Dysregulation of MYD88 Regulatory Isoforms in Waldenstrom's Macroglobulinemia

Mutations in MYD88 are found in 95% Waldenstrom's Macroglobulinemia (WM) patients, nearly all of which correspond to the c.978T>C mutation resulting in an p.Leu265Pro substitution at the protein level. MYD88 is an adaptor protein for the Toll like receptors and IL1R signaling. Activation of these receptors triggers MYD88 homodimerization and downstream signaling to NF-kB through IRAK1/4 and BTK. Chronic MYD88 activation is associated with the increased transcription of the shorter MYD88 isoforms that lack the ability to interact with IRAK4 and serve as dominate negative regulators of MYD88 signaling (Ruland, Nature Immunol 2011). The c.978T>C mutation that is characteristic of WM corresponds to a stop loss in these regulatory isoforms. A novel mutation was recently observed in one of our patients during routine screening for MYD88 mutations that is silent in the primary transcript that encodes for p.Leu265Pro but corresponds to p.V199E in the regulatory isoforms. This isoform specific damaging mutation underscores the importance of isoform regulation in MYD88 in WM. To further study possible dysregulation within the isoforms of MYD88, we collected next generation RNA sequencing data from 55 MYD88 mutant and 22 MYD88 wild type (WT) WM patients. For comparison we also sequenced RNA from 16 healthy donor (HD) plasma cells, 9 HD CD19+CD27- peripheral blood B-cells and 9 HD CD19+CD27+ memory B-cells. Isoform transcripts per million (TpM) were calculated using Salmon (https://combine-lab.github.io/salmon/). Total MYD88 expression in TpM was used to calculate the percentage of total transcription devoted to each MYD88 isoform per patient (Figure 1). Clear differences based on MYD88 mutation status were observed even though no significant differences in MYD88 overall gene expression were observed between WM and HD B-cells. In spite of the constitutive signaling resulting for mutant MYD88, the short regulatory isoforms (ENST00000495303; ENST00000443433) were not well expressed. Both regulatory isoforms didn't present any statistically significant differences between groups, but in the first of the two regulatory isoforms (ENST00000495303) expression was lower in MYD88 WT WM patients (p=0.01). Our findings therefore provide evidence of isoform dysregulation in MYD88 WT MYD88 Mutated WM, and affirm that the somatic MYD88 mutations p.Leu265Pro and p.V199E play an important role in MYD88 isoform dysregulation in WM.

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