MLL1 Modulates IgM and Inflammatory Cytokines in Waldenstrom's Macroglobulinemia

Waldenström macroglobulinemia (WM) is a low-grade B-cell lymphoma associated with the accumulation of lymphoplasmacytic cells in the bone marrow (BM) and a high level of monoclonal IgM protein in the serum. Genome-wide studies revealed genomic alternations in WM, with the most prevalent being a point mutation in the myeloid differentiation primary response gene 88 (MyD88) resulting in an amino acid substitution (L265P) that is present in over 90% of WM patients. MyD88 is an effector of Toll-like receptor (TLR) signaling pathway that mediates inflammatory cytokine expression and secretion upon activation. Here, we found that activation of the TLR-MyD88 pathway in WM cells carrying the MyD88 L265P somatic mutation increases the expression of IL-6 and CCL2, two cytokines known to participate in the pathobiology of several neoplasms including B cell malignancies. Furthermore, IL-6 has been shown to modulate IgM secretion and malignant B cell growth in WM. We demonstrate that an active ERK1/2 pathway, which is elevated in WM patient samples, is required to maintain IL-6 and CCL2 expression in BCWM.1 (p=0.0339) and RPCI-WM1 (p=0.0005) cells. Further characterization identified a change in the chromatin landscape in response to TLR-MyD88 activation in WM cells. We found increased levels of trimethylation of histone 3 lysine 4 (H3K4me3) at the promoters of the inflammatory cytokines IL-6 and CCL2 by chromatin immunoprecipitation (ChIP) assay followed by qPCR (ChIP-qPCR). The H3K4me3 modification is catalyzed by six related homologs of the yeast histone methyltransferase (HMT) family. Further analysis identified the Mixed-lineage leukemia 1 (MLL1) as the enzyme bound to these promoters in response to TLR-MyD88 stimulation of WM cells. Analysis of WM cell lines and primary WM patient cells showed that MLL1 and its binding partner, menin, are expressed at significantly higher levels in CD19⁺CD138⁺cells from WM patients compared to CD19⁺B cells from peripheral blood of healthy donors (p<0.001). We also found an increase in H3K4me3 deposition on IL-6 and CCL2 promoters during early (1-3 hr) and late (12-24 hr) kinetics following TLR-MyD88 stimulation. This also coincided with increased deposition of MLL1 on these cytokine promoters by ChIP-qPCR. Disruption of menin-MLL1 using the MI-2 or MI-503 menin-MLL1 inhibitors significantly reduced IL-6 and CCL2 expression in WM cell lines (BCWM.1 p=0.039; RPCI-WM1 p=0.0041). This also results in significantly reduced IgM expression (p<0.0001) and secretion at inhibitor dose (5 μM) that has no effect on cell proliferation. Finally, MLL1 knockdown using RNAi significantly reduces IgM expression in BCWM.1 (p<0.0001), MWCL (p=0.0016) and RPCI-WM1 (p<0.0001) and IgM secretion in BCWM.1 (p<0.0001) and MWCL-1 (p<0.0001). Taken together, these results identify a novel role for menin-MLL1 in regulating inflammatory cytokines and IgM expression and secretion in WM and provide the rationale for targeting these molecules in WM patients.