Functional Evaluation and Mechanism Study of Kel As an Oncogenic Driver in Acute Erythroleukemia

Abstract

Background Acute erythroleukemia (AEL) is defined as an distinct subtype (<5%) of AML in FAB classification, characterized by predominant erythropoiesis. It is associated with a poor prognosis, with a median overall survival (OS) of 3-14 months from diagnosis. No recurrent cytogenetic abnormality is specific of AEL. Through "Oncomine" Database, we found that KEL was significantly up-regulated in AEL patients, compared with other types of AML and normal patients. It has been reported that KEL promoter exhibited a strong transcriptional erythroid activity in K562 cells, but its role in the development of AEL remains unclear. Circular RNAs (circRNAs) have been implicated in various biological processes. Here we found one circRNA transcripted from KEL gene (circ-KEL), which also had a high expression in AEL, may play a role of coordinate regulation with KEL. The present study aimed to investigate the potential modulation of KEL in the development and progression of AEL. Method In our study, we collected blood samples of 11 AEL patient, 20 other types of AML patients and 20 normal patients. qPCR and Western blot analysis were performed to examine the expression of KEL and circ-KEL. We set up the erythroid differentiation models in vitro using two AEL cells lines (K562 and HEL) , induced with 50 μM hemin from day1 to day 7. The oncogenic functions of KEL were further measured in AEL cell lines by performing RNA interfering (RNAi), immunofluorescence (IF) staining, CCK8 assay and flow cytometry. The expression levels of γ-globin and FUT1 as well as the erythroid surface markers TER119 and GPA were examined to evaluate their function in erythroid differentiation. The proliferative ability of AEL cells was determined by CCK-8 assay. AEL animal models were established using NCG mice transplanted with K562 (NC and KEL over-expression). The tumor formation rate, body weight and survival curve were investigated to evaluate the oncogenic functions of KEL in-vivo. Protein antibody microarray was used to analyze the activation of related pathways after KEL over-expression in AEL cells. Moreover, we explored the molecular mechanisms of KEL and verified the interactions among GATA1 and H3K27ac as well as the PKC/RSK2/CREB pathway by performing ChIP, RIP, dual-luciferase reporter assay and Western blotting. Result KEL was specifically higher expressed in AEL clinical samples/cells lines, consistent with the results of the database. The over-expression of KEL could up-regulate the expression of GATA1, thus promoting the erythroid differentiation of AEL cells. Moreover, protein antibody microarray results showed that the up-regulation of KEL in AEL cells leads to the activation of multiple proliferation and PDL1 related pathways. After subsequent verification, it was verified that PKC/RSK2/CREB pathway was significantly activated. Meanwhile, the expression of PDL1 was highly correlated with KEL. We found that the higher expression of KEL in AEL cells could up-regulate PDL1 expression through activating the phosphorylation of relative transcript factors on PDL1 DNA promoter. In-vivo experiment showed that KEL over-expression may accelerate the leukemia progression. In addition, we discovered that JQ1 could be used as a inhibitor of KEL, which could effectively decrease the expression of GATA1 and PDL1. A number of circRNAs have been reported to act as microRNA (miRNA) sponges, which regulate target gene expression by interacting with miRNA. Our results revealed that circ-KEL could serve as a sponge for miR-671 and up-regulate miR-617 functional target KEL, thus promote the erythroid differentiation. Conclusion Our study reveals the oncogenic role and a novel regulatory mechanism of KEL in AEL progression. For the first time, we demonstrated that KEL was specifically highly expressed in AEL cells, leading to the dysregulation of erythroid differentiation through up-regulating GATA1, and on the other hand, promoting cell proliferation via PKC/RSK2/CREB pathway. Circ-KEL which also had a high expression in AEL, may play a role of coordinate regulation with KEL. These findings may provide a promising strategy for future diagnosis, biomarker discoveries and treatment for AEL patients. Figure Disclosures No relevant conflicts of interest to declare.