Abstract
Abstract Background: Anti-CD20 monoclonal antibodies (mAbs) are an essential component of the treatment of patients with CD20-positive non-Hodgkin's lymphoma and chronic lymphocytic leukemia (CLL). Anti-CD20 mAbs mediate their antitumor effects by activating the immune system or by direct apoptotic signaling in target cells. In a previous preclinical study, we have shown that treatment of B-lymphoma cell lines with anti-CD20 mAbs, rituximab and obinutuzumab, resulted in upregulated expression of the transcription factor early growth factor -1 (EGR-1) (Dalle et al. 2011). However, the role of EGR-1 in response to passive immunotherapies has not been explored so far. Furthermore, EGR-1 has been described as a calcium (Ca2+) regulated transcription factor and CD20 is hypothesized to regulate transmembrane Ca2+ flux. In this study we aim to assess the role of EGR-1 and Ca2+ flux in the cytotoxic activity of anti-CD20 mAbs. Methods: EGR-1 modulation and cell death induction by anti-CD20 mAbs rituximab and obinutuzumab were investigated in cells expressing endogenous and exogenous CD20. The cytotoxic effect of anti-CD20 mAbs was evaluated in SCID mice and in B-lymphoma cell lines overexpressing EGR-1 or knocked down for EGR-1. The impact of anti-CD20 mAbs on Ca2+ flux was investigated by flow cytometry using Indo-1 AM stained cells. Ca2+ channel blocker agent nifedipine was used to investigate the role of Ca2+ flux on obinutuzumab efficacy. Results: EGR1 expression is rapidly upregulated in CD20+ cells following rituximab and obinutuzumab exposure. Decreasing EGR-1 expression by shRNA abolished the direct cytotoxic effect of obinutuzumab both in vitro and in vivo, indicating that EGR-1 is required for CD20-mediated apoptosis. Additionally, the overexpression of EGR-1 resulted in enhanced cytotoxic activity of obinutuzumab both in vitro and in vivo. Rescuing EGR-1 expression in EGR-1 knocked-down cells restored sensitivity to obinutuzumab. Moreover, our results indicate that both rituximab and obinutuzumab could induce calcium influx in the presence of suboptimal concentrations of ionomycin. Blocking Ca2+ flux with the calcium channel blocker nifedipine or the Ca2+ chelating agent EGTA abolished EGR-1 induction by anti-CD20 mAbs. In vivo, nifedipine treatment interfered with obinutuzumab antitumor activity against established Granta (a human mantle cell lymphoma line) xenografts in SCID mice. Conclusion: EGR-1 plays a major role in the direct cytotoxic activity of anti-CD20 monoclonal antibodies and should be evaluated as a new biomarker to predict response to anti-CD20 treatment. Our data also show that calcium channel blockers interfere with the antitumor activity of obinutuzumab in preclinical models. Citation Format: Ivana Spasevska, Jade Villé, Kamel Chettab, Eva-Laure Matera, Charles Dumontet. Induction of apoptosis by anti-CD20 antibodies requires the induction of EGR-1 and calcium influx [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4594. doi:10.1158/1538-7445.AM2017-4594
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