Pb1705: the histone modifier setd6 is epigenetically regulated in b-cell acute lymphoblastic leukemia and interferes with proliferation and gene expression

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: Acute lymphocytic leukemia (ALL) affects both children and adults. It is the most common pediatric cancer, the most frequent cause of death from cancer under 20 and a life threat if not treated immediately. Despite the remarkable developments in the treatment of ALL, resistance and relapse remain the leading cause of death. Although cancer has long been recognized as a genomic disease, the importance of epigenetic mechanisms in neoplasia has been acknowledged more recently. DNA methylation and histone modifications are the main epigenetic marks and participate in fundamental biological processes. The relevance of these marks in cancer initiation and progression is supported by evidence revealing that several epigenetic modifiers are recurrently mutated or dysregulated in multiple cancers, including B-ALL. That is why targeting epigenetic regulators have emerged as an effective therapeutic strategy for cancer treatment. The histone methyltransferase SETD6 is a protein to consider since it is involved in controlling cell adhesion and migration and cancer cell survival. Aims: To investigate the role of SETD6 methylation in B-ALL cell lines and its implication in cell proliferation and gene expression. Methods: The methylation status of the SETD6 promoter was studied in silico and validated in 4 different B-ALL cell lines (RCH-ACV, P30-OHK, REH, MHH-CALL4) by bisulfite sequencing. SETD6 mRNA and protein levels were tested by qPCR and Western Blot, respectively. Then, we generated SETD6 overexpression and knockdown models by viral transduction to test: 1) proliferation using MTT and EdU assays, 2) cell cycle and apoptosis rates by flow cytometry with propidium iodide (PI) and Annexin V, respectively, and 3) differences in their transcriptional profiles when compared to empty vector models by RNA-seq experiments. Finally, SETD6 methylation, expression and survival in B-ALL were analyzed using public data from the TARGET project (Phase II). Results: An in-silico analysis showed that SETD6 promoter methylation and its mRNA levels negatively correlated in hematological cell lines. These results were further experimentally validated in four B-ALL cell lines. Furthermore, we observed that SETD6 expression was restored in hypermethylated cell lines after AZA treatment. MTT assays proved that cells in which SETD6 is downregulated grew significantly less than the corresponding controls (p < 0.001), which were additionally supported by EdU and PI assays (p < 0.050 for all instances). This was in line with increased proliferative rate in the SETD6 overexpressing cells (p = 0.020). The RNA-seq studies performed with the different cellular models led us to identify differentially expressed genes. Of note, these genes are associated with cell morphogenesis, cytoskeleton reorganization and motility. Similar results were obtained after the analysis of B-ALL patients’ data. Such analysis also revealed that SETD6 methylation and expression levels negatively correlate, as seen in cell lines, and of interest, patients with higher expression of SETD6 had shorter overall survival than those with lower expression of this protein (p < 0.001). Summary/Conclusion: The expression of SETD6 is regulated by DNA methylation and variations in its protein levels result in changes in proliferation and gene expression profiles in B-ALL models. Overall, our results and the analysis of patients’ public data show that SETD6 is a potential biomarker for B-ALL patients, especially for those with bad outcome, and could be involved in pathways promoting cancer progression. Keywords: Methylation, Epigenetic, Proliferation, Gene expression