Citations

Key Points IMiDs inhibit TrxR-mediated intracellular decomposition of H2O2 and caused oxidative stress in MM cells. MM cells with lower antioxidative capacity were more vulnerable to lenalidomide-induced H2O2 accumulation and its associated cytotoxicity.

Key Points The incidence of mutations within the MAPK pathway, the CRBN pathway, and TP53 is significantly increased in drug-refractory MM. Mutations in CRBN might contribute to IMiD resistance in drug-refractory MM.

Key Points High-throughput RNAi screening identified lenalidomide sensitizer genes, including RSK2, RAB, peroxisome, and potassium channel family members. Knockdown or inhibition of RSK2 synergized with lenalidomide to induce myeloma cytotoxicity and downregulation of interferon regulatory factor 4 and MYC.

SummaryWe constructed a multiple myeloma (MM)‐specific gene panel for targeted sequencing and investigated 72 untreated high‐risk (del17p) MM patients. Mutations were identified in 78% of the patients. While the majority of studied genes were mutated at similar frequency to published literature, the prevalence of TP53 mutation was increased (28%) and no mutations were found in FAM46C. This study provides a comprehensive insight into the mutational landscape of del17p high‐risk MM. Additionally, our work demonstrates the practical use of a customized sequencing panel, as an easy, cheap and fast approach to characterize the mutational profile of MM.

Key PointsWe identified CRBN-binding proteins and confirmed recent work implicating the Ikaros transcription factors as important members. Low IZKF1 levels predict lack of IMiD responsiveness and shorter OS; IZKF1 expression is a potential predictive biomarker for clinic application.

Abstract The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their antitumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the antimyeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells, but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone, and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion, further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to, and putatively resistant to, lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity and a possible biomarker for the clinical assessment of antimyeloma efficacy.

Abstract The molecular target(s) cooperating with proteasome inhibition in multiple myeloma (MM) remain unknown. We therefore measured proliferation in MM cells transfected with 13 984 small interfering RNAs in the absence or presence of increasing concentrations of bortezomib. We identified 37 genes, which when silenced, are not directly cytotoxic but do synergistically potentiate the growth inhibitory effects of bortezomib. To focus on bortezomib sensitizers, genes that also sensitized MM to melphalan were excluded. When suppressed, the strongest bortezomib sensitizers were the proteasome subunits PSMA5, PSMB2, PSMB3, and PSMB7 providing internal validation, but others included BAZ1B, CDK5, CDC42SE2, MDM4, NME7, RAB8B, TFE3, TNFAIP3, TNK1, TOP1, VAMP2, and YY1. The strongest hit CDK5 also featured prominently in pathway analysis of primary screen data. Cyclin-dependent kinase 5 (CDK5) is expressed at high levels in MM and neural tissues with relatively low expression in other organs. Viral shRNA knockdown of CDK5 consistently sensitized 5 genetically variable MM cell lines to proteasome inhibitors (bortezomib and carfilzomib). Small-molecule CDK5 inhibitors were demonstrated to synergize with bortezomib to induce cytotoxicity of primary myeloma cells and myeloma cell lines. CDK5 regulation of proteasome subunit PSMB5 was identified as a probable route to sensitization.

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.

AbstractAs multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, pristimerin. Strikingly, the early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-κB activation via inhibition of IKKα or IKKβ, whereas proteosome inhibitors instead suppress NF-κB function by impairing degradation of ubiquitinated IκB. By inhibiting both IKK and the proteosome, pristimerin causes overt suppression of constitutive NF-κB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-κB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC50 < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib—providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-κB inhibitors as therapeutics for human multiple myeloma and related malignancies.