Abstract
The receptor-interacting protein kinase 3 (RIP3) associates with RIP1 in a necrosome complex that can induce necroptosis, apoptosis, or cell proliferation. 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. 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. Transfection of RIP3 in the WEHI-3b leukemia cell line or in the mouse embryonic fibroblasts also resulted in increased cell death. Surprisingly, re-expression of a RIP3 mutant with an inactive kinase domain (RIP3-kinase dead (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. The expression of RIP3-KD induced p65/RelA nuclear factor-κB (NF-κB) subunit caspase-dependent cleavage, and a non-cleavable p65/RelA D361E mutant rescued these cells from apoptosis. p65/RelA cleavage appears to be at least partially mediated by caspase-6. These data indicate that RIP3 silencing in leukemia cells results in suppression of the complex regulation of the apoptosis/necroptosis switch and NF-κB activity.
Highlights
The role of RIP3 in necroptosis and inflammation has been extensively studied, but its role in cancer remains poorly understood
A previous study in chronic lymphocytic leukemia (CLL) showed that malignant lymphoid cells were resistant to tumor necrosis factor-a (TNFa þ Z-VAD-induced necroptosis and expressed reduced levels of RIP3 and cylindromatosis (CYLD), which regulates receptor-interacting protein kinase 1 (RIP1).7 Another study on childhood acute lymphoblastic leukemia reported that RIP1 was necessary to mediate the inhibitor of apoptosis proteinmediated sensitization of blast cells to chemotherapy.[8]
acute myeloid leukemia (AML) CD34 þ blast cells expressed significantly reduced RIP3 mRNA compared with CD34 þ cells from healthy donors (Figure 1a), whereas the expression of RIP1 did not differ significantly (Figure 1b)
Summary
The role of RIP3 in necroptosis and inflammation has been extensively studied, but its role in cancer remains poorly understood. A previous study in chronic lymphocytic leukemia (CLL) showed that malignant lymphoid cells were resistant to tumor necrosis factor-a (TNFa þ Z-VAD-induced (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) necroptosis and expressed reduced levels of RIP3 and cylindromatosis (CYLD), which regulates RIP1.7 Another study on childhood acute lymphoblastic leukemia reported that RIP1 was necessary to mediate the inhibitor of apoptosis proteinmediated sensitization of blast cells to chemotherapy.[8] Autocrine TNFa loops that activate NF-kB through RIP1 have been described in various cancer cell lines.[9,10]. The expression of a RIP3 mutant with an inactivated kinase domain (RIP3-kinase dead (RIP3-KD)) in myeloid cell lines resulted in massive and early apoptosis and the caspase-mediated cleavage of p65/RkelA at a caspase-6 putative consensus site.
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