The ERK1/2 Regulator WNK2 Shows Novel Alternative Splicing Aberrations That Support Tumor Growth in MYD88 Mutated Waldenström's Macroglobulinemia

Background: In previous studies, we and others showed that Waldenström's Macroglobulinemia (WM) patients carry MYD88 (MYD88^MUT) and CXCR4 (CXCR4^MUT) in 95-97% and 30-40% of cases, respectively. However, in transgenic mouse studies by us and others, MYD88^MUT alone was insufficient to drive oncogenesis. Our previous transcriptome analysis showed that WNK2 was one of the top dysregulated genes in MYD88^MUT WM, occurring in either CXCR4 mutated or chromosome 6q deleted patients (Guerrera ML et al, Haematologica 2018). WNK2 is a serine/threonine protein kinase that negatively regulates ERK1/2 activation; has known tumor suppressor function and is silenced by aberrant epigenomic regulation in certain solid cancers. ERK1/2 is a critical pro-survival molecule triggered by MYD88^MUT and CXCR4^MUT signaling whose activation is accompanied by the release of inflammatory cytokines and can mediate ibrutinib resistance in WM (Chen et al, Blood 2018).

Methods: We investigated by combined transcriptome, PacBio IsoSeq, methylome and ATAC-sequencing analysis WNK2 regulation in WM patients subtyped by MYD88 and CXCR4 mutation status. CRISPR activation (CRISPRa) dCas9-VPR system was used to overexpress and characterize WNK2 isoform expression and function in MYD88^MUT BCWM.1 WM and TMD8 ABC-DLBCL cells.

Results: WNK2 was markedly overexpressed MYD88^MUTCXCR4^WT versus MYD88^MUTCXCR4^MUT patients (N=47; p<0.001). Methylome studies showed that promoter CpG hypermethylation of WNK2 in malignant WM cells from MYD88^MUTCXCR4^MUT patients, and hypomethylation at a putative enhancer in MYD88^MUTCXCR4^WT patients were significantly correlated with WNK2 expression. We identified that the Ensembl WNK2-207 isoform which is characterized by two spliced out exons and lacks the regulatory kinase domain was over-expressed in MYD88^MUTCXCR4^WT WM patients. ATAC-seq data from 44 untreated WM and 9 healthy controls showed a differentially open chromatin site in MYD88^MUTCXCR4^WT versus MYD88^MUTCXCR4^MUT patients which encompassed an enhancer upstream of the WNK2-207 transcriptional start site (TSS). Moreover, this observed open chromatin site positively correlated with increased WNK2-207 expression in MYD88^MUTCXCR4^WT patients. Analysis of PacBio IsoSeq data from 12 WM patients showed that all WNK2 isoforms shared the same exon splicing events leading to loss of the same two exons identified in WNK2-207. The regulatory function for these spliced out exons is unknown. RNA-Seq analysis of 253 WM patents confirmed these IsoSeq predictions (Figure 1).

We next sought to functionally characterize WNK2 expression in MYD88 mutated lymphoma cells. We used a CRISPR activation (CRISPRa) dCas9-VPR system and overexpressed WNK2 in MYD88 mutated BCWM.1 WM and TMD8 ABC-DLBCL cells. We targeted the canonical and the annotated TSS for Ensembl WNK2-207 as well as WNK-203, the second most abundant transcript in WM, and transduced the stably dCas9 expressing cells with at least 5 single guide RNAs (sgRNA) per TSS. We obtained sustained upregulation of the target at the expression and protein levels in the cells activating the canonical and WNK-207 specific TSS. Notably, the activation of the canonical TSS induced the expression of WNK2 long and short isoforms. Overexpression of the different WNK2 isoforms consistently reduced phosphorylation of ERK1/2 (Figure 2). CRISPRa cells stably expressing WNK2-207 in both BCWM.1and TMD8 also showed greater viability vs. vector only expressing cells.

Conclusions: Taken together, our findings identify aberrant WNK2 transcription mediated by methylation and chromatin access in MYD88^MUT WM patients that differentially aligns with CXCR4 mutation status. Moreover, we identified an exon splicing event unique to WNK2-207 leading to loss of expression for two exons that was aberrantly shared by all other WNK2 transcripts. Expression of WNK2-207 bearing the spliced-out exons domains provided a growth advantage to MYD88^MUT lymphoma cells. The findings provide a critical framework for understanding epigenomic regulation in WM, and identify a role for aberrantly expressed WNK2 in the oncogenesis of MYD88^MUT WM.

Yang:Blueprint Medicines: Current Employment. Castillo:Cellectar: Consultancy; AstraZeneca: Research Funding; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy. Sarosiek:ADC Therapeutics: Research Funding; BeiGene: Consultancy. Branagan:Adaptive Biotechnologies: Consultancy; Genzyme: Consultancy; Janssen/Pharmacyclics: Consultancy; CSL Behring: Consultancy; BeiGene: Consultancy; Karyopharm Therapeutics: Consultancy. Tedeschi:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Beigene: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau. Cairoli:Gilead Sciences: Other: Travel, Accommodations; Novartis, Celgene: Speakers Bureau; Celgene, AbbVie, Daiichi Sankyo, Novartis: Consultancy. Sewastianik:Affini-T Therapeutics: Current Employment. Anderson:Raqia: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder ; Starton: Membership on an entity's Board of Directors or advisory committees; Window: Membership on an entity's Board of Directors or advisory committees; Precision Biosciences: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; NextRNA: Other: Scientific founder ; Dynamic Cell Therapy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi:Adaptive Biotechnology: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Takeda Oncology: Consultancy; Amgen: Consultancy; GSK: Consultancy; Karyopharm: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Legend: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy. Treon:BeiGene: Consultancy; BMS: Research Funding; X4: Consultancy, Research Funding; Janssen/Pharmacyclics: Consultancy, Research Funding; Abbvie: Consultancy.