Identification Of The Novel Fusion Gene KANSL1-MTCP1/CMC4 In An Acute Myeloid Leukemia Patient With T(X;17)(q28;q21) And Its Mechanism Study

Background/Aim: We first discovered a novel chromosomal translocation t(X;17)(q28;q21)in the bone marrow(BM)sample of a patient with de novo acute monocytic leukemia.The patient relapsed and died within one year after chemotherapy.In view of the significance of leukemia-related translocation on diagnosis,prognosis,pathogenesis and treatment,it is important to identify the fusion gene involved in t(X;17)(q28;q21)and study its mechanism in leukemogenesis.Methods: RNA-seq,PCR and Sanger Sequencing was performed to identify and validate the fusion gene involved of t(X;17)(q28;q21)in the the acute monocytic leukemia patient.Using qRT-PCR method,the expression of involved genes(KANSL1,MTCP1 and CMC4)were detected in various types of leukemia cell lines and bone marrow samples of different subtypes of leukemia patients.Immunofluorescence assay was performed on NIH3T3 cells to detect the subcellular localization of the involved genes.MTCP1 and CMC4 genes were overexpressed in the mouse myeloid progenitor cell line 32 D,and the related stable cell lines were established,including MTCP1-32 D and CMC4-32 D.Using sh RNA,the expression KANSL1 was knock down in MTCP1-32 D cells and the MTCP1-sh KANSL1-32 D stable cell line was also established.The effect of aberrant expression of involved genes on proliferation and differentiation was detected by cell counting and flow cytometry.Furthermore,to explore the capacity of aberrant expression of involved genes on clonogenic growth,colony forming assay and serial replating assay were performed on both mouse progenitor cell line 32 D and BM hematopoietic progenitor cells.Results: RNA-seq analysis showed that the 5’-UTR of KANSL1 gene(17q21)was fused to the CDS region of MTCP1/CMC4 gene(Xq28),which formed four different fusion transcripts.All fusion transcripts were verified by PCR and Sanger Sequencing.Structure analyses of the fusion transcripts indicated that no fusion protein was generated of KANSL1-MTCP1/CMC4 fusion.However,qRT-PCR results revealed that the expression of KANSL1 gene was downregulated,and the MTCP1 and CMC expression was significantly up-regulated in the bone marrow sample of the patient.Immunofluorescence assay showed that under the expression of KANSL1-MTCP1/CMC4 fusion gene,the localization of MTCP1 and CMC4 protein was not altered.The results of cell counting showed that the stable cell lines MTCP1-32 D and MTCP1-sh KANSL1-32 D presented the proliferation advantage comparing with the CMC4-32 D and the Vector-32 D.Flow cytometry showed that MTCP1-32 D and MTCP1-sh KANSL1-32 D inhibited G-CSF induced cell differentiation,while CMC4-32 D could only partiallly inhibite the differentiation.Colony forming and serial replating assay results showed that MTCP1-32 D and MTCP1-sh KANSL1-32 D had advantages over clonogenic growth,while CMC4-32 D gradually lost its proliferative ability.The same results were found in mouse lineage-negative cells.Conclusion: In this study,we identified a novel fusion gene KANSL1-MTCP1/CMC4 resulting from a rare chromosomal translocation t(X;17)(q28;q21)in an acute monocytic leukemia patient.The main biological function of this fusion gene is the aberrant expression of MTCP1 accompanied with down-regulation of KANSL1 gene expression,sebsuquently presented the capacity of clonogenic preoliferation and inhibition of myeloid cell differentiation,thus participating in the leukemogenesis.