Demethylation and upregulation of an oncogene post hypomethylating treatment

BackgroundWhile hypomethylating agents (HMA) are currently used to treat myelodysplastic syndrome (MDS) and patients with cancer, their effects on reactivation and/or upregulation of oncogenes are generally not well elucidated. SALL4 is a known oncogene that plays an important role in MDS. In this study, we examined the impact of HMA on SALL4 methylation and expression. MethodsPaired bone marrow samples from a cohort of MDS patients on the BMT-AZA trial, collected before and after four cycles of azacytidine (AZA) treatment, were used to explore the relationship between changes in SALL4 expression, treatment response and clinical outcome with a follow-up of up to 40 months. No/low-SALL4 expressing leukemic cell lines were used to study the relationship between SALL4 methylation and expression. A novel locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island critical for SALL4 expression. ResultsIn MDS patients, we noted SALL4 upregulation after AZA treatment in 40% of the cases. Significantly, patients with SALL4 upregulation had a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a 500bp CpG island within the 5UTR-Exon1-Intron1 region was critical for SALL4 expression. Importantly, in cell lines and patients, we confirmed that HMA treatment led to demethylation of the same CpG region and upregulation of SALL4 expression. ConclusionsCRISPR-DiR was useful to define the critical region important for gene activation. Along with analysis of patient samples, we demonstrated that demethylation and upregulation of an oncogene after HMA treatment can indeed occur and should be further studied.