Comprehensive Integration of Whole Genome, Transcriptome and Methylation Profiling Reveals Novel Gene Dysregulation Including IL15, SOCS6 and CARD11 Associated with MYD88 and CXCR4 Genotype Status in WM

Background: Waldenstrom's Macroglobulinemia/IgM lyphoplasmacytic lymphoma is a B-cell lymphoma defined by highly recurring mutations in MYD88 (95-97%) and CXCR4 (30-40%) by whole genome, PCR and Sanger sequencing (Treon et al NEJM 2012, Hunter et al Blood 2013, Xu et al, BJH 2016). Using next generation RNA sequencing studies (RNASeq) of these same group of patients, we subsequently observed that MYD88 and CXCR4 mutation status were the primary determinants of differential gene expression in WM (Hunter et al, Blood 2016). We have now integrated enhanced reduced representation bisulfite sequencing (ERRBS) data into the existing WGS and RNASeq data to present of more complete picture of genomic regulation in WM.

Methods: CD19+ selected bone marrow samples from 54 patients with WM, and CD19- peripheral blood mononuclear samples were used for tumor and germline controls, respectively. WGS and RNASeq were conducted as previously described (Hunter et al, Blood 2014; 2016) and methylation profiling was conducted using ERRBS at the Weil Cornell Medical Epigenomics Core and analyzed using Bismark. Results were filtered for methylation sites supported by at least 10 reads across all of the samples. Differential methylation analysis was conducted at the individual site level and aggregated promoter level using an established edgeR protocol. Differential methylation data was then compared with Salmon derived gene and isoform expression data which was available for 49/54 (91%) of the samples.

Results: Clinical characteristics of patients were as follows: Median age 60 (range 40-87 years); BM involvement 43% (range 4-95%); serum IgM 3590 (range 416-8320 ug/dl). Most patients (67%) were untreated. WGS data revealed 33 (61%), 18 (33%), and 3 (6%) were MYD88^Mutant/CXCR4^Wild-Type, MYD88^Mutant/CXCR4^Mutant and MYD88^Wild-Type/CXCR4^Wild-Type, respectively. As was observed in our RNASeq analysis, pairwise multidimensional scaling of the methylation status of the top 2,000 high variance promoters revealed segregation of the MYD88^Mutant/CXCR4^Wild-Type from both MYD88^Mutant/CXCR4^Mutant and MYD88^Wild-Type/CXCR4^Wild-Type (Figure 1A). Log fold change in methylation in differentially methylated promoters from MYD88^Mutant/CXCR4^Mutant and MYD88^Wild-Type/CXCR4^Wild-Type samples relative to MYD88^Mutant/CXCR4^Wild-Type were negatively correlated with log-fold change in gene expression (Rho = -0.463, p <0.0001; Figure 1B). Promoter level analysis revealed 556 differentially methylated promoters in MYD88^Mutant/CXCR4^Mutant of which 440 (78%) had increased levels of methylation relative to MYD88^Mutant/CXCR4^Wild-Type samples. Affected genes included IL15, GNAO1, HIF1A, SOCS6, PIK3R5, IRF8 and CD38. For MYD88^Wild-Type/CXCR4^Wild-Type samples, 126 promoters were significantly differentially methylated with only 28 (22%) demonstrating increased methylation relative to MYD88^Mutant/CXCR4^Wild-Type samples. Affected promoters included NRIP1, FNBP1L, and PTK2. Single site analysis revealed 127 and 1 significantly differentially methylated CpG loci for MYD88^Mutant/CXCR4^Mutant and MYD88^Wild-Type/CXCR4^Wild-Type, respectively. This analysis revealed a large intronic CpG island in CARD11 that was demethylated only in MYD88^Mutant/CXCR4^Wild-Type patients and a methylated site in the PPP1CC promoter found in MYD88^Wild-Type/CXCR4^Wild-Type.

Conclusions: The studies provide the first comprehensive insights into epigenomic regulation of WM and show that MYD88 and CXCR4 mutation status drives methylation and confers a distinct transcriptomic profile that includes genes such as IL15, SOCS6, CARD11, HIF1A, and PIK3R5 with important pro-survival and immune regulatory roles.

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