Downregulation Of The Histone Methyltransferase SETD2 Promotes Imatinib Resistance In Chronic Myeloid Leukemia

Objective:To measure the expression level of SETD2 in imatinib sensitive and imatinib resistant chronic myeloid leukemia(CML)cell lines,investigate the explicit mechanisms through which SETD2 influences the sensitivity of CML cells to imatinib and explore SETD2-associated small molecule,which could raise the sensitivity of imatinib.Methods:Immunoblotting and quantitative Real-Time PCR assays were performed to measure the expression level of SETD2 in imatinib sensitive and imatinib resistant CML cells.To respectively knock down SETD2 in imatinib sensitive CML cells through shRNA and overexpress SETD2 in imatinib resistant CML cells by amaxa nucleofector,after detecting the efficiency by qRT-PCR and immunoblotting assays,cell proliferation and cell apoptosis assays were performed to analyze the sensitivity of CML cells to imatinib upon on SETD2 knockdown or overexpression.The percentage and quantity of CD34~+CD38~-leukemic stem cells(LSKs)were assessed by flow cytometry and colony formation assays.To explore the underlying mechanisms,RNA-sequencing and chromatin immunoprecipitation were performed to clarify the putative downstream targets of SETD2-H3K36me3,then followed by verification through rescue assays.The reversed impact of small-molecule inhibitor JIB-04 on imatinib insensitivity and CD34~+CD38~-stem cell accumulation caused by SETD2 downregulation were assessed by cell proliferation,cell apoptosis,flow cytometry and colony formation assays.Results:Compared with imatinib sensitive cells,SETD2 was significantly downregulated in imatinib-resistant CML cells at post-translational level and could be successfully restored by protease inhibitor MG132 treatment.The decrement of SETD2promoted imatinib resistance in CML cells,accompanied by the enhancement of CD34~+CD38~-stem cells,which could be eventually reversed by SETD2 overexpression.A subset of genes involved in stemness regulation,especially ERG and MYCN,were upregulation induced by SETD2 downregulation.The knockdown of ERG and/or MYCN successfully reversed the effects of imatinib resistance resulted from SETD2knockdown,and the treatment of small-molecule inhibitor JIB-04 could effectively raise the sensitivity of imatinib in SETD2 downregulated cells.Conclusion:SETD2 downregulation potentiates imatinib resistance in CML cell lines through the overexpression of ERG and MYCN.Knock down of ERG and/or MYCN,as well as the use of small-molecule inhibitor JIB-04 all could efficiently reverse the effect of imatinib resistance.