AT7519, a Novel Small Molecule Multi-Cyclin Dependent Kinase Inhibitor, Induces Apoptosis in Multiple Myeloma VIA GSK3 beta

Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM

Poly(ADP-ribose) polymerase family member 14 (PARP14) is a novel effector of the JNK2-dependent pro-survival signal in multiple myeloma

Copyright @ 2013 Macmillan Publishers Limited. This is the author's accepted manuscript. The final published article is available from the link below.Regulation of cell survival is a key part of the pathogenesis of multiple myeloma (MM). Jun N-terminal kinase (JNK) signaling has been implicated in MM pathogenesis, but its function is unclear. To elucidate the role of JNK in MM, we evaluated the specific functions of the two major JNK proteins, JNK1 and JNK2. We show here that JNK2 is constitutively activated in a panel of MM cell lines and primary tumors. Using loss-of-function studies, we demonstrate that JNK2 is required for the survival of myeloma cells and constitutively suppresses JNK1-mediated apoptosis by affecting expression of poly(ADP-ribose) polymerase (PARP)14, a key regulator of B-cell survival. Strikingly, we found that PARP14 is highly expressed in myeloma plasma cells and associated with disease progression and poor survival. Overexpression of PARP14 completely rescued myeloma cells from apoptosis induced by JNK2 knockdown, indicating that PARP14 is critically involved in JNK2-dependent survival. Mechanistically, PARP14 was found to promote the survival of myeloma cells by binding and inhibiting JNK1. Moreover, inhibition of PARP14 enhances the sensitization of MM cells to anti-myeloma agents. Our findings reveal a novel regulatory pathway in myeloma cells through which JNK2 signals cell survival via PARP14, and identify PARP14 as a potential therapeutic target in myeloma.Kay Kendall Leukemia Fund, NIH, Cancer Research UK, Italian Association for Cancer Research and the Foundation for Liver Research

p21WAF1/CIP1 Upregulation through the Stress Granule-Associated Protein CUGBP1 Confers Resistance to Bortezomib-Mediated Apoptosis

p21(WAF1/CIP1) is a well known cyclin-dependent kinase inhibitor induced by various stress stimuli. Depending on the stress applied, p21 upregulation can either promote apoptosis or prevent against apoptotic injury. The stress-mediated induction of p21 involves not only its transcriptional activation but also its posttranscriptional regulation, mainly through stabilization of p21 mRNA levels. We have previously reported that the proteasome inhibitor MG132 induces the stabilization of p21 mRNA, which correlates with the formation of cytoplasmic RNA stress granules. The mechanism underlying p21 mRNA stabilization, however, remains unknown.We identified the stress granules component CUGBP1 as a factor required for p21 mRNA stabilization following treatment with bortezomib ( =  PS-341/Velcade). This peptide boronate inhibitor of the 26S proteasome is very efficient for the treatment of myelomas and other hematological tumors. However, solid tumors are sometimes refractory to bortezomib treatment. We found that depleting CUGBP1 in cancer cells prevents bortezomib-mediated p21 upregulation. FISH experiments combined to mRNA stability assays show that this effect is largely due to a mistargeting of p21 mRNA in stress granules leading to its degradation. Altering the expression of p21 itself, either by depleting CUGBP1 or p21, promotes bortezomib-mediated apoptosis.We propose that one key mechanism by which apoptosis is inhibited upon treatment with chemotherapeutic drugs might involve upregulation of the p21 protein through CUGBP1

Effects of PS-341 on the activity and composition of proteasomes in multiple myeloma cells.

Multiple myeloma is a B-cell malignancy for which no curative therapies exist to date, despite enormous research efforts. The remarkable activity of the proteasome inhibitor bortezomib (PS-341, Velcade) observed in clinical trials of patients with relapsed refractory myeloma has led to investigations of the role of the ubiquitin-proteasome pathway in the pathogenesis of myeloma. Here we report a biochemical analysis of proteasome activity and composition in myeloma cells exposed to PS-341 in the presence or absence of cytokines present in the bone marrow milieu. We observed that the myeloma cell lines MM1.S, RPMI8226, and U266 contain active immunoproteasomes, the amount of which is enhanced by IFN-gamma and tumor necrosis factor-alpha. Using a radiolabeled active site-directed probe specific for proteasome catalytic subunits, we show that PS-341 targets the beta5 and beta1 subunits in a concentration-dependent manner. Furthermore, PS-341 also targeted the corresponding catalytic subunits of the immunoproteasome, beta5i and beta1i, respectively. These data suggest that PS-341 targets both normal and immunoproteasome species to a similar extent in myeloma cells