Abstract 876: CD40-induced growth inhibition of Burkitt lymphoma: A possible role for NADPH oxidase by upregulation of p67phox
Authors: Dahan Mazal Iris , Antony Smitha , Juhasz Agnes , Jiang Guojian , Konate Mariam , Lu Jiamo , Meitzler Jennifer , Wu YongZhong , Roy Krishnendu , Doroshow James H. 
Published: August 2019

Abstract
CD40 stimulation together with IL4 has been shown to promote growth of normal B cells in vitro. In contrast to this, CD40 stimulation in several B cell lymphoma cell lines has been demonstrated to be growth inhibitory. Though the mechanism is not completely known, CD40-stimulated cells exhibit increased levels of reactive oxygen species (ROS). In B lymphocytes, a major source of ROS production is the NADPH oxidase (NOX) that is comprised of a membrane-bound Nox2 catalytic subunit and four cytosolic regulatory components, with the p67phox cytosolic protein being the activator factor. Upon stimulation, by either cross linking of the B-cell receptor or following treatment with PMA, the active NADPH oxidase generates superoxide. We have investigated the role of NADPH oxidase in CD40-mediated growth inhibition in Burkitt lymphoma (BL) cells. For this study, we first evaluated the effect of CD40 stimulation on the expression of Nox2 and p67phox in BL cells that demonstrate different levels of endogenous Nox2 and p67phox. Accordingly, Daudi and Raji cell lines, with endogenously high Nox2 and low p67phox, NAMALWA cells which have high Nox2 and no p67phox expression, and Ramos cells that contain no Nox2 or p67phox were analyzed. Treatment with anti-CD40 or CD40 ligand (CD40L) for 24 h induced up-regulation of p67phox in Daudi, Ramos, and NAMALWA cells, but not in the Raji line. No significant change in the expression of Nox2 was observed. Additionally, Daudi, Ramos and NAMALWA cells showed higher production of ROS after CD40 stimulation and activation with anti-IgM or PMA, in a chemiluminescence assay. To determine whether the change in ROS level was related to the upregulation of p67phox, Daudi cells that express either stable p67phox shRNA or control shRNA were generated. Treatment with CD40L for 24 h demonstrated significant ROS production only in control shRNA and not in the p67shRNA cells, confirming that the ROS production is NOX2-p67phox mediated. This enhanced ROS production was observed following treatment with CD40L alone and was enhanced further in conjunction with anti-IgM or PMA. However, cells with the p67phox shRNA showed detectable level of ROS only after activation with anti IgM, albeit to much lower levels than the control. Co-treatment of Daudi cells with anti-CD40 or CD40L and IL4 further increased the expression of p67phox. Although the basal level of ROS in the cells was unchanged upon co-treatment, the ROS generated following stimulation with anti-IgM or PMA was significantly enhanced. Preliminary MTT studies confirmed the growth inhibition of CD40-stimulated Daudi cells but not of Raji cells, suggesting a role for p67phox-dependent, ROS-related inhibition of lymphoma cell proliferation. Experiments are currently ongoing to delineate further the role of CD40 stimulation and Nox2-p67phox mediated ROS in affecting the growth of Burkitt lymphomas and signaling pathways downstream of CD40.
Citation Format: Mazal Iris Dahan, Smitha Antony, Agnes Juhasz, Guojian Jiang, Mariam Konate, Jiamo Lu, Jennifer Meitzler, YongZhong Wu, Krishnendu Roy, James H. Doroshow. CD40-induced growth inhibition of Burkitt lymphoma: A possible role for NADPH oxidase by upregulation of p67phox [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 876.