RESUMO
Identifying biomarkers associated with disease progression and drug resistance are important for personalized care. We investigated the expression of 121 curated genes, related to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs) responsiveness. We analyzed 28 human multiple myeloma (MM) cell lines with known drug sensitivities and 130 primary MM patient samples collected at different disease stages, including newly diagnosed (ND), on therapy (OT), and relapsed and refractory (RR, collected within 12 months before the patients' death) timepoints. Our findings led to the identification of a subset of genes linked to clinical drug resistance, poor survival, and disease progression following combination treatment containing IMIDs and/or PIs. Finally, we built a seven-gene model (MM-IMiD and PI sensitivity-7 genes [IP-7]) using digital gene expression profiling data that significantly separates ND patients from IMiD- and PI-refractory RR patients. Using this model, we retrospectively analyzed RNA sequcencing (RNAseq) data from the Mulltiple Myeloma Research Foundation (MMRF) CoMMpass (n = 578) and Mayo Clinic MM patient registry (n = 487) to divide patients into probabilities of responder and nonresponder, which subsequently correlated with overall survival, disease stage, and number of prior treatments. Our findings suggest that this model may be useful in predicting acquired resistance to treatments containing IMiDs and/or PIs.
RESUMO
We generated eight multiple myeloma cell lines resistant to bortezomib; five acquired PSMB5 mutations. In 1,500 patients such mutations were rare clinically. To better understand disruption of proteasomes on multiple myeloma viability and drug sensitivity, we systematically deleted the major proteasome catalytic subunits. Multiple myeloma cells without PSMB5 were viable. Drug-resistant, PSMB5-mutated cell lines were resensitized to bortezomib by PSMB5 deletion, implying PSMB5 mutation is activating in its drug resistance function. In contrast, PSMB6 knockout was lethal to multiple myeloma cell lines. Depleting PSMB6 prevented splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8, and PSMB10; however, PSMB6 engineered without splicing function or catalytic activity, also restored viability, inferring the contribution of PSMB6 to proteasome structure to be more important than functional activity. Supporting this, bortezomib sensitivity was restored in drug-resistant multiple myeloma cell lines by low level expression of mutated PSMB6 lacking splicing function. Loss of PSMB8 and PSMB9 was neither lethal nor restored bortezomib sensitivity. Significant codependency of PSMB5, PSMB6, and PSMB7 expression was observed. We demonstrated elevated levels of PSMB6 and 7, but not 8 and 9, in some, but not all, serial patient samples exposed to proteasome inhibitors. In summary, we show PSMB6 and PSMB7, but not PSMB5, to be essential for multiple myeloma cell survival, this dependency is structural and that upregulation or activating mutation of PSMB5, 6, and 7 confers proteasome inhibitor resistance, while depletion confers sensitivity. IMPLICATIONS: These findings support modulation of PSMB5, PSMB6, or PSMB7 expression as a new therapeutic strategy.
Assuntos
Mieloma Múltiplo/genética , Inibidores de Proteassoma/uso terapêutico , Diferenciação Celular , Sobrevivência Celular , Humanos , Inibidores de Proteassoma/farmacologiaRESUMO
Seventy-six FDA-approved oncology drugs and emerging therapeutics were evaluated in 25 multiple myeloma (MM) and 15 non-Hodgkin's lymphoma cell lines and in 113 primary MM samples. Ex vivo drug sensitivities were mined for associations with clinical phenotype, cytogenetic, genetic mutation, and transcriptional profiles. In primary MM samples, proteasome inhibitors, dinaciclib, selinexor, venetoclax, auranofin, and histone deacetylating agents had the broadest cytotoxicity. Of interest, newly diagnosed patient samples were globally less sensitive especially to bromodomain inhibitors, inhibitors of receptor tyrosine kinases or non-receptor kinases, and DNA synthesis inhibitors. Clustering demonstrated six broad groupings of drug sensitivity linked with genomic biomarkers and clinical outcomes. For example, our findings mimic clinical observations of increased venetoclax responsiveness in t(11;14) patients but also identify an increased sensitivity profile in untreated patients, standard genetic risk, low plasma cell S-Phase, and in the absence of Gain(1q) and t(4;14). In contrast, increased ex vivo responsiveness to selinexor was associated with biomarkers of poor prognosis and later relapse patients. This "direct to drug" screening resource, paired with functional genomics, has the potential to successfully direct appropriate individualized therapeutic approaches in MM and to enrich clinical trials for likely responders.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Mieloma Múltiplo/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Humanos , Hidrazinas/farmacologia , Mieloma Múltiplo/genética , Medicina de Precisão/métodos , Sulfonamidas/farmacologia , Triazóis/farmacologia , Células Tumorais CultivadasRESUMO
To understand immunomodulatory drug (IMiD) resistance in multiple myeloma (MM), we created isogenic human multiple myeloma cell lines (HMCLs) sensitive and resistant to lenalidomide, respectively. Four HMCLs were demonstrated to be resistant to all IMiDs including lenalidomide, pomalidomide, and CC-220, but not to Bortezomib. In three HMLCs (MM.1.SLenRes, KMS11LenRes and OPM2LenRes), CRBN abnormalities were found, including chromosomal deletion, point mutation, and low CRBN expression. The remaining HMCL, XG1LenRes, showed no changes in CRBN but exhibited CD147 upregulation and impaired IRF4 downregulation after lenalidomide treatment. Depletion of CD147 in XG1LenRes and three additional HMCLs had no significant impact on MM viability and lenalidomide response. Further analysis of XG1LenRes demonstrated increased IL6 expression and constitutive STAT3 activation. Inhibition of STAT3 with a selective compound (PB-1-102) re-sensitized XG1LenRes to lenalidomide. Since XG1LenRes harbors a truncated IRF4 that is not downregulated by lenalidomide, we targeted IRF4/MYC axis with a selective inhibitor of the bromodomain of CBP/EP300 (SGC-CBP30), which restored lenalidomide response in XG1LenRes. This strategy also appeared to be more broadly applicable as SGC-CBP30 could re-sensitize two resistant HMCLs with low but detectable CRBN expression to lenalidomide, suggesting that targeting CBP/E300 is a promising approach to restore IMiD sensitivity in MM with detectable CRBN expression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fatores Reguladores de Interferon/antagonistas & inibidores , Lenalidomida/farmacologia , Mieloma Múltiplo/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Biomarcadores Tumorais , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Citocinas , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Humanos , Imunomodulação/efeitos dos fármacos , Lenalidomida/uso terapêutico , Modelos Biológicos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Ligação Proteica , Ubiquitina-Proteína LigasesRESUMO
Bortezomib is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. Consequently, we employed CRISPR targeting 19,052 human genes to identify unbiased targets that contribute to bortezomib resistance. Specifically, we engineered an RPMI8226 multiple myeloma cell line to express Cas9 infected by lentiviral vector CRISPR library and cultured derived cells in doses of bortezomib lethal to parental cells. Sequencing was performed on surviving cells to identify inactivated genes responsible for drug resistance. From two independent whole-genome screens, we selected 31 candidate genes and constructed a second CRISPR sgRNA library, specifically targeting each of these 31 genes with four sgRNAs. After secondary screening for bortezomib resistance, the top 20 "resistance" genes were selected for individual validation. Of these 20 targets, the proteasome regulatory subunit PSMC6 was the only gene validated to reproducibly confer bortezomib resistance. We confirmed that inhibition of chymotrypsin-like proteasome activity by bortezomib was significantly reduced in cells lacking PSMC6. We individually investigated other members of the PSMC group (PSMC1 to 5) and found that deficiency in each of those subunits also imparts bortezomib resistance. We found 36 mutations in 19S proteasome subunits out of 895 patients in the IA10 release of the CoMMpass study (https://themmrf.org). Our findings demonstrate that the PSMC6 subunit is the most prominent target required for bortezomib sensitivity in multiple myeloma cells and should be examined in drug-refractory populations. Mol Cancer Ther; 16(12); 2862-70. ©2017 AACR.
Assuntos
Antineoplásicos/uso terapêutico , Bortezomib/uso terapêutico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Mieloma Múltiplo/tratamento farmacológico , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
FAM46C is one of the most recurrently mutated genes in multiple myeloma; however its role in disease pathogenesis has not been determined. Here we demonstrate that wild-type (WT) FAM46C overexpression induces substantial cytotoxicity in multiple myeloma cells. In contrast, FAM46C mutations found in multiple myeloma patients abrogate this cytotoxicity, indicating a survival advantage conferred by the FAM46C mutant phenotype. WT FAM46C overexpression downregulated IRF4, CEBPB, and MYC and upregulated immunoglobulin (Ig) light chain and HSPA5/BIP Furthermore, pathway analysis suggests that enforced FAM46C expression activated the unfolded protein response pathway and induced mitochondrial dysfunction. CRISPR-mediated depletion of endogenous FAM46C enhanced multiple myeloma cell growth, decreased Ig light chain and HSPA5/BIP expression, activated ERK and antiapoptotic signaling, and conferred relative resistance to dexamethasone and lenalidomide treatments. Genes altered in FAM46C-depleted cells were enriched for signaling pathways regulating estrogen, glucocorticoid, B-cell receptor signaling, and ATM signaling. Together these results implicate FAM46C in myeloma cell growth and survival and identify FAM46C mutation as a contributor to myeloma pathogenesis and disease progression via perturbation in plasma cell differentiation and endoplasmic reticulum homeostasis. Cancer Res; 77(16); 4317-27. ©2017 AACR.
Assuntos
Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas/genética , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Chaperona BiP do Retículo Endoplasmático , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Nucleotidiltransferases , Transdução de SinaisRESUMO
To identify molecular targets that modify sensitivity to lenalidomide, we measured proliferation in multiple myeloma (MM) cells transfected with 27 968 small interfering RNAs in the presence of increasing concentrations of drug and identified 63 genes that enhance activity of lenalidomide upon silencing. Ribosomal protein S6 kinase (RPS6KA3 or RSK2) was the most potent sensitizer. Other notable gene targets included 5 RAB family members, 3 potassium channel proteins, and 2 peroxisome family members. Single genes of interest included I-κ-B kinase-α (CHUK), and a phosphorylation dependent transcription factor (CREB1), which associate with RSK2 to regulate several signaling pathways. RSK2 knockdown induced cytotoxicity across a panel of MM cell lines and consistently increased sensitivity to lenalidomide. Accordingly, 3 small molecular inhibitors of RSK2 demonstrated synergy with lenalidomide cytotoxicity in MM cells even in the presence of stromal contact. Both RSK2 knockdown and small molecule inhibition downregulate interferon regulatory factor 4 and MYC, and provides an explanation for the synergy between lenalidomide and RSK2 inhibition. Interestingly, RSK2 inhibition also sensitized MM cells to bortezomib, melphalan, and dexamethasone, but did not downregulate Ikaros or influence lenalidomide-mediated downregulation of tumor necrosis factor-α or increase lenalidomide-induced IL-2 upregulation. In summary, inhibition of RSK2 may prove a broadly useful adjunct to MM therapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Mieloma Múltiplo/genética , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/isolamento & purificação , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Talidomida/análogos & derivados , Inibidores da Angiogênese/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Lenalidomida , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Talidomida/farmacologia , Células Tumorais CultivadasRESUMO
Cereblon (CRBN) mediates immunomodulatory drug (IMiD) action in multiple myeloma (MM). Using 2 different methodologies, we identified 244 CRBN binding proteins and established relevance to MM biology by changes in their abundance after exposure to lenalidomide. Proteins most reproducibly binding CRBN (>fourfold vs controls) included DDB1, CUL4A, IKZF1, KPNA2, LTF, PFKL, PRKAR2A, RANGAP1, and SHMT2. After lenalidomide treatment, the abundance of 46 CRBN binding proteins decreased. We focused attention on 2 of these-IKZF1 and IKZF3. IZKF expression is similar across all MM stages or subtypes; however, IKZF1 is substantially lower in 3 of 5 IMiD-resistant MM cell lines. The cell line (FR4) with the lowest IKZF1 levels also harbors a damaging mutation and a translocation that upregulates IRF4, an IKZF target. Clinical relevance of CRBN-binding proteins was demonstrated in 44 refractory MM patients treated with pomalidomide and dexamethasone therapy in whom low IKZF1 gene expression predicted lack of response (0/11 responses in the lowest expression quartile). CRBN, IKZF1, and KPNA2 levels also correlate with significant differences in overall survival. Our study identifies CRBN-binding proteins and demonstrates that in addition to CRBN, IKZF1, and KPNA2, expression can predict survival outcomes.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fatores Imunológicos/farmacologia , Mieloma Múltiplo/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Anti-Inflamatórios/farmacologia , Western Blotting , Ensaios Clínicos Fase II como Assunto , Dexametasona/farmacologia , Citometria de Fluxo , Seguimentos , Humanos , Fator de Transcrição Ikaros/metabolismo , Imunoprecipitação , Lenalidomida , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Prognóstico , Estudos Prospectivos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Taxa de Sobrevida , Talidomida/análogos & derivados , Talidomida/farmacologia , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases , alfa Carioferinas/metabolismoRESUMO
Cereblon (CRBN) mediates immunomodulatory drug (IMiD) action in multiple myeloma (MM). We demonstrate here that no patient with very low CRBN expression responded to IMiD plus dexamethasone therapy. In 53 refractory MM patients treated with pomalidomide and dexamethasone, CRBN levels predict for decreased response rates and significant differences in PFS (3.0 vs. 8.9 months, p<0.001) and OS (9.1 vs. 27.2 months, p=0.01) (lowest quartile vs. highest three quartiles). While higher CRBN levels can serve as a surrogate for low risk disease, our study demonstrates that low CRBN expression can predict resistance to IMiD monotherapy and is a predictive biomarker for survival outcomes.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Peptídeo Hidrolases/genética , Proteínas Adaptadoras de Transdução de Sinal , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Dexametasona/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Mieloma Múltiplo/patologia , Prognóstico , Análise de Sobrevida , Talidomida/administração & dosagem , Talidomida/análogos & derivados , Resultado do Tratamento , Ubiquitina-Proteína LigasesRESUMO
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.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Peptídeo Hidrolases/genética , Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Farmacológicos/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Ácidos Borônicos/administração & dosagem , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Lenalidomida , Modelos Biológicos , Peptídeo Hidrolases/metabolismo , Pirazinas/administração & dosagem , Pirazinas/farmacologia , RNA Interferente Pequeno/farmacologia , Talidomida/administração & dosagem , Talidomida/análogos & derivados , Talidomida/farmacologia , Talidomida/uso terapêutico , Ubiquitina-Proteína LigasesRESUMO
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.