Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia–reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia–reperfusion injury model, the high-dose TNF model, and in A20−/− mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia–reperfusion injury model and no benefit in A20−/− mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.

IntroductionThe SET protein is a potent physiologic inhibitor of protein phosphatase 2A (PP2A) 1 that was isolated from a chromosomal rearrangement at 9q34 in a patient with acute undifferentiated leukemia. 2 The SET protein is overexpressed in chronic myelogenous leukemia (CML) cells, and SET protein levels are further elevated during blast crisis. 3 SET overexpression in CML cells correlates with decreased PP2A activity. 3 This indicates that many of the SET oncogenic activities may be manifest through inhibition of PP2A. PP2A plays a role in many cellular processes, including cell cycle regulation, cell proliferation, apoptosis, development, cytoskeleton dynamics, cell motility, and stem cell self-renewal. 4 In addition, PP2A is a critical tumor suppressor gene that regulates multiple important oncogenic signal transduction pathways. [5][6][7] PP2A inhibition is essential for cell transformation and tumor formation, 8,9 but overexpression of PP2A inhibitory proteins in chronic lymphocytic leukemia (CLL) has not been reported.Of the nearly 84 000 annual cases of leukemia in the Western world, B-cell CLL is the most common, accounting for ϳ 30% of adult leukemia cases. 10 Characterized by accumulation of monoclonal mature B cells, 11 the CLL clinical course is heterogeneous, with some patients experiencing an aggressive course that demands early treatment and others experiencing long survival without disease-related symptoms or ever requiring treatment. 11 Aberrant apoptosis in CLL cells correlates with arrest either in the G 0 or early G 1 phases of the cell cycle. 12,13 This defective apoptosis in CLL cells is partly the result of aberrant signaling through the Akt kinase and the ERK MAPK pathways, in which phosphorylated-Akt is necessary for survival of the leukemia cells. 14,15 The observation of aberrantly activated Akt and downstream pathways in CLL cells also suggests that the normal regulator of these pathways, PP2A, is unable to perform its normal role.We thus sought to determine whether SET is overexpressed in CLL cells relative to normal B cells. We found that SET is significantly overexpressed in CLL cells and related non-Hodgkin lymphoma (NHL) cell line cells. In freshly isolated CLL patient samples, higher cellular levels of the SET correlated with more aggressive disease requiring earlier treatment. Antagonism of SET using shRNA-mediated knockdown or pharmacologic antagonism with novel cell-permeable SET antagonist peptides induced apoptosis, reduced cellular levels of Mcl-1, and caused death of CLL and NHL cells, but normal B cells were scarcely affected by SET antagonism. We also found that pharmacologic SET antagonism in vivo inhibited growth of B-cell NHL tumor xenografts in SCID mice. Methods GeneralAll reagents were from Sigma-Aldrich unless noted otherwise. Anti-SET antibody was from Santa Cruz Biotechnology. Anti-␤-actin, total c-Myc, pS62 c-Myc, and Mcl-1 were from Abcam. All primary antibodies were used at a 1:1000 dilution, except for ␤-actin, which was used at 1:10 000. All secondary ...

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HA associated with fludarabine therapy appears to be uncommon, but it can be severe and fatal, especially if a patient is re-treated with this drug after a previous episode of HA. The mechanism of this toxicity is unknown, but it may be caused by the release of a suppressed auto-antibody to a native red cell antigen.