Que, Qiang

Abstract Bortezomib and related proteasome inhibitors have profound pre-clinical and clinical activity in Multiple Myeloma (MM). However, the critical molecular target(s) modulated by proteosome inhibition remains unknown. The overall response rate to bortezomib in relapsed and refractory MM patients is consistently 35%–50%, suggesting specific molecular targets or resistance mechanisms are inherent in patients. None of the known effects of bortezomib has been shown to correlate with clinical responses in patients or to explain the unique sensitivity of MM (as opposed to other malignancies) to these reagents. Therefore, in this study, we have conducted a small interfering RNA (siRNA) screen of the druggable genome in human myeloma cells to identify genes that modulate sensitivity to bortezomib. KMS11 human myeloma cells were reverse transfected with 13,984 synthetic short interfering RNAs (siRNA) against each of 6992 druggable targets (2 siRNA/gene with a single-siRNA per well/384 well format). At 24 hours post transfection, bortezomib was added at IC10, 30, 60 and 90. Cell viability was measured by ATP-dependent luminescence assay 72 hours post drug treatment. The resulting data from primary screening was evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >99% global transfection efficiency, <0.25 CV values across the dataset with minimal plate-to-plate and set-to-set variation observed. From analysis of the primary screening data, a core panel of 320 (4.6% of druggable targets in library) bortezomib sensitizers were identified. 187 (58%) of these were further confirmed in a secondary screening experiment by multiple (2 to 4) independent siRNA. Among these 187 genes (2.6% of the genes investigated), some known targets, such as proteasome subunits are evident. Other genes belonging to the same functional gene families (e.g. TNF receptor family, Ras family, kinases, oncogenes and transporter proteins) or functioning within the same network of signal transduction (e.g. NF-kB, Wnt and estrogen receptor pathways) are evident. Our primary screen also identified genes that attenuate bortezomib-induced cytotoxicity. Members in this group include genes implicated in the negative regulation of cell proliferation and survival, such as NF-kB inhibitors, JNK pathway activators, tumor suppressors, apoptosis inducers and phosphatases. These studies enable dissection of the molecular mechanism of bortezomib in myeloma cells and may serve to point to biomarkers for response prediction and novel chemotherapeutic combination therapies.

AbstractA paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein–coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.