Search

Angiogenesis has been postulated to be critical for the pathogenesis of multiple myeloma, a neoplastic disease characterized by abnormal proliferation of malignant plasma cells in the bone marrow (BM). Cleavage of the N- and C-terminal regions of circulating chromogranin A (CgA, CHGA), classically an antiangiogenic protein, can activate latent antiangiogenic and proangiogenic sites, respectively. In this study, we investigated the distribution of CgA-derived polypeptides in multiple myeloma patients and the subsequent implications for disease progression. We show that the ratio of pro/antiangiogenic forms of CgA is altered in multiple myeloma patients compared with healthy subjects and that this ratio is higher in BM plasma compared with peripheral plasma, suggesting enhanced local cleavage of the CgA C-terminal region. Enhanced cleavage correlated with increased VEGF and FGF2 BM plasma levels and BM microvascular density. Using the Vk*MYC mouse model of multiple myeloma, we further demonstrate that exogenously administered CgA was cleaved in favor of the proangiogenic form and was associated with increased microvessel density. Mechanistic studies revealed that multiple myeloma and proliferating endothelial cells can promote CgA C-terminal cleavage by activating the plasminogen activator/plasmin system. Moreover, cleaved and full-length forms could also counter balance the pro/antiangiogenic activity of each other in in vitro angiogenesis assays. These findings suggest that the CgA-angiogenic switch is activated in the BM of multiple myeloma patients and prompt further investigation of this CgA imbalance as a prognostic or therapeutic target. Cancer Res; 76(7); 1781–91. ©2016 AACR.

Amino-biphosphonates (like pamidronate) activate human Vγ9/Vδ2 T lymphocytes and promote their cytotoxicity against multiple myeloma cells. T-cell receptor (TCR)–mediated effector functions of γδ cells are enhanced upon triggering of the activating receptor NKG2D by MICA, a stress-inducible antigen expressed by epithelial and some hematopoietic tumors, including multiple myeloma. Here we show that MICA was expressed not only by myeloma cell lines and by 6 of 10 primary multiple myeloma cells from patients but also by bone marrow plasma cells from all (six of six) patients with preneoplastic gammopathy (monoclonal gammopathy of undetermined significance, MGUS), a direct precursor of multiple myeloma. Moreover, compared with multiple myeloma plasma cells, MICA was expressed by MGUS plasma cells at significantly (P < 0.05) higher levels. MICA expressed by myeloma cell lines contributed to killing and IFN-γ production by Vγ9/Vδ2 cells only upon pamidronate treatment, suggesting a dual interaction between Vγ9/Vδ2 lymphocytes and multiple myeloma plasma cells involving both TCR triggering and NKG2D-mediated signals. Finally, MICA enhanced killing of freshly derived, pamidronate-treated multiple myeloma cells from patients by γδ cells, as indicated by the significantly (P < 0.05) higher γδ cytotoxicity against MICA-positive rather than MICA-negative multiple myeloma cells. Our results indicate that MICA expressed by monoclonal plasma cells is functional and correlates with disease stages, suggesting a role for the molecule in the immune surveillance against multiple myeloma. Moreover, pamidronate-activated Vγ9/Vδ2 lymphocytes can be exploited in the immune therapy of early stages multiple myeloma and possibly of premalignant disease.

B-cell receptor (BCR) signaling is a central pathogenetic mechanism in CLL. Most CLL cells express both IgM and IgD receptors, and increased responsiveness to IgM stimulation correlates with poor disease outcome and with expression of IGHV-unmutated immunoglobulin receptors (U-CLL). In contrast, the relevance of IgD signaling remains poorly understood. The aim of the present study is to characterize functional differences between IgM and IgD signaling in CLL in terms of protection from in vitro apoptosis, BCR signaling activation and CCL3/4 chemokine secretion and to explore the mechanism through which CCL3/4 are regulated, with a focus on their transcriptional repressor BCL6. CLL cells were stimulated with anti-IgM and anti-IgD; 48-hour cell viability and BCR expression were analyzed by flow cytometry; phosphorylation of the BCR-signaling related molecules HS1 and ERK after 2, 5, 15, 30, 60 minutes of stimulation were analyzed by Western Blot and flow cytometry; BCL6 protein expression by Western Blot and CCL3/4 secretion by ELISA were tested after 3, 6, 9, 12, 24 hours of stimulation. Anti-IgM stimulation protected CLL cells from in vitro apoptosis to a greater extent than anti-IgD and U-CLL were more responsive to anti-IgM as compared to cells carrying IGHV-mutated receptors (M-CLL) (p = 0.0022; n = 13 M-CLL vs. n = 11 U-CLL). In line with this finding, U-CLL expressed higher levels of surface IgM, as compared to M-CLL (p In conclusion, IgM stimulation induces a long-lived signaling response, increased CLL-cell survival and CCL3/4 secretion, in particular in U-CLL; in contrast, IgD signaling is short-lived and has minimal effects on protection from apoptosis and chemokine secretion. BCL6 protein downregulation induced by anti-IgM parallels CCL3/4 production, suggesting a functional link between BCL6 expression and CCL3/4. Taken together, these evidences suggest that the differential functional outcomes of IgM and IgD signaling might be related to the duration of the signaling response and support the relevance of IgM signaling in CLL pathogenesis. Citation Format: Elisa ten Hacken, Cristina Scielzo, Susan O'Brien, William G. Wierda, Michael J. Keating, Paolo Ghia, Federico Caligaris-Cappio, Jan A. Burger. Duration of signaling through IgM and IgD receptors differentially influences cell survival and BCL6-regulated CCL3/4 chemokine production in Chronic Lymphocytic Leukemia (CLL). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1023. doi:10.1158/1538-7445.AM2015-1023