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Targeting protein kinase C (PKC) isoforms by the small molecule inhibitor enzastaurin has shown promising pre-clinical activity in a wide range of tumor cells. In this study, we further delineated its mechanism of action in multiple myeloma (MM) cells and found a novel role of b-catenin in regulating growth and survival of tumor cells. Inhibition of PKC leads to rapid accumulation of b-catenin by preventing the phosphorylation required for its proteasomal degradation. Specifically, b-catenin was dephosphorylated at Ser33,37,41 and accumulated in a dose- and time-dependent manner in all cell lines tested (including primary MM cells and 10 MM cell lines, 3 colon cancer, HeLa, as well as HEK 293 cells). Microarray analysis and siRNA-mediated gene silencing in MM cells revealed that accumulated b-catenin activates early ER stress signaling via eIF2a, CHOP and p21, leading to immediate inhibition of proliferation. Conversely, knock-down of components of the ER stress response pathway by siRNA (i.e., CHOP) abrogated the inhibitory effect of enzastaurin on MM cell proliferation. Importantly, accumulated b-catenin also contributes to enzastaurin-induced cell death, since inhibition of b-catenin by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a b-catenin -dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; b-catenin induction by enzastaurin led to p73 (but not p53) activation, which was also abrogated by b-catenin -specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of b-catenin in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. In summary, our data reveal a novel role of b-catenin in ER stress-mediated growth inhibition and a new pro-apoptotic mechanism triggered by b-catenin upon inhibition of PKC isoforms, and further demonstrate that p73 represents a novel therapeutic target in MM. Based on these and previous data, enzastaurin is currently under clinical investigation in a variety of hematologic malignancies including MM.

We have previously shown that the novel orally available small molecule inhibitor of PKC enzastaurin (Eli Lilly and Company) inhibits MM cell growth, survival and angiogenesis both in vitro and in vivo. To date, however, the downstream effects contributing to growth inhibition and cell death remain to be determined. Here, we performed global gene expression profiling on enzastaurin treated MM cells and identified 200 Genes to be differentially regulated with a > 2-fold cut off. Strikingly, two major groups of up-regulated probe sets were associated with either of two pathways - endoplasmatic reticulum (ER)-stress response or WNT-signaling. Importantly, MM cells, producing high levels of paraprotein, are highly susceptible to perturbation of ER function and protein folding. Moreover, PKC isoforms have been reported to directly regulate the canonical WNT pathway via phosphorylation of b-catenin (CAT), leading to its ubiquination and proteasomal degradation. Specifically, we fist evaluated the role of enzastaurin in mediating ER-stress in MM cells. The transcriptional up-regulation of genes involved in ER-stress (GADD153/CHOP, GADD34, ATF3), triggered by enzastaurin at 3h, was confirmed by western blot analysis, accompanied by induction of the molecular ER chaperone BiP/grp78, phosphorylation of eIF2a consistent with PERK activation, and up-regulation of p21. These events were preceded by an early (1h) increase of intracellular calcium levels, a hallmark of ER-stress, assessed by FLUO4 staining. These data suggest an important role of ER-stress response in the early growth inhibition of MM cells caused by enzastaurin. Second, we delineated effects of enzastaurin on WNT pathway in MM and other tumor cell lines. Upon enzastaurin treatment, CAT was dephosphorylated at Ser33, 37, 41 in a dose- and time-dependent manner in all cell lines tested (10 MM, 3 colon cancer, HeLa, as well as human embryonic kidney 293 cells). Consequently, accumulation of CAT occurred in both cytosolic and nuclear fractions of treated MM cells, associated with activated TOPflash LUC-reporter system, confirming nuclear transactivating activity. Specific inhibition of CAT by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a CAT-dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; CAT induction by enzastaurin led to p73 (but not p53) activation and was also abrogated by CAT-specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of CAT in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. Our studies therefore indicate that ER-stress response contributes to the immediate inhibition of proliferation by enzastaurin, followed by CAT accumulation leading to p73 activation, contributing to enzastaurin-mediated cell death. These findings provide a novel link between CAT and p53-family members. Moreover p73, which is only rarely mutated in human cancers, represents a novel therapeutic target in MM.