Integrin-β7 is part of an extensive family of glycoproteins and is overexpressed in the MF subgroups in Multiple Myeloma (MM), contributing to drug resistance and poor survival. ITGB7 promotes adhesion and proliferation of myeloma cells in bone-marrow stroma by supplementing growth factors like VEGF. Here, we investigate the possible epigenetic mechanism of ITGB7 overexpression in the MF subgroup, comprising of t(14;16) and t(14;20) myeloma. Newly diagnosed MM (NDMM) patient bone marrow aspirates underwent CD138 cell selection to enrich tumor cells to >98%. Samples consisted of those with a t(14;16) (n=17), t(14;20) (n=7), t(4;14) (n=9), t(11;14) (n=10) and hyperdiploidy (n=19, separated into D1 (n=12) or D2 (n=7 subgroups). NDMM patient samples were compared to plasma cells isolated from age-matched healthy donors (n=4), to determine differential changes in epitranscripts. We performed unbiased genome-wide reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs) in CpG islands, which were validated by Infinium MethylationEPIC arrays (Illumina) in conjunction with gene expression array data (U133 Plus 2.0, Affymetrix) to determine the epitranscriptomic profile in all samples. RRBS was performed using 75 bp reads to a minimum of 20 M reads per sample. Data from MM cells at the Blueprint consortium were also used for annotation of epigenetic marks. Using the RRBS data we identified 26 hypomethylated, overexpressed genes in the MF group samples, which were not present in the other subgroups. In comparison, only 1 gene, FUT7, was hypermethylated and down-regulated in the MF cluster. Most interestingly, we identified ITGB7 amongst the hypomethylated, overexpressed genes. With an in-depth analysis we identified 4 significant (p<0.05) DMRs across intragenic regions of ITGB7, within a 2.5 kb region. Mean methylation across the DMRs reduced from 58% to 22% (p<0.01) in t(14;16) and 39% (p=0.02) in t(14;20) with concomitant overexpression of ITGB7 in the t(14;16), 19.5-fold increase, and the t(14;20), 23.4-fold increase, subgroups. These 4 DMRs belong to CpG islands, annotated to be the part of possible intragenic enhancer site with promoter like activity, and enriched for open chromatin structures. The DMRs also align with the hotspot of H3K4me1 marks and a putative binding site of activating transcription factors such as AP1/2 or Sp1. This suggests that the identified DMRs could be within the enhancer that regulates ITGB7 overexpression through DNA-hypomethylation. ITGB7 is a known oncogenic factor in high-risk MM, contributing to cell adhesion, migration and homing. Here we show using combined DNA methylation and expression data that ITGB7 is regulated through hypomethylation of the an enhancer region in the MF subgroup in MM.

Citation Format: Samrat Roy Choudhury, Cody Ashby, Ruslana Tytarenko, Yan Wang, Purvi H. Patel, Aneta Mikulasova, Michael Bauer, Shayu Deshpande, Faith E. Davies, Gareth J. Morgan, Brian A. Walker. Intragenic DNA-hypomethylation promotes overexpression of ITGB7 in MF subgroup of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5324.