Multi-Omics DNA Methylation Analysis of MYD88 Wild-Type Waldenstrom Macroglobulinemia Reveals Potential Epigenetic Influence on Oncogenic Pathways

Introduction MYD88 L265P mutations (MUT) are found in 90% of patients with Waldenstrom macroglobulinemia (WM) and lead to the activation of pro-survival signaling. Limited literature has demonstrated that patients with MYD88 wild-type (WT) WM have poorer responses to conventional therapies and a higher likelihood of histological transformation. Increasingly the role of non-coding genomics in lymphomagenesis has demonstrated importance and currently, the epigenetic landscape of MYD88 WT WM remains underexplored. This study utilized a multi-omics approach to assess DNA methylation in MYD88 WT WM compared to MYD88 MUT patients and controls. Methods Bone marrow samples were prospectively collected from 20 subjects [3 MYD88 WT/CXCR4 WT WM, 12 MYD88 MUT WM and 5 normal controls] and positive selection for CD19+ and/or CD138+ B cells was performed. The analysis focused on the comparison between MYD88 WT WM to normal controls and MYD88-WT to MUT WM. Genome-wide DNA methylation analysis was performed, and CpG methylation ratios were segmented into 200-bp regions. Differentially methylated regions (DMRs) were determined using a Q-value <0.05 and absolute methylation difference ≥10%. Next, RNA-seq data was interrogated and differentially expressed genes (log2fold change>0.5 or <-0.5, false discovery rate<0.05) with corresponding promoter hypo- or hypermethylation were assessed. Ingenuity Pathway Analysis was used to determine potentially epigenetically regulated pathways using a corrected p<0.05. Results Comparing MYD88 WT WM samples to controls, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (29129 vs 6300), 3'untranslated region (UTR) (2068 vs 1551), 5'UTR (10169 vs 735), intron (30344 vs 24511), exon (2224 vs 6855), CpG island (30372 vs 1240) and CpG shore (14477 vs 5872) (all p<0.001). Next, mRNA analysis demonstrated that 214 genes were differentially up-/downregulated with concordant promoter hypo-/hypermethylation. On the assessment of promoter-methylation-based pathways, multiple pathways involved in cell signaling, proliferation, gene regulation, cytokine and immune signaling and metabolism were found to be deregulated. Most pathways were found to be negatively enriched in MYD88 WT patients compared to controls including several members of interleukin (IL) signaling (IL-2, IL-3, IL,7) and regulators of cell proliferation and signaling including mTOR, PI3K/AKT, and p53. Positive enrichment of the PTEN, FGFR3 and ERK/MAPK pathways were observed. Underlying multiple pathways was promoter hypermethylation with corresponding mRNA downregulation of AKT3 and PIK3CA. Furthermore, promoter hypomethylation with transcript upregulation of PTEN was observed. Next, in comparison of MYD88 WT to MUT samples, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (24829 vs 2544), 3'UTR (3534 vs 378), 5'UTR (6111 vs 605), intron (52292 vs 5360), exon (21066 vs 2187), CpG island (18334 vs 1551) and CpG shore (16351 vs 1957) (all p<0.001). Assessing mRNA expression, 354 genes were up-/downregulated with concordant promoter hypo-/hypermethylation. Pathway analysis revealed positive enrichment of the mitotic G1 phase to G1/S transition pathway with upregulation of the CDK4, LYN, and PSMA6 transcripts in MYD88 WT compared to MUT samples. Further, negative enrichment of cell signaling regulators including the RHO GTPase family, IL pathways (IL-4, IL-8, IL-13, IL-17) and integrin signaling were found in MYD88 WT compared to MUT samples. Underlying multiple pathways were downregulation with promoter hypermethylation of BCL2, an important regulator of apoptosis. Conclusion Genome-wide methylation analysis of MYD88 WT WM revealed a unique DNA methylation profile, with potential epigenetic alteration of key oncogenic transcripts and pathways. The negative enrichment of cell signaling and cytokine/inflammatory pathways in MYD88 WT samples compared to controls and MYD88 MUT WM suggests an epigenetic mechanism for immune evasion and potentially underlies the reduced response to conventional WM-directed therapies. Overall, this study reveals that MYD88 WT WM is epigenetically distinct from MYD88 MUT WM and regulators such as methylation may underlie the unique clinical and phenotypic properties observed.