was significantly higher than that in K562 cells.5.3.The role of CRYAB in IM-resistance of CML:Transfection of siCRYAB downregulated the expression of CRYAB.CRYAB downregulation significantly increased cell sensitivity to IM and IM-induced apoptosis in K562/G01 cells.6.Overexpression of miR-23a induced by chidamide modulates the resistance of IM in CML cells.6.1.After treated with chidamide,the expression of miR-23a showed an increasing trend as detected by qRT-PCR in K562/G01 cells.6.2.After the intervention of chidamide,the sensitivity of K562/G01 to IM was enhanced and the apoptosis rate of K562/G01 cells induced by imatinib was significantly increased.6.3.After downregulation of miR-23a expression in K562/G01 cells,there was no significant difference in IM-sensitivity and IM-induced apoptosis with the intervention of chidamide.Conclusion:1.Our study indicates that the expression of miR-23a was significantly different in CML patients with different disease stages and different efficacy of IM,suggesting that miR-23a may be closely related to the disease progression of CML and the drug resistance of IM in CML.2.Reversing the expression of miR-23a in IM-resistant cells significantly increased the sensitivity of CML cells to IM and IM-induced apoptosis,suggesting that miR-23a may plays an important role in IM resistance mechanisms in CML and is expected to be a new target for CML treatment.3.CRYAB is the target gene of miR-23a,revealing that miR-23a mediate the resistance of CML cells to IM by regulating the expression of CRYAB.4.Chidamide can enhance the sensitivity of CML drug resistant cells to IM,moreover,the mechanism of the regulation might be through regulating miR-23a expression.Aims:Due to the wide application of TKIs,the life time of most CP patients can reach a stage comparable to the general population,where leukemia is no longer the leading cause of death.Based on this,the Sokal scoring system established in the pre-TKI era was considered to have some defects.Therefore,the EUTOS long-term survival(ELTS)scoring system was established in TKI era.At present,the clinical application value of this new scoring system has been confirmed in CML patients in western countries,and it is found that ELTS score can identify the high-risk group of CML death.However,its prognostic value and therapeutic guidance role in Chinese CML patients are not very clear.This study aims to investigate the predictive value of the ELTS scoring system for the curative effect and survival outcome of Chinese CML-CP patients receiving first-line TKI therapy,and to compare with the Sokal scoring system established in the pre-TKI era.It provides a basis for clinicians to carry out early intervention and guide treatment selection.Methods:The clinical data of 303 CML-CP patients treated with first-line TKIs from October 2005 to August 2015 were retrospectively analyzed.Risk stratification was assessed according to Sokal,ELTS scores.The predictive value of Sokal score and ELTS score system was compared and analyzed,including CCyR(complete cytogenetic response),MMR(major molecular response),MR4.5(molecular response of 4.5)and other indicators.Failure-and progression-free survivals(FFS,PFS),overall survival(OS),and CML-related survival(CML-OS)were calculated using the Kaplan-Meier estimator and log-rank tests.T-test and Pearson’s chi-squared test were used to compare the IM and 2G-TKI cohorts.Potential predictive covariates for diverse responses and outcomes were tested in univariable analyses and those with P<0.05 were included in multivariable analyses.The cumulative incidences of outcomes were calculated with Kaplan-Meier estimator and log-rank tests.P<0.05 represents a statistically significant difference.This study used SPSS26.0 software for statistical data analysis.Results:1.Compared to the Sokal score system,the ELTS score system identifies more patients as low-risk and fewer as high-risk:Among 303 evaluable CML-CP patients,158(52.1%),94(31.0%),and 51(16.9%)were classified as low-,intermediate-,or high risk using the Sokal score.Similar assignments using the ELTS score were 191(63%),83(27.4%),and 29(9.6%).Compared with the Sokal score system,the ELTS score system is closer to the current efficacy and survival distribution trend of CML patients.2.Prediction of sokal and ELTS scores for efficacy and survival outcomes in all enrolled patients:Kaplan-Meier curves were plotted to explore whether the Sokal or ELTS score was a better predictor.The results showed that Sokal score was significantly associated with the cumulative incidence of CCyR and MMR(P<0.001),but not the deep molecular response MR4.5.However,only the ELTS score was significantly associated with the cumulative incidence of MR4.5.The analysis of the survival outcome showed the Sokal score was significantly associated with the cumulative incidence of FFS,PFS and OS in the high-risk group.The ELTS score was significantly associated with the cumulative incidence of FFS,PFS and OS in all groups.Only risk stratification using ELTS scores can effectively distinguish the incidence of CML-OS in patients with various risk groups(P<0.001).Moreover,lower hemoglobin in concentration and higher WBC counts were significantly associated with lower probabilities of molecular responses and/or inferior outcomes.3.Prediction of Sokal and ELTS scores for efficacy and survival outcomes in IM cohort:Both scores were significantly associated with the cumulative incidence of CCyR,MMR,FFS,PFS and survival.However,only the ELTS score was significantly associated with the cumulative incidence of MR4.5 and CML-OS.Lower hemoglobin in concentration and higher WBC counts were significantly associated with lower probabilities of molecular responses and/orinferior outcomes.4.Prediction of Sokal and ELTS scores for efficacy and survival outcomes in 2G-TKI cohort:Only the ELTS score predicted the cumulative incidence of CCyR(P<0.001),MMR(P=0.001),MR4.5(P=0.004),FFS(P=0.02),PFS(P=0.02)and OS(P=0.03).We found that the ELTS score was a better overall response and outcome predictor,especially in subjects receiving 2G-TKI therapy.Conclusion:1.The Sokal and ELTS scoring systems are effective assessment tools for predicting the efficacy and survival outcomes of first-line TKI treatment.The ELTS scoring system is better in predicting the deep molecular response MR4.5.2.In terms of survival outcomes,the ELTS scoring system was able to identify high and intermediate-risk groups with lower CML-OS,which may benefit better with 2G-TKI treatment.3.The ELTS score is a better overall response and outcome predictor in subjects receiving first-line TKI therapy.