Citations

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. Disclosures Ansell: Mayo Clinic Rochester: Employment; Mayo Clinic Rochester: Employment; Regeneron: Research Funding; Seattle Genetics: Research Funding; Trillium: Research Funding; Seattle Genetics: Research Funding; Regeneron: Research Funding; Trillium: Research Funding; Bristol-Myers Squibb: Research Funding; Mayo Clinic Rochester: Employment; Trillium: Research Funding; Affimed: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Trillium: Research Funding; Mayo Clinic Rochester: Employment; Bristol-Myers Squibb: Research Funding; Mayo Clinic Rochester: Employment; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; LAM Therapeutics: Research Funding; LAM Therapeutics: Research Funding.

Abstract Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.

Abstract Many phytochemicals possess antioxidant and cancer-preventive properties, some putatively through antioxidant response element–mediated phase II metabolism, entailing mutagen/oxidant quenching. In our recent studies, however, most candidate phytochemical agents were not potent in inducing phase II genes in normal human lung cells. In this study, we applied a messenger RNA (mRNA)–specific gene expression–based high throughput in vitro screening approach to discover new, potent plant-derived phase II inducing chemopreventive agents. Primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBECs) were exposed to 800 individual compounds in the MicroSource Natural Products Library. At a level achievable in humans by diet (1.0 μM), 2,3-dihydroxy-4-methoxy-4′-ethoxybenzophenone (DMEBP), triacetylresveratrol (TRES), ivermectin, sanguinarine sulfate, and daunorubicin induced reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 (NQO1) mRNA and protein expression in NHBE cells. DMEBP and TRES were the most attractive agents as coupling potency and low toxicity for induction of NQO1 (mRNA level, ≥3- to 10.8-fold that of control; protein level, ≥ two- to fourfold that of control). Induction of glutathione S-transferase pi mRNA expression was modest, and none was apparent for glutathione S-transferase pi protein expression. Measurements of reactive oxygen species and glutathione/oxidized glutathione ratio showed an antioxidant effect for DMEBP, but no definite effect was found for TRES in NHBE cells. Exposure of NHBE cells to H2O2 induced nuclear translocation of nuclear factor erythroid 2–related factor 2, but this translocation was not significantly inhibited by TRES and DMEBP. These studies show that potency and low toxicity may align for two potential NQO1-inducing agents, DMEBP and TRES.