RESUMO
ABSTRACT: To establish a strict p53-dependent gene-expression profile, TP53-/- clones were derived from TP53+/+ and TP53-/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53-/- clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation-coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Pirimidinas , Tiofenos , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linhagem Celular Tumoral , Apoptose , Antineoplásicos/uso terapêuticoRESUMO
A strategy combining targeted therapies is effective in B-cell lymphomas (BCL), such as mantle cell lymphoma (MCL), but acquired resistances remain a recurrent issue. In this study, we performed integrative longitudinal genomic and single-cell RNA-sequencing analyses of patients with MCL who were treated with targeted therapies against CD20, BCL2, and Bruton tyrosine kinase (OAsIs trial). We revealed the emergence of subclones with a selective advantage against OAsIs combination in vivo and showed that resistant cells were characterized by B-cell receptor (BCR)-independent overexpression of NF-κB1 target genes, especially owing to CARD11 mutations. Functional studies demonstrated that CARD11 gain of function not only resulted in BCR independence but also directly increased the transcription of the antiapoptotic BCL2A1, leading to resistance against venetoclax and OAsIs combination. Based on the transcriptional profile of OAsIs-resistant subclones, we designed a 16-gene resistance signature that was also predictive for patients with MCL who were treated with conventional chemotherapy, underlying a common escape mechanism. Among druggable strategies to inhibit CARD11-dependent NF-κB1 transduction, we evaluated the selective inhibition of its essential partner MALT1. We demonstrated that MALT1 protease inhibition led to a reduction in the expression of genes involved in OAsIs resistance, including BCL2A1. Consequently, MALT1 inhibition induced synergistic cell death in combination with BCL2 inhibition, irrespective of CARD11 mutational status, both in vitro and in vivo. Taken together, our study identified mechanisms of resistance to targeted therapies and provided a novel strategy to overcome resistance in aggressive BCL. The OAsIs trial was registered at www.clinicaltrials.gov #NCT02558816.
Assuntos
Linfoma Difuso de Grandes Células B , Linfoma de Célula do Manto , Adulto , Humanos , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular Tumoral , Mutação com Ganho de Função , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma de Célula do Manto/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Although treatment of multiple myeloma (MM) with daratumumab significantly extends the patient's lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/r MM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high-affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38, including those with a lower sensitivity to daratumumab. In a killing assay where multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity toward MM cells compared with daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in 2 therapeutic mouse models, unlike daratumumab. Finally, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/r MM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently being developed in a phase 1 clinical study.
Assuntos
Anticorpos Biespecíficos , Mieloma Múltiplo , Animais , Camundongos , ADP-Ribosil Ciclase 1/metabolismo , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Linfócitos T/patologiaRESUMO
Innovative therapeutic strategies have emerged over the past decade to improve outcomes for most lymphoma patients. Nevertheless, the aggressive presentation seen in high-risk mantle cell lymphoma (MCL) patients remains an unmet medical need. The highly proliferative cells that characterize these tumors depend on nucleotide synthesis to ensure high DNA replication and RNA synthesis. To take advantage of this vulnerability, STP-B, a clinically available small molecule selectively targeting CTP synthase 1 (CTPS1) has been recently developed. CTPS1 is a key enzyme of the pyrimidine synthesis pathway mediated through its unique ability to provide enough CTP in highly proliferating cells. Herein, we demonstrated that CTPS1 was expressed in all MCL cells, and that its high expression was associated with unfavorable outcomes for patients treated with chemotherapy. Using aggressive MCL models characterized by blastoid morphology, TP53 mutation or polyresistance to targeted therapies, we showed that STP-B was highly effective at nanomolar concentrations in vitro and in vivo, irrespective of these high-risk features. Inhibition of CTPS1 rapidly leads to cell cycle arrest in early S-phase accompanied by inhibition of translation, including of the anti-apoptotic protein MCL1. Consequently, CTPS1 inhibition induced synergistic cell death in combination with the selective BCL2 inhibitor venetoclax, both in vitro and in vivo. Overall, our study identified CTPS1 as a promising target for MCL patients and provided a mechanism-based combination with the BCL2 inhibitor venetoclax for the design of future chemotherapy-free treatment regimens to overcome resistance.
Assuntos
Sinergismo Farmacológico , Linfoma de Célula do Manto , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Humanos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes , Carbono-Nitrogênio Ligases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/patologia , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/genética , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aggressive B-cell malignancies, such as mantle cell lymphoma (MCL), are microenvironment-dependent tumors and a better understanding of the dialogs occurring in lymphoma-protective ecosystems will provide new perspectives to increase treatment efficiency. To identify novel molecular regulations, we performed a transcriptomic analysis based on the comparison of circulating MCL cells (n=77) versus MCL lymph nodes (n=107) together with RNA sequencing of malignant (n=8) versus normal B-cell (n=6) samples. This integrated analysis led to the discovery of microenvironment-dependent and tumor-specific secretion of interleukin-32 beta (IL32ß), whose expression was confirmed in situ within MCL lymph nodes by multiplex immunohistochemistry. Using ex vivo models of primary MCL cells (n=23), we demonstrated that, through the secretion of IL32ß, the tumor was able to polarize monocytes into specific MCL-associated macrophages, which in turn favor tumor survival. We highlighted that while IL32ß-stimulated macrophages secreted several protumoral factors, they supported tumor survival through a soluble dialog, mostly driven by BAFF. Finally, we demonstrated the efficacy of selective NIK/alternative-NFkB inhibition to counteract microenvironment-dependent induction of IL32ß and BAFF-dependent survival of MCL cells. These data uncovered the IL32ß/BAFF axis as a previously undescribed pathway involved in lymphoma-associated macrophage polarization and tumor survival, which could be counteracted through selective NIK inhibition.
Assuntos
Fator Ativador de Células B , Interleucinas , Linfoma de Célula do Manto , Proteínas Serina-Treonina Quinases , Adulto , Humanos , Linhagem Celular Tumoral , Interleucinas/metabolismo , Linfoma de Célula do Manto/patologia , Macrófagos/metabolismo , NF-kappa B/metabolismo , Microambiente Tumoral , Fator Ativador de Células B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinase Induzida por NF-kappaBRESUMO
BH3 mimetics are promising drugs for hematologic malignancies that trigger cell death by promoting the release of proapoptotic BCL2 family members from antiapoptotic proteins. Multiple myeloma is considered to be a disease dependent mainly on MCL1 for survival, based mostly on studies using cell lines. We used a BH3-mimetic toolkit to study the dependency on BCL2, BCLXL, or MCL1 in malignant plasma cells from 60 patients. Dependencies were analyzed using an unbiased BH3 mimetics cell-death clustering by k-means. In the whole cohort of patients, BCL2 dependency was mostly found in the CCND1 subgroup (83%). Of note, MCL1 dependence significantly increased from 33% at diagnosis to 69% at relapse, suggesting a plasticity of the cellular dependency favoring MCL1 dependencies at relapse. In addition, 35% of overall patient samples showed codependencies on either BCL2/MCL1 or BCLXL/MCL1. Finally, we identified a group of patients not targeted by any of the BH3 mimetics, predominantly at diagnosis in patients not presenting the common recurrent translocations. Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection from cell death observed by BAK knock-down, as well as the complete and early disruption of MCL1/BAK complexes on A1210477 treatment. Interestingly, this complex was also dissociated in A1210477-resistant cells, but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. In conclusion, our study opens the way to rationally use venetoclax and/or MCL1 BH3 mimetics for clinical evaluation in myeloma at both diagnosis and relapse.
Assuntos
Antineoplásicos , Materiais Biomiméticos , Mieloma Múltiplo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fragmentos de Peptídeos , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas , Antineoplásicos/química , Antineoplásicos/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Mantle cell lymphoma (MCL) is a lymphoproliferative disorder characterized by the t(11;14)(q13;q32) CCND1/IGH translocation. This lymphoma is however extremely heterogeneous in terms of molecular alterations. Moreover, the course of the disease can vary greatly between indolent forms with slow progression and aggressive conditions rapidly pejorative. The identification of early markers allowing to predict individual patients outcome has however been unsuccessful so far. The LyMa trial treated homogeneously a cohort of young MCL patients. This appeared as a good opportunity to search for biomarkers of response to therapy. DNA extracted from diagnostic paraffin-embedded lymph node biopsies from 100 patients with newly diagnosed MCL, homogeneously treated in this prospective clinical trial, were investigated for copy number alterations and copy neutral loss of heterozygosity using the Oncoscan SNP-array scanning the whole genome. An independent confirmatory cohort was used to strengthen the possibly relevant anomalies observed. Here we describe the recurrent anomalies identified with this technique. Deletions of 17p(TP53) and 9p(CDKN2A) were more frequent in refractory or early relapsing patients (10%), but had no significant impact in univariate analysis on progression-free (PFS) or overall survival (OS). Regardless of the presence of TP53 or CDKN2A deletions, gains in 7p22 (8,5%) were associated with better PFS in univariate but not in multivariate analysis including MCL International Prognostic Index and treatment. Gains of 11q(CCDN1), suggesting gains of the CCND1/IGH fusion, were associated with worse OS and PFS in univariate and multivariate analyses. This worse prognosis impact was confirmed by FISH in an independent confirmatory cohort. This work, using a whole genome approach, confirms the broad genomic landscape of MCL and shows that gains of the CCND1/IGH fusion can be considered as a new prognostic structural variant. Genomic abnormalities of prognostic impact could be useful to strengthen or de-escalate treatment schedules or choosing targeted therapies or CART-cells.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Variações do Número de Cópias de DNA , Genoma Humano , Linfoma de Célula do Manto/patologia , Recidiva Local de Neoplasia/patologia , Adulto , Idoso , Terapia Combinada , Ciclina D1/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Seguimentos , Humanos , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/terapia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/terapia , Prognóstico , Estudos Prospectivos , Transplante de Células-Tronco , Taxa de Sobrevida , Translocação Genética , Proteína Supressora de Tumor p53/genética , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Despite recent advances in the treatment of multiple myeloma, the disease constantly relapses and is still considered as incurable. The current knowledge about the biological mechanisms underlying resistance to the different class of drugs in multiple myeloma remains poor. The primary objective of the MYRACLE (Myeloma Resistance And Clonal Evolution) cohort, a multicenter prospective cohort of patients with multiple myeloma, is to address this limitation. We here describe the study background, design and methods used for this cohort. METHODS/DESIGN: All patients (> 18 year old) diagnosed with de novo or relapsed multiple myeloma and treated in two hematology department from west of France are included in the MYRACLE cohort. Patients provide a signed informed to be included in the study. All subjects are followed until refusal to participate in the study or death. The MYRACLE cohort prospectively collects data on socio-economic status, medical status, imaging, prognosis factors, MM therapies and associated events (resistance, safety issues). Patients also complete standardized quality of life questionnaires. In addition, bone marrow samples will be collected at time of diagnosis and relapses to perform biomarkers analysis and functional assays exploring mechanisms underlying drug resistance. DISCUSSION: The "real-life" MYRACLE cohort offers the opportunity to prospectively collect epidemiological, medical, QoL and biological data from MM patients during the course of the disease (at time of diagnosis and subsequent relapses). At mid-tem, this integrative cohort will be unique at producing a large variety of data that can be used to conceive the most effective personalized therapy for MM patients. Additionally, the MYRACLE cohort will allow integrating the medical care of MM patients in a health and pharmacoeconomic perspective.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Feminino , França , Humanos , Masculino , Prognóstico , Estudos Prospectivos , Qualidade de Vida , Inquéritos e Questionários , Análise de Sobrevida , Resultado do TratamentoRESUMO
Mantle cell lymphoma (MCL) accumulates in lymphoid organs, but disseminates early on in extranodal tissues. Although proliferation remains located in lymphoid organs only, suggesting a major role of the tumor ecosystem, few studies have assessed MCL microenvironment. We therefore cocultured primary circulating MCL cells from 21 patients several weeks ex vivo with stromal or lymphoid-like (CD40L) cells to determine which interactions could support their proliferation. We showed that coculture with lymphoid-like cells, but not stromal cells, induced cell-cycle progression, which was amplified by MCL-specific cytokines (insulin-like growth factor-1, B-cell activating factor, interleukin-6, interleukin-10). Of interest, we showed that our model recapitulated the MCL in situ molecular signatures (ie, proliferation, NF-κB, and survival signatures). We further demonstrated that proliferating MCL harbored an imbalance in Bcl-2 family expression, leading to a consequent loss of mitochondrial priming. Of interest, this loss of priming was overcome by the type II anti-CD20 antibody obinutuzumab, which counteracted Bcl-xL induction through NF-κB inhibition. Finally, we showed that the mitochondrial priming directly correlated with the sensitivity toward venetoclax and alkylating drugs. By identifying the microenvironment as the major support for proliferation and drug resistance in MCL, our results highlight a selective approach to target the lymphoma niche.
Assuntos
Linfoma de Célula do Manto/patologia , Linfoma de Célula do Manto/terapia , Terapia de Alvo Molecular , Microambiente Tumoral , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD20/imunologia , Ligante de CD40/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Tecido Linfoide/patologia , Masculino , Mesoderma/patologia , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteína bcl-X/metabolismoRESUMO
(1) Background: TP53 deficiency remains a major adverse event in Multiple Myeloma (MM) despite therapeutic progresses. As it is not possible to target TP53 deficiency with pharmacological agents, we explored the possibility of activating another p53 family member, p73, which has not been well studied in myeloma. (2) Methods: Using human myeloma cell lines (HMCLs) with normal or abnormal TP53 status, we assessed TP73 methylation and expression. (3) Results: Using microarray data, we reported that TP73 is weakly expressed in 47 HMCLs and mostly in TP53 wild type (TP53wt) HMCLs (p = 0.0029). Q-RT-PCR assays showed that TP73 was expressed in 57% of TP53wt HMCLs (4 out of 7) and 11% of TP53 abnormal (TP53abn) HMCLs (2 out of 18) (p = 0.0463). We showed that TP73 is silenced by methylation in TP53abn HMCLs and that decitabine increased its expression, which, however, remained insufficient for significant protein expression. Alkylating drugs increased expression of TP73 only in TP53wt HMCLs but failed to synergize with decitabine in TP53abn HMCLs. (4) Conclusions: Decitabine and melphalan does not appear as a promising combination for inducing p73 and bypassing p53 deficiency in myeloma cells.
Assuntos
Azacitidina/análogos & derivados , Melfalan/farmacologia , Mieloma Múltiplo/metabolismo , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/deficiência , Azacitidina/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Cisplatino/farmacologia , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Decitabina , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína Tumoral p73/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The aim of this study was to assess the efficiency of p53 reactivation and induction of massive apoptosis (PRIMA-1(Met)) in inducing myeloma cell death, using 27 human myeloma cell lines (HMCLs) and 23 primary samples. Measuring the lethal dose (LD50) of HMCLs revealed that HMCLs displayed heterogeneous sensitivity, with an LD50 ranging from 4 µM to more than 200 µM. The sensitivity of HMCLs did not correlate with myeloma genomic heterogeneity or TP53 status, and PRIMA-1(Met) did not induce or increase expression of the p53 target genes CDKN1A or TNFRSF10B/DR5. However, PRIMA-1(Met) increased expression of NOXA in a p53-independent manner, and NOXA silencing decreased PRIMA1(Met)-induced cell death. PRIMA-1(Met) depleted glutathione (GSH) content and induced reactive oxygen species production. The expression of GSH synthetase correlated with PRIMA-1(Met) LD50 values, and we showed that a GSH decrease mediated by GSH synthetase silencing or by and L-buthionine sulphoximine, an irreversible inhibitor of γ-glutamylcysteine synthetase, increased PRIMA-1(Met)-induced cell death and overcame PRIMA-1(Met) resistance. PRIMA-1(Met) (10 µM) induced cell death in 65% of primary cells independent of the presence of del17p; did not increase DR5 expression, arguing against an activation of p53 pathway; and synergized with L-buthionine sulphoximine in all samples. Finally, we showed in mouse TP53(neg) JJN3-xenograft model that PRIMA-1(Met) inhibited myeloma growth and synergized with L-buthionine sulphoximine in vivo.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Glutationa/metabolismo , Mieloma Múltiplo/patologia , Quinuclidinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Quinuclidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Mantle Cell Lymphoma (MCL) is often associated with progression, temporary response to therapy and a high relapse rate over time resulting in a poor long-term prognosis. Because MCL is classified as an incurable disease, therapeutic resistance is of great interest. However, knowledge about the biological mechanisms underlying resistance associated with MCL therapies and about associated predictors remains poor. The REFRACT-LYMA Cohort, a multicenter prospective cohort of patients with MCL, is set up to address this limitation. We here describe the study background, design and methods used for this cohort. METHODS/DESIGN: The REFRACT-LYMA Cohort Study aims at including all patients (>18 years old) who are diagnosed with MCL in any stage of the disease and treated in specialized oncology centers in three public hospitals in Northwestern France. Any such patient providing a signed informed consent is included. All subjects are followed up indefinitely, until refusal to participate in the study, emigration or death. The REFRACT-LYMA follow-up is continuous and collects data on socio-economic status, medical status, MCL therapies and associated events (resistance, side effects). Participants also complete standardized quality of life (QOL) questionnaires. In addition, participants are asked to donate blood samples that will support ex vivo analysis of expression and functional assays required to uncover predictive biomarkers and companion diagnostics. If diagnostic biopsies are performed during the course of the disease, extracted biological samples are kept in a dedicated biobank. DISCUSSION: To our knowledge, the REFRACT-LYMA Cohort Study is the first prospective cohort of patients with MCL for whom "real-life" medical, epidemiological and QOL data is repeatedly collected together with biological samples during the course of the disease. The integrative cohort at mid-term will be unique at producing a large variety of data that can be used to conceive the most effective personalized therapy for MCL patients. Additionally, the REFRACT-LYMA Cohort puts the medical care of MCL patients in a health and pharmacoeconomic perspective.
Assuntos
Linfoma de Célula do Manto/terapia , Qualidade de Vida , Projetos de Pesquisa , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Terapia Combinada , Feminino , Seguimentos , França , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Estudos Prospectivos , Taxa de Sobrevida , Adulto JovemRESUMO
BACKGROUND: The aim of this study was to evaluate the efficacy of the p53-reactivating drugs RITA and nutlin3a in killing myeloma cells. METHODS: A large cohort of myeloma cell lines (n = 32) and primary cells (n = 21) was used for this study. This cohort contained cell lines with various TP53 statuses and primary cells with various incidences of deletion of chromosome 17. Apoptosis was evaluated using flow cytometry with Apo2.7 staining of the cell lines or via the loss of the myeloma-specific marker CD138 in primary cells. Apoptosis was further confirmed by the appearance of a subG1 peak and the activation of caspases 3 and 9. Activation of the p53 pathway was monitored using immunoblotting via the expression of the p53 target genes p21, Noxa, Bax and DR5. The involvement of p53 was further studied in 4 different p53-silenced cell lines. RESULTS: Both drugs induced the apoptosis of myeloma cells. The apoptosis that was induced by RITA was not related to the TP53 status of the cell lines or the del17p status of the primary samples (p = 0.52 and p = 0.80, respectively), and RITA did not commonly increase the expression level of p53 or p53 targets (Noxa, p21, Bax or DR5) in sensitive cells. Moreover, silencing of p53 in two TP53(mutated) cell lines failed to inhibit apoptosis that was induced by RITA, which confirmed that RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, apoptosis induced by nutlin3a was directly linked to the TP53 status of the cell lines and primary samples (p < 0.001 and p = 0.034, respectively) and nutlin3a increased the level of p53 and p53 targets in a p53-dependent manner. Finally, we showed that a nutlin3a-induced DR5 increase (≥ 1.2-fold increase) was a specific and sensitive marker (p < 0.001) for a weak incidence of 17p deletion within the samples (≤ 19%). CONCLUSION: These data show that RITA, in contrast to nutlin3a, effectively induced apoptosis in a subset of MM cells independently of p53. The findings and could be of interest for patients with a 17p deletion, who are resistant to current therapies.
Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Concentração Inibidora 50 , Mitocôndrias/metabolismo , Transporte Proteico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ativação Transcricional/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoAssuntos
Apoptose/efeitos dos fármacos , Mieloma Múltiplo/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirróis/farmacologia , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Expressão Gênica , Humanos , Mieloma Múltiplo/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Multiple myeloma is a plasma cell malignancy that is heterogeneous with respect to its causative molecular abnormalities and the treatment response of patients. The Bcl-2 protein family is critical for myeloma cell survival. ABT-737 is a cell-permeant compound that binds to Bcl-2 and Bcl-x(L) but not to Mcl-1. Using a myeloma cell line collection (n = 25) representative of different molecular translocations, we showed that ABT-737 effectively kills a subset of cell lines (n = 6), with a median lethal dose ranging from 7 ± 0.4 nM to 150 ± 7.5 nM. Of interest, all sensitive cell lines harbored a t(11;14). We demonstrated that ABT-737-sensitive and ABT-737-resistant cell lines could be differentiated by the BCL2/MCL1 expression ratio. A screen of a public expression database of myeloma patients indicates that the BCL2/MCL1 ratio of t(11;14) and hyperdiploid patients was significantly higher than in all other groups (P < .001). ABT-737 first induced the disruption of Bcl-2/Bax, Bcl-2/Bik, or Bcl-2/Puma complexes, followed by the disruption of Bcl-2 heterodimers with Bak and Bim. Altogether, the identification of a subset of cell lines and primary cells effectively killed by ABT-737 alone supported the evaluation of ABT-263, an orally active counterpart to ABT-737, for the treatment of t(11;14) and hyperdiploid groups of myeloma harboring a Bcl-2(high)/Mcl-1(low) profile.
Assuntos
Antineoplásicos/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/tratamento farmacológico , Nitrofenóis/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/uso terapêutico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Células Tumorais CultivadasAssuntos
Deleção Cromossômica , Cromossomos Humanos Par 5 , Mutação , Síndromes Mielodisplásicas/genética , Proteína Supressora de Tumor p53/genética , Progressão da Doença , Feminino , Humanos , Lenalidomida/uso terapêutico , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/classificação , Síndromes Mielodisplásicas/diagnóstico , PrognósticoRESUMO
Secondary plasma cell leukemia (sPCL) is a rare form of aggressive plasma cell malignancy arising mostly at end-stage refractory multiple myeloma and consequently presenting limited therapeutic options. We analyzed 13 sPCL for their sensitivity to BH3 mimetics targeting either BCL2 (venetoclax) or BCLXL (A1155463) and showed that 3 sPCL were efficiently killed by venetoclax and 3 sPCL by A1155463. Accordingly, BH3 profiling of 2 sPCL sensitive to BCLXL inhibition confirmed their high BCLXL primed profile. While targeting BCLXL using BH3 mimetics induces platelets on-target drug toxicity, the recent development of DT2216, a clinical-stage BCLXL proteolysis targeting chimera PROTAC compound, provides an alternative strategy to target BCLXL. Indeed, DT2216 specifically degrades BCLXL via VHL E3 ligase, without inducing thrombocytopenia. We demonstrated in human myeloma cell lines and sPCL that sensitivity to DT2216 strongly correlated with the sensitivity to A1155463. Interestingly, we showed that low doses of DT2216 (nM range) were sufficient to specifically degrade BCLXL after 48 hours of treatment, consistent with VHL expression, in all cell lines but irrespectively to DT2216 sensitivity. In myeloma cells, DT2216 induced apoptotic cell death and triggered BAX and BAK activation. In conclusion, our study demonstrated that patients with sPCL addicted to BCLXL, a small but a very challenging group, could potentially receive therapeutic benefit from DT2216. Clinical trials of DT2216 in this subset of sPCL patients are warranted.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Melanoma/genética , Mimetismo Molecular , Domínios e Motivos de Interação entre Proteínas , Sulfonamidas/farmacologia , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Humanos , Melanoma/diagnóstico , Melanoma/tratamento farmacológico , Sulfonamidas/uso terapêuticoRESUMO
The purpose of this study was to identify the pathways associated with the ability of CD138(+) human myeloma cells to form colonies in a serum-free semi-solid human collagen-based assay. Only 26% (7 of 27) of human myeloma cell lines were able to spontaneously form colonies. This spontaneous clonogenic growth correlated with the expression of the NOTCH ligand JAG2 (p<0.001). Blocking JAG-NOTCH interactions with NOTCH-Fc chimeric molecules impaired self-colony formation, indicating a role for JAG-NOTCH pathway in colony formation. In two cell lines, silencing of JAG2 blocked both colony formation and in vivo tumor formation in immunocompromised mice. RT-PCR and flow cytometry analysis revealed that JAG2 is often expressed by CD138(+) primary cells. Our results indicate that spontaneous clonogenic growth of myeloma cells requires the expression of JAG2.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Receptores Notch/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-2 , Ligantes , Proteínas de Membrana/genética , Camundongos , Camundongos SCID , Mieloma Múltiplo/genética , Transdução de Sinais , Sindecana-1/metabolismoRESUMO
The level of the Mcl-1 pro-survival protein is highly regulated, and the down-regulation of Mcl-1 expression favors the apoptotic process. Mcl-1 physically interacts with different BH3-only proteins; particularly, Noxa is involved in the modulation of Mcl-1 expression. In this study, we demonstrated that Noxa triggers the degradation of Mcl-1 at the mitochondria according to the exclusive location of Noxa at this compartment. The Noxa-induced degradation of Mcl-1 required the E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Because the USP9X deubiquitinase was recently demonstrated to be involved in Mcl-1 protein turnover by preventing its degradation through the removal of conjugated ubiquitin, we investigated whether Noxa affected the deubiquitination process. Interestingly, Noxa over-expression caused a decrease in the USP9X/Mcl-1 interaction associated with an increase in the Mcl-1 polyubiquitinated forms. Additionally, Noxa over-expression triggered an increase in the Mule/Mcl-1 interaction in parallel with the decrease in Mule/USP9X complex formation. Taken together, these modifications result in the degradation of Mcl-1 by the proteasome machinery. The implication of Noxa in the regulation of Mcl-1 proteasomal degradation adds complexity to this process, which is governed by multiple interactions.