Endoplasmic Reticulum Stress Is a Target for Therapy in Waldenstrom's Macroglobulinemia (WM).

Abstract

Background: WM is an incurable low-grade lymphoplasmacytic lymphoma with limited options of therapy. Proteasome inhibition has been shown to induce components of the proapoptotic/terminal unfolded protein response (UPR), a signaling pathway activated by accumulation of misfolded proteins within the endoplasmic reticulum (ER). We previously found that UPR gene expression is related to disease activity in WM with a particular role for GRP78/Bip as a prognostic factor. We therefore examined tunicamycin (Sigma, St Louis, MO), a potent inducer of ER stress, for potential anti-tumor effects in WM.Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEC1, RL) and primary CD19+ selected LPC cells from WM patients were incubated with tunicamycin (0.01–10 uM) for 24–72 hours and evaluated by MTT, thymidine uptake, and Apo2.7/PI staining for effects on proliferation and survival. Since bone marrow stromal cells (BMSC) confer growth and resistance to conventional treatments, we also tested the effect of tunicamycin on WM cells co-cultured with BMSC. Immunoblotting for caspases was also performed and expression of UPR genes determined using relative quantitative RT-PCR reaction following (0.5–16 hrs) culture with tunicamycin.Results: WM cells inherently expressed the ER chaperones GRP78/Bip and GRP94/gp96. Tunicamycin rapidly induced components of the proapoptotic/terminal UPR, including PERK, the ER stress-specific eIF-2alpha kinase; ATF6, an ER stress-induced transcription factor; and its proapoptotic target, CHOP/GADD153. Tunicamycin also induced significant cytotoxicity, and inhibited DNA synthesis with an IC50 of 0.5–1 ug/mL in all cell lines, as well as primary LPC from 3/3 WM patients. Furthermore, tunicamycin induced apoptosis in WM cells, with an increase in the sub-G1 population notable at 12 hrs. Tunicamycin induced apoptosis was preceded by caspase-12 cleavage, followed then by caspase-8, -9 and PARP cleavage. Importantly, co-culture of WM cells with the survival factors IL-6, IGF-1 as well as BMSC did not inhibit tunicamycin induced cytotoxicity. Lastly, tunicamycin did not induce cytotoxicity in healthy donor peripheral blood mononuclear or hematopoietic stem cells.Conclusion: These pre-clinical studies provide a framework for further evaluation of ER stress inducing agents as therapeutic agents in WM.