Abstract 1342: RIP3 is downregulated in human myeloid leukemia cells and modulates apoptosis and caspase-mediated p65/RelA cleavage
Authors: Nugues Anne-Lucie , Bouafia Hassiba , Hetuin Dominique , Berthon Celine , Loyens Anne , Bertrand Elisabeth , Jouy Nathalie , Idziorek Thierry , Quesnel Bruno 
Published: May 2015

Abstract
Introduction. Impairment in cell death pathways represents a general characteristic of most cancer cells. The receptor-interacting protein kinase 3 (RIP3) associates with RIP1 in a necrosome complex that can induce necroptosis, apoptosis, or cell proliferation. The role of RIP3 in necroptosis and inflammation has been extensively studied, but its role in cancer remains poorly understood Methods. We analyzed the expression of RIP1 and RIP3 in CD34+ leukemia cells from a cohort of patients with acute myeloid leukemia (AML) and CD34+ cells from healthy donors. To analyze the potential advantages for myeloid malignant cells due to reduced RIP3 expression, we induced the expression of RIP3 in the DA1-3b mouse leukemia cell line. Results. RIP3 expression was significantly reduced in most AML samples, whereas the expression of RIP1 did not differ significantly. When re-expressed in the mouse DA1-3b leukemia cell line, RIP3 induced apoptosis, and necroptosis in the presence of caspase inhibitors. Surprisingly, the re-expression of a RIP3 mutant with an inactive kinase domain (RIP3-KD) induced significantly more and earlier apoptosis than wild-type RIP3 (RIP3 WT), indicating that the RIP3 kinase domain is an essential regulator of apoptosis/necroptosis in leukemia cells. The induced in vivo expression of RIP3-KD, but not RIP3 WT prolonged the survival of mice injected with leukemia cells. RIP3-KD-induced cell death but not RIP3 WT was significantly antagonized by an IKKβSSEE constitutively active mutant, showing that RIP3-KD-induced apoptosis, but not RIP3 WT-induced apoptosis, was dependent on NF-κB activity. The expression of RIP3-KD induced p65/RelA NF-κB subunit caspase-dependent cleavage, and a non-cleavable p65/RelA D361E mutant rescued cells from apoptosis. The protective effect of the p65/RelA D361E mutant against apoptosis was specific to RIP3-KD-induced cell death because no change in cell death was observed when apoptosis was instead induced by treatment with imatinib or DMSO. The p65/RelA D361E mutant was generated by mutating the INFD putative consensus recognition site for caspase-6. The caspase-6 inhibitor Z-VEID-fmk partially reduced the cell death induced by RIP3-KD and slightly reduced p65/RelA cleavage. p65/RelA cleavage appears to be at least partially mediated by caspase-6. Conclusions. These data indicate that RIP3 silencing in leukemia cells results in suppression of the complex regulation of the apoptosis/necroptosis switch and the modulation of the NF-κB pathway through the caspase-mediated cleavage of p65/RelA.
Citation Format: Anne-Lucie Nugues, Hassiba Bouafia, Dominique Hetuin, Celine Berthon, Anne Loyens, Elisabeth Bertrand, Nathalie Jouy, Thierry Idziorek, Bruno Quesnel. RIP3 is downregulated in human myeloid leukemia cells and modulates apoptosis and caspase-mediated p65/RelA cleavage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1342. doi:10.1158/1538-7445.AM2014-1342