Effects Of Different Aberrations In The CRLF2 Gene On The Biological Characteristics And Drug Sensitivities Of B Cell Acute Lymphocytic Leukaemia Cells

Objective: To investigate the effects and mechanism of action of upregulated CRLF2 expression resulting from different aberrations of the CRLF2 gene on the proliferation,cell cycle,and sensitivity to chemotherapeutic drugs in the B-cell acute lymphocytic leukaemia(B-ALL)cell line Nalm6.Methods: Fluorescent protein-expressing plasmids harbouring an empty vector(Vector)and four different aberrations of the CRLF2 gene(i.e.,CRLF2 overexpression,CRLF2-IK6,P2RY8-CRLF2,and CRLF2 mutant [F232C])were constructed,respectively.Lentiviral vector system was used to package the GFP-labelled CRLF2,CRLF2-IK6,P2RY8-CRLF2,and F232 C into lentiviruses and the control virus.Meanwhile,Nalm6 cell lines with stable infection of the CRLF2,CRLF2-IK6,P2RY8-CRLF2,and F232 C lentiviruses as well as the Nalm6 cell line with stable infection the control virus were established.Quantitative PCR(q PCR)and Western Blot were used to validate the upregulation of CRLF2 m RNA and protein in the Nalm6 cell lines after transfection.CCK-8 and flow cytometry were used to determine the effects of upregulated CRLF2 expression resulting from different aberrations of the CRLF2 gene on cell proliferation and cell cycle.Western Blot was used to examine the effects of the different CRLF2 aberrations on cell proliferation and expression of cell-cycle proteins.High-throughput drug sensitivity testing was used to determine the effects of different aberrations of the CRLF2 gene on the sensitivity of cells to 19 chemotherapeutic drugs.Transcriptome sequencing technology(RNA-seq)was used to compare changes in gene expression resulting from different aberrations of the CRLF2 gene.Results: B-ALL cell lines infected with CRLF2,CRLF2-IK6,P2RY8-CRLF2,and F232 C lentiviruses and the B-ALL cell line infected with the control virus were successfully established.All four aberrations of the CRLF2 gene upregulated CRLF2 expression and promoted the proliferation of Nalm6 cells.The results from RNA-seq revealed the upregulation of the JAK/STAT pathway caused by the CRLF2 mutant(F232C),and Western blot showed that the expression of p-STAT5 protein was significantly increased in the F232C-overexpressing cells.In the Nalm6 cell lines with overexpression of CRLF2-IK6,P2RY8-CRLF2,and F232 C,the expression of pathways such as systemic lupus erythematosus(SLE)and transcriptional misregulation were upregulated,as detected by RNA-seq.In contrast,the expression of pathways such as hematopoietic cell lineage and osteoclast differentiation were downregulated in most Nalm6 cell lines.Of the five cell lines,the half-maximal inhibitory concentration(IC50or GI50)of dexamethasone was significantly higher in the F232C-overexpressing cell line compared with the control group and the cell lines overexpressing CRLF2,CRLF2-IK6,and P2RY8-CRLF2,indicating the presence of resistance to dexamethasone.Conclusions: The overexpression of CRLF2,CRLF2-IK6,P2RY8-CRLF2,and F232 C could all promote the proliferation of B-ALL cells and activate the JAK/STAT signalling pathway.Meanwhile,they also led to reduction in sensitivities towards various chemotherapeutic drugs.In particular,cells expressing the CRLF2 mutant(F232C)exhibited significant resistance to dexamethasone.Data presented herein provide a novel perspective for future research on dexamethasone resistance in B-ALL.