Epigenetic regulation of the RAB5B GTPase expression in B-cell acute lymphoblastic leukemia

Ras analog in brain (Rab) proteins are important parts of small GTPase signaling pathways in human malignancies. RAB5B is the GTPase, which promotes cellular proliferation and is overexpressed in a large number of human cancers, including leukemia. Regulation of RAB5B transcription in human hematological malignancies is mostly unknown. Here, we analyze regulation of RAB5B transcription in human B-cell acute lymphoblastic leukemia (B-ALL). The use of global chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq), showed increased occupancy of IKAROS tumor suppressor protein at the promoter of RAB5B gene in several B-ALL cell lines and primary B-ALL cells. IKAROS direct binding to promoter of RAB5B was confirmed by quantitative chromatin immunoprecipitation (qChIP). The role of IKAROS in transcriptional regulation of RAB5BB in B-ALL was assessed by gain-of-function and loss-of-function experiments. Luciferase reporter assay showed that IKAROS directly represses transcription of RAB5B. Overexpression of IKAROS in B-ALL cells via retroviral transduction resulted in increased IKAROS binding to the RAB5B promoter and decreased RAB5B transcription. This was associated with enrichment in constitutive heterochromatin marker, H3K9me3 and the loss of active chromatin markers, H3K4me3 and H3K27ac. In contrast, shRNA-mediated knockdown of IKAROS inhibited IKAROS binding to the RAB5B promoter and resulted in increased RAB5B expression, along with increased enrichment of H3K4me3 and H3K27ac active chromatin markers, and the loss of H3K9me3 marker at RAB5B promoter. Primary B-ALL cells that carry deletion of one IKAROS allele have increased expression of RAB5B, associated with strong enrichment of H3K4me3 and H3K27ac, as well as the absence of H3K9me3 at RAB5B promoter. In conclusion, presented data suggest that IKAROS functions as a direct transcriptional repressor of RAB5B in B-ALL and that the mechanism of RAB5B repression by IKAROS involves formation of heterochromatin at the RAB5B promoter. Results uncovered a novel mechanism through which IKAROS exerts its tumor suppressor effect and regulate transcription and cellular proliferation in leukemia. Supported by NIH/NCI R01 CA278226 grant.