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 ...
