Research On Relationship And Mechanism Of Histone Methyltransferase KMT2C And BCR-ABL1-independent Resistance In Chronic Myeloid Leukemia

Chronic Myeloid Leukemia(CML)is accounting for about 15%of adult leukemia,which is the most common type of chronic leukemia.It can occur at any age and the incidence of the disease increases with age.In the world,the CML incidence is 1?2 cases per 100,000,about 0.36-1 cases per 100,000 in China,and mainly in the elderly.The presence of BCR-ABL1 tyrosine kinase inhibitor(TKI)-imatinib(IM)has completely changed the way of CML treatment,so that the annual mortality rate of CML patients has decreased from 10~20%to 1?2%,and the 10 year survival time has also increased from 10?20%to 80-90%.The continuous emergence of TKI drugs has brought good prospects for the maintenance of long-term remission in CML.IM has transformed CML from a deadly malignant disease to a chronic disease.However,about 20?40%newly diagnosed CML patients in chronic phase,will eventually need to change the treatment because of adverse drug reactions or drug resistance.The main causes of drug resistance can be divided into BCR-ABL1-dependent resistance and BCR-ABL1-independent resistance.Dependent resistance is mainly associated with more than 100 ABL kinase region mutations.In the Independent resistance,although TKI successfully inhibit the activity of BCR-ABL1 kinase,the activation of the accessory signal pathway can make up for the loss of tyrosine kinase activity,to promote the prolifeation of CML cells,leading to TKI resistance.However,less related studies have been made for independent resistance,and its mechanism is still not clear.For the reasons above,this study aimed at to explore the BCR-ABL1-independent resistance in CML.By means of high-througput sequencing and CRISPR/Cas9,we used clinical information,bone marrow/peripheral blood detection and in vitro cell model construction to preliminarily discuss the signaling pathways and related mechanisms.Part I:Clinical study of early molecular response with imatinib resistance in chronic myeloid leukemiaAim:To explore the relationship between the early molecular response and imatinib resistance in CML-CP patients.Methods:1.Sanger sequencing was used to detect the mutation of BCR-ABL1 kinase domain in patients with IM resistance,and to identify the causes of IM resistance.2.Analyzing the relationship between the early molecular response and IM resistance.3.Statistical analysis:Age and time were described as the media.Chi square test was used to analyze the difference between the groups.The difference of parameters between groups was analyzed by homogeneity of variance and t test.P value of 0.05 was Lused to determine statistical significance.SPSS20.0 statistical software was used for statistical analysis.Results:1.In 198 IM-resistant patients without mutation,the proportion of patients with taking hydroxyurea,melphalan or interferon as initial therapy was higher(43%).And along with the non IM drugs use,the proportion of IM-resistant patients with no kinase domain mutation was higher.2.The longer the initial use of non IM drugs.the higher risk for the patients with BCR-ABL13mon>10%.and the higher proportion of patients with no mutation in kinase domain.3.The possibility of detecting kinase region mutations was higher in 34 patients with BCR-ABL13mon?10%and BCR-ABL16mon>1%.Conclusions:1.CML patients initial treatment with interferon,hydroxyurea and melphalan,may cause the BCR-ABL1-independent resistance.2.BCR-ABL1-independent resistance may cause poor molecular response at 3 months.but not affect the occurrence time of IM resistance.3.Increasing BCR-ABL1 levels at 6 months is suggested the possibility of secondary resistance due to mutation in BCR-ABL1 kinase region.Part II:Screening of genes related with BCR-ABL1-independent resistance in chronic myeloid leukemia by high-throughput sequencingAim:To explore genetic abnormality related with BCR-ABL1-independent resistance by Ihigh-throughput sequencing.Methods:1.Genes related with BCR-ABL1-independent resistance were screened with oral mucosa as the control and using high-throughput sequencing.2.Sanger sequencing was used to verify screened gene mutations in 347 CML patients with IM resistance.3.Analyzing the expression level of screened genes to further analyze the possible causes of BCR-ABL1-independent resistance.4.Statistical anzalysis:Data were described as mean�standard deviation.Chi square test was used to analyze the difference between the groups.The difference of parameters between groups was analyzed by homogneity of variance and t test.P value of 0.05 was used to determine statistical significance.SPSS20.0 statistical software was used for statistical analysis.Results:1.19 IM-resistant CML patients with no mutations in kinase domain were screened for 578 tumor related genes by high-throughput sequencing.The results showed that KMT2C K339N and RUNX1 H214P might lead to dysfunction of related genes after screening for multiple conditions.2.In 198 IM-resistant CML.patients with no mutations.25 cases with KMT2C K339N were detected.The frequency of KMT2C K339N was significantly higher than that with mutation in kinase domain.While the mutation frequency of RUNX1 H214P between the two groups was not significantly different.3.There was no correlation between the expression level of KMT2C mRNA and BCR-ABLl-independent resistance.Conclusions:1.KMT2C K339N do not change with the expression level of KMT2C mRNA.This showes that KMT2C K339N may cause the abnormal function of KMT2C protein.2.KMT2C K339N activates related signaling pathways leading to BCR-ABL1-independent resistance in CML patients.Part III:To analyzing the relationship between H3K4 monomethylation regulated by KMT2C and BCR-ABL1-independent resistance in chronic myeloid leukemia Aim:To further clarify the relationship and mechanism between KMT2C K339N and BCR-ABL1-independent resistance.Methods:1.The relationship between KMT2C K339N and the level of H3K4 methylation was analyzed by Western Blot.2.The relationship between KMT2C K339N and PI3K/AKT signaling pathways was analyzed by Real-time RT-PCR and Western Blot.3.Statistical analysis:Data were described as mean1standard deviation.The difference of parameters between groups was analyzed by homogeneity of variance and t test.P value of 0.05 was used to determine statistical significance.SPSS20.0 statistical software was used for statistical analysis.Results:1.The level of H3K4 methylation analysis showed that the level of H3K4mel in KAMT2CK39N patients was significantly lower than that in KMT2CWT patients,and there was no significant difference between the H3K4me2 and H3K4me3 in the two groups.2.The levels of PTEN and P21 in KMT2CK339N patients were significantly decreased,and the levels of AKT Thr308.AKT Selr473 and XIAP increased significantly,while the expression levels of PI3K p110?,AKT,BCL-2,BAX and P27 showed not significant difference.Conclusions:1.KMT2C K339N activates the signal pathway by regulating the level of H3K4mel,causing BCR-ABL1-independent resistance.2.The downregulation of H3K4mel decreases PTEN expression,and then phosphorylates AKT,regulates the expression level of' P21 and XIAP,and ultimately leads to BCR-ABL1-independent resistance.Part IV:Construction of KMT2CK339N K562 cell model by CRISPR/Cas9 and related mechanism study Aim:To construct the KMT2CK339N K562 cell model and further clarify the related mechanism.Methods:1.KMT2CK339N K562 cell model was constructed by CRISPR/Cas9 and ssODN.2.Ceel proliferation ability of KMT2CK339N K562 was further clarified by using CCK8 cell proliferation test,EdU cell proliferation test and methyl cellulose clone formation test.3.The apoptosis situation of KMT2CK339N K562 was further clarified by using the apoptosis detection of Annexin V-FITC/PI double staining cells.4.Real-time RT=PCR and Western Blot were used to further clarify the relationship among KMT2C K339N.H3K4 methylation and PI3K/AKT pathway.5.Statistical analysis:Data were described as mean�standard deviation.The difference of parameters between groups was analyzed by homogeneity of variance and t test.P value of 0.05 was used to determine statistical significance.SPSS20.0 statistical software was used for statistical analysis.Results:1.K562 cell was constructed as KMT2CK339N K562 cell model by Lusiing CRISPR/Cas9 and ssODN.2.The proliferation ability of KMT2Ck339N K562 cells increased and the level of apoptosis decreased.3.The level of H3K4 monomethylation in KMT2CAK339N K562 cells decreased,while the levels of PTEN,P21,BCL-2,BAX and P27 decreased significantly,and the levels of AKT Thr308,AKT Ser473.and X:IAP increased significantly,while the expression levels of PI3K p110? and AKT were not changed significantly.Conclusions:KMT2C K339N inhibits PTEN expression through down regulating H3K4mel level,and then activates PI3K/AKT signaling pathway,which affects CML cell apoptosis and proliferation,and finally leads to BCR-ABL1-independent resistance.