The ERK1/2 Regulator WNK2 Shows Differential Expression and Isoform Usage, Primarily Driven By Aberrant Methylation, in MYD88 Mutated Waldenström's Macroglobulinemia
Authors: Guerrera Maria Luisa , Yang Guang , Liu Xia , Tsakmaklis Nickolas , Demos Maria G , Kofides Amanda , Munshi Manit , Xu Lian , Patterson Christopher J , Castillo Jorge J. , Sarosiek Shayna , Flynn Catherine A , Meid Kirsten , Gustine Joshua , Sewastianik Tomasz , Carrasco Ruben D. , Treon Steven P , Hunter Zachary R 
Published: November 2021

Abstract
MYD88 mutations (MYD88 MUT) are present in 95-97% of patients with Waldenström's Macroglobulinemia (WM). Transgenic mouse studies show that MYD88 MUT alone is insufficient to drive oncogenesis. We previously reported the occurrence of non-overlapping somatic mutations in either CXCR4 (CXCR4 MUT) or deletions in 6q (del6q) in MYD88 mutated (MYD88 MUT) WM patients, suggesting either event may serve as a secondary driver of WM oncogenesis. Intersecting the lists of genes differentially modulated by either CXCR4 MUT or del6q revealed WNK2 as a top hit (Guerrera ML et al, Haematologica 2018). WNK2 is a serine/threonine protein kinase that negatively regulates ERK1/2 activation and has known tumor suppressor function in several solid tumors, wherein its expression is primarily silenced by aberrant promoter methylation. ERK1/2 pathway plays an important pro-survival role, its activation is accompanied by the release of inflammatory cytokines and can mediate ibrutinib resistance in WM (Chen et al, Blood 2018). We therefore sought to investigate the expression of WNK2, and clarify the mechanisms underlying its aberrant expression by genomic subtypes in WM.
To study the regulation of WNK2 gene expression, we analyzed our previously published RNA-Seq data (Hunter ZH et al, Blood 2016) and validated findings in 47 WM patients by RT-qPCR. Compared to healthy donor (HD) B and plasma cells (PC), we observed marked upregulation of WNK2 expression in bone marrow (BM) CD19-selected tumor cells from MYD88 MUTCXCR4 WT patients (p&lt;0.0001). Conversely, WNK2 expression was silenced in tumor cells from MYD88 MUTCXCR4 MUT and MYD88 WTCXCR4 WT (p&lt;0.001 vs HD PC) patients, with less pronounced suppression of WNK2 also observed in MYD88 MUTdel6q cases. Immunohistochemistry analysis on patient BM biopsies confirmed the findings observed at the mRNA level per patient genotype.
To assess the impact of alternative splice variants on overall gene expression, we then studied the relative composition of the 16 documented isoforms (Figure 1). RNA-Seq showed a prevalence of protein-coding transcripts, particularly those coding for isoforms missing the ERK regulatory kinase domain, in MYD88 MUT vs MYD88 WT WM. By RT-PCR, we validated the preferential use of the protein-coding WNK2-208, WNK2-203 and WNK2-205 transcripts in 32 of 47 WM samples with detectable total WNK2 levels. Of those, WNK2-208 and WNK2-203 lack the region coding for the functional kinase domain. Analysis of known post-translational modification (PTM) sites annotated for the canonical isoform WNK2-201 in the 3 isoforms of interest demonstrated that C-terminal PTM sites are highly conserved across all isoforms even in the absence of the kinase domain, alluding to novel protein regulatory sites yet to be characterized.
Analysis of CpG methylation using enhanced reduced representation bisulfite sequencing from 62 WM patients identified 3 clusters of differentially methylated CpG islands (CpGs) between MYD88 MUTCXCR4 WT and MYD88 MUTCXCR4 MUT patients. Three CpGs in the WNK2 promoter, four in intron 1 and one in intron 11 constituted clusters 1, 2 and 3, respectively (Figure 1). Compared to healthy donor memory B-cell and PC, we observed aberrant hypermethylation in cluster 1 in MYD88 MUTCXCR4 MUT and hypomethylation at a putative enhancer in cluster 2 in MYD88 MUTCXCR4 WT patients, while del6q cases showed a mixed methylation pattern. A higher degree of methylation, particularly in cluster 2, significantly correlated with reduction in total and transcript-specific WNK2 mRNA levels that affected expression of transcripts encoding for kinase domain-containing isoforms. Furthermore, we validated the differential methylation status of 3 of 4 methylated CpGs in cluster 2 in 10 patients, including MYD88 MUTCXCR4 WT, MYD88 MUTCXCR4 MUT and MYD88 MUTdel6q, and 3 MYD88 MUT(BCWM.1, TMD-8) and MYD88 WT (Ramos) cell lines by means of bisulfite PCR followed by Sanger sequencing. Taken together, our findings reveal that the ERK1/2 regulator WNK2 shows differential expression and isoform usage in distinct genomic subsets of MYD88 MUT WM; is primarily driven by aberrant methylation; and may serve as a key driver of disease progression in WM.
Figure 1 Figure 1.

Disclosures
Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.