Class of 2012 Abstract / Specific Aims: Autologous stem cell transplant (ASCT) is the current gold standard following induction therapy to improve survival of multiple myeloma (MM). Lenalidomide (LEN) is used for treatment of MM before ASCT, but exposure may impair autologous peripheral blood stem cell (PBSC) mobilization. Chemomobilization with cyclophosphamide (CTX) has not been evaluated in this setting. CTX + filgrastim was investigated to determine if LEN-associated mobilization impairment can be abrogated. Methods: 36 pts (group A=12 pts who received ≥2 cycles of LEN and group B=24 pts without LEN) were analyzed retrospectively. Baseline characteristics were matched (p>0.05 for all variables). All pts received CTX (median group B, 1.5g/m2; median group A, 3gm/m2(p=0.18)) and filgrastim 10µg/kg/day. Primary outcomes include number of CD34+ cells collected and number of leukapheresis sessions. Secondary outcomes include failure to collect CD34+ cells and total CD34+ cells collected after second leukapheresis. Main Results: Total median number of CD34+ cells collected in group B=9.15x106/kg CD34+ cells and group A=7.43x106/kg CD34+ cells (p=0.159). Median number of apheresis sessions in group B=2 and group A=3 (p=0.42). Two of 12 pts with antecedent LEN usage failed to collect while no patient without previous LEN exposure failed to collect (p=0.105). Total number of CD34+ cells collected after 2 apheresis sessions for group B=8.13x106/kg CD34+ cells and group A=3.34x106/kg CD34+ cells (p=0.06). Conclusions: Chemomobilization with CTX + filgrastim yields robust PBSC collections irrespective of antecedent lenalidomide. There was a trend towards lesser PBSC collection in LEN-treated pts.

No description available.

Multiple myeloma (MM) is an incurable progressive hematologic malignancy with heterogeneous evolution. It is a disease characterized by abnormal clonal proliferation of plasma cells in the bone marrow producing monoclonal immunoglobulins and causing a number of organ dysfunctions. Most patients relapse after treatment. Therefore, the use of methods of analysis of serum and urinary samples in order to detect as early as possible the presence of monoclonal immunoglobulins, before the occurrence of relapses, may aid in the treatment of patients with MM, improving its quality and prolonging survival. In this study we compared the effectiveness of the techniques of immunofixation (IF) and electrophoresis (EP) in the detection of MM relapses. For this, 52 patients under treatment in the University Hospital of Santa Maria (HUSM) were monitored, being detected the relapse of the disease in nine of these patients. A retrospective analysis of serum proteins of the nine patients, between January 2012 and July 2014, showed that IF was more effective than EP in early detection of relapse, regardless of the present class of immunoglobulins. The precocity of IF in relation to EP in detecting MM relapses in the nine patients studied, ranged from 2.0 to 18.8 months, with a mean of 6.6 months. Thus, we suggest the implementation of IF in the Clinical Analysis Laboratory (CAL) of HUSM to help onco-hematology physicians in the diagnosis and supportive care for patients with MM. / O mieloma múltiplo (MM) é uma neoplasia hematológica progressiva, com evolução heterogênea e ainda incurável. É uma doença caracterizada pela proliferação clonal anormal de plasmócitos na medula óssea produzindo imunoglobulinas monoclonais e ocasionando uma série de disfunções orgânicas. A maioria dos pacientes recidivam após o tratamento. Portanto, a utilização de métodos de análise de amostras séricas e urinárias, que permitam detectar o mais precocemente possível a presença de imunoglobulinas monoclonais, antes da ocorrência das recidivas, pode auxiliar no tratamento de pacientes com MM, melhorando a qualidade e ampliando o tempo de sobrevida dos mesmos. No presente estudo foram comparadas as técnicas de imunofixação (IF) e eletroforese (EF) quanto a sua eficácia na detecção das recidivas do MM. Para isso, foram monitorados 52 pacientes em tratamento junto ao Hospital Universitário de Santa Maria (HUSM), sendo detectada a recidiva da doença em nove destes pacientes. A análise retrospectiva de proteínas séricas dos nove pacientes, no período entre janeiro de 2012 e julho de 2014, mostrou que a IF foi mais eficaz do que a EF em detectar precocemente as recidivas, independentemente da classe de imunoglobulinas presente. Nos nove pacientes recidivados, a precocidade da IF, em relação à EF, na detecção das recidivas do MM variou de 2,0 a 18,8 meses, com um tempo médio de 6,6 meses.

In spite of recent therapeutic advances, multiple myeloma (MM) remains a malignancy with very low curability. This has been partly attributed to the existence of a drug-resistant subpopulation known as cancer stem cells (CSCs). MM-CSCs are equipped with the necessary tools that render them highly resistant to virtually all conventional therapies. In this study, the growth inhibitory effects of withanolide D (WND), a steroidal lactone isolated from Withania somnifera, on drug-sensitive tumoral plasma cells and drug-resistant MM cells have been investigated. In MTT/XTT assays, WND exhibited similar cytostatic effects between drug-resistant and drug-sensitive cell lines in the nM range. WND also induced cell death and apoptosis in MM-CSCs and RPMI 8226 cells, as examined by the calcein/ethidium homodimer and annexin V/propidium iodide stainings, respectively. To determine whether P-glycoprotein (P-gp) efflux affected the cytostatic activity of WND, P-gp was inhibited with verapamil and results indicated that the WND cytostatic effect in MM-CSCs was independent of P-gp efflux. Furthermore, WND did not increase the accumulation of the fluorescent P-gp substrate rhodamine 123 in MM-CSCs, suggesting that WND may not inhibit P-gp at the tested relevant doses. Therefore, the WND-induced cytostatic effect may be independent of P-gp efflux. These findings warrant further investigation of WND in MM-CSC animal models.

Le myélome multiple (MM) est une néoplasie B caractérisée par l’accumulation d’un clone plasmocytaire dans la moelle osseuse. Des études ont démontré que les modifications épigénétiques comme la méthylation de l’ADN jouent un rôle dans la régulation d’expression de différents gènes associés au cancer. Dans une étude récente, nous avons pu décrire un score génique de méthylation de l’ADN permettant de prédire la sensibilité des cellules de MM aux inhibiteurs de DNMT (DNA methyltranfexrase) (Moreaux, et al 2012). Parmi les gènes dont l’expression est inhibée par les inhibiteurs de DNMT et associés avec un pronostic péjoratif chez les patients atteints de MM, nous avons identifié RECQ1. RECQ1 est une hélicase de la famille RECQ qui s’associe aux origines de réplication durant la phase S du cycle cellulaire et joue un rôle important dans l’élongation des fourches de réplication. RECQ1 est fortement exprimé dans différents types de tumeurs solides et l’inhibition de RECQ1 conduit à la catastrophe mitotique et inhibe la croissance de tumeurs solides. Le but de notre projet a été de caractériser la fonction de RECQ1 dans la physiopathologie du MM et les mécanismes de résistance aux traitements. Afin d’étudier le rôle biologique de RECQ1 dans les plasmocytes tumoraux, nous avons utilisé des vecteurs lentiviraux pour induire de façon inductible la surexpression ou l'inhibition de RECQ1. La déplétion de RECQ1 dans les cellules de MM entraîne une inhibition de la croissance, une induction significative d’apoptose et la formation de foyers 53BP1 indiquant la présence de cassures d’ADN double brin. Une forte expression de RECQ1 étant associée à un mauvais pronostic et la déplétion de RECQ1 conduisant à une induction de cassures d’ADN double brin, nous nous sommes demandé si l’inhibition de l’expression de RECQ1 pourrait sensibiliser les cellules de MM aux agents génotoxiques utilisés dans le traitement du MM. La déplétion de RECQ1 sensibilise, de façon significative, les cellules de MM au melphalan suggérant que l’association d’un inhibiteur de DNMT pour cibler RECQ1 et du melphalan pourrait avoir un effet synergique chez les patients RECQ1++. La surexpression de RECQ1 protège les lignées cellulaires de myélome contra l'apoptose induite par melphalan et bortézomib. De plus, l'épuisement RECQ1 sensibilise les cellules de myélome de traitement est démontré que RECQ1 interagit avec des protéines impliquées dans différentes voies de réparation des dommages de l’ADN : PARP1 (NHEJ/BER), RAD51 (HR), MSH2 et MSH6 (Mismatch repair). RECQ1 interagit avec PARP1 dans la fraction chromatinienne des cellules de MM mais pas avec RAD51 ni MSH2. Cette interaction est significativement induite en présence de melphalan. Des inhibiteurs de PARP sont actuellement en développement préclinique ou en essai clinique. De façon intéressante, la déplétion de RECQ1 sensibilise significativement les cellules de MM à un inhibiteur de PARP in vitro suggérant que l’association d’un inhibiteur de DNMT pour cibler RECQ1 et d’un inhibiteur de PARP pourrait avoir un intérêt thérapeutique dans le MM. Nous avons également confirmé que des doses sous-létales d’inhibiteur de DNMT sensibilisent les cellules de MM au melphalan in vitro. / Multiple myeloma (MM) is a plasma cell cancer with poor survival, characterized by the clonal expansion of multiple myeloma cells (MMCs), primarily in the bone marrow. Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the genes downregulated by DNMT inhibitor. RECQ helicase are DNA unwinding enzymes involved in the maintenance of chromosome stability. RECQ1 silencing in cancer cells results in mitotic catastrophe and prevents tumor growth in murine models. RECQ1 is significantly overexpressed in primary myeloma cells compared to normal plasma cells and in myeloma cell lines compared to primary myeloma cells of patients. High RECQ1 expression is associated with a poor prognosis in two independent cohorts of patients. RECQ1 knock down inhibits growth of myeloma cells and induces apoptosis. Given the known role of RECQ1 in replication and DNA repair activation, the effect of RECQ1 depletion in DNA damage response was investigated. RECQ1 depletion induced spontaneous accumulation of DNA double strand breaks (DSBs) evidenced by the phosphorylation of ATM and H2AX histone and detection of 53BP1 foci. Using an alkaline comet assay, a significant increase in DNA strand breaks was confirmed in RECQ1 depleted cell lines compared to control. RECQ1 depletion was associated with CHK1 and CHK2 phosphorylation in MM cells. Since RECQ1 depletion is associated with DNA damage response activation and DNA strand breaks formation, a link between RECQ1 expression and drug sensitivity was hypothesized. RECQ1 overexpression significantly protects myeloma cell lines from melphalan and bortezomib-induced apoptosis. Furthermore, RECQ1 depletion sensitizes myeloma cells to treatment. Using immunoprecipitation, RECQ1 was shown to interact with PARP1 but not RAD51 or MSH2. An increased association of the two proteins was found upon DNA damages induced by melphalan. In agreement, RECQ1 depletion sensitizes myeloma cell lines to PARP inhibitor. We identified RECQ1 as a miR-203 target. Interestingly, aberrant methylation of miR-203 was reported in MM cells and treatment with 5-aza-2’-deoxycitidine led to promoter demethylation and miR-203 re-expression. Furthermore, anti-miR-203 treatment induced a significant increase of RECQ1 mRNA level in MM cells.In conclusion, RECQ1 represent a biomarker of drug resistance in MM, which is targeted by DNMT inhibitors. This suggests association of alkylating agents and/or PARP inhibitors with DNMT inhibitor may represent a therapeutic approach in RECQ1high patients associated with a poor prognosis.

Introdução: Os Centros de Referência (CR) são serviços especializados, parte de um projeto inovador de parceria entre a academia e os gestores do Sistema Único de Saúde (SUS). O principal objetivo de um CR é promover a assistência através de acompanhamento multidisciplinar, facilitando o acesso ao medicamento fornecido pelas Secretarias Estaduais de Saúde (SES). Objetivos: avaliar a efetividade da assistência de pacientes com Mieloma Múltiplo (MM) em um CR no âmbito do SUS (Centro de Referência para Mieloma Múltiplo do Hospital de Clínicas de Porto Alegre [CRMM-HCPA]); comparar a qualidade de vida entre portadores de MM atendidos no CRMM-HCPA e em outros serviços de saúde que não são CR. Metodologia: Foi realizado um estudo prospectivo de 6 meses, com participantes que recebiam a Talidomida pela SES-RS, e eram tratados pelo CRMM-HCPA, em comparação com os que recebiam tratamento em outras instituições, fora de um CR. Foram incluídos 32 participantes da pesquisa, sendo 19 do CRMM-HCPA e 13 participantes de outras instituições. Para análise da efetividade do CRMM-HCPA foi considerado como desfecho principal o tempo, a partir do diagnóstico, em que os participantes da pesquisa levaram para realização do Transplante Autólogo de Células Tronco Hematopoiéticas (TACTH), por ser considerado este a terapia “padrão ouro” para o MM. Para análise deste tempo, foram aplicados questionários específicos do estudo e para análise da qualidade de vida foi aplicado o questionário SF-36. Resultados: Na análise de qualidade de vida encontramos diferença significativa em relação ao item aspecto social (do SF-36), relacionado à realização de atividades sociais (P = 0,02). O tempo (em meses) a partir do diagnóstico até o transplante de medula óssea, em cada grupo, foi medido apenas em participantes da pesquisa com idade \65 anos (n=25), dos quais, nesta análise, 15 eram do HCPA e 10 das outras instituições. Encontramos diferença significativa (P=0,036) em relação ao tempo em que os participantes da pesquisa foram encaminhados para realizarem o transplante autólogo, demonstrando que os do CRMM (HCPA) são encaminhados para o transplante em tempo significativamente inferior (mediana: 9 meses; IIQ: 8,5 a 14,5) do que são atendidos nas demais instituições (mediana: 24 meses; IIQ: 16 a 24). Conclusões: Os participantes do CRMM demonstraram mais facilidade em executar atividades sociais, com menos interferências relacionadas a problemas físicos ou emocionais. O CRMM demonstrou ser uma estratégia de tratamento mais efetiva do que outros serviços de saúde do SUS, que não são CR, uma vez que permitiu uma redução do tempo para a realização do transplante. / Introduction:Within the Brazilian Unified Health System (SUS), Referral Centers (RCs) are care facilities that provide specialized services as part of an innovative partnership project between universities and SUS managers. The main goal of RCs is to promote care through a multidisciplinary approach, facilitating access to medicines supplied by state health departments (SHDs).Objectives:To evaluate the effectiveness of care provided to patients with multiple myeloma (MM) in a RC within SUS (Hospital de Clínicas de Porto Alegre Referral Center for Multiple Myeloma, CRMM-HCPA) and to compare quality of life between patients with MM treated at CRMM-HCPA and other non-RC health care facilities. Methods: A 6-month prospective cohort study was conducted in patients receiving thalidomide from the Rio Grande do Sul SHD (SHD-RS) and treated at CRMM-HCPA, as compared to patients receiving treatment at other non-RC health care facilities. Thirty-two patients were included in the study, 19 from CRMM-HCPA and 13 from other institutions. To analyze the effectiveness of CRMM-HCPA, the main outcome measure was the time from diagnosis to referral for autologous hematopoietic stem cell transplantation (HSCT), as this is considered the gold-standard treatment for MM. Time from diagnosis to referral for autologous HSCT was assessed using questionnaires specifically designed for this study, and quality of life was assessed using the SF-36 questionnaire. Results: On quality of life analysis, there was a significant difference in the “social functioning” domain of the SF-36 questionnaire, which relates to performance of social activities (P=0.02). The time (in months) from diagnosis to referral for autologous HSCT, in each group, was measured only in patients aged \65 years (n=25); of these, 15 were from CRMM-HCPA and 10 from other institutions. In this analysis, there was a significant difference (P=0.036) in time between diagnosis and referral for autologous HSCT, which was significantly shorter for patients treated at CRMM-HCPA (median, 9 months; IQR, 8.5–14.5) than for those treated elsewhere (median, 24 months; IQR, 16–24).Conclusions:Patients treated at CRMM-HCPA demonstrated greater ease in performing social activities, with less interference from physical or emotional problems. CRMM-HCPA offers a more effective treatment strategy as compared with other non-RC health care facilities, as it enabled a reduction in time to transplantation.

Orientador: Carmino Antonio de Souza / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T12:06:26Z (GMT). No. of bitstreams: 1 Beltrame_LuizPaulo_D.pdf: 1273755 bytes, checksum: a809e73238738768c971673f3daef900 (MD5) Previous issue date: 2006 / Resumo: As elevadas taxas de mortalidade após transplante alogênico de medula óssea (TMO) com regime de condicionamento pré-transplante mieloablativo, levou-nos a iniciar a realização destes procedimentos utilizando-se regime de condicionamento pré-transplante não-mieloablativo, para pacientes considerados de maior risco. Esta modadlidade de tratamento passou a ser oferecida para pacientes com neoplasias hematológicas que devido à idade mais avançada, e/ou presença de comorbidades, eram inelegíveis para serem submetidos a um TMO convencional, e também para aqueles que estavam sendo submetidos a um segundo TMO. Entre julho de 2001 e outubro de 2005, 26 pacientes (leucemia mielóide aguda, n=1; leucemia mielóide crônica, n=1; Linfoma de Hodgkin, n=4; Linfoma não-Hodgkin agressivo, n=6; mieloma múltiplo, n=14) receberam TMO de um doador relacionado HLA-idêntico com células progenitoras periféricas (CPP), após um condicionamento pré-TMO não-mieloablativo. O regime de condicionamento consistia de fludarabina, 30 mg/m2/dia por 3 dias, e 2 Gy de irradiação corporal total. A profilaxia da doença do enxerto-contra-hospedeiro foi feita com ciclosporina-A por 56 dias, e micofenolato mofetil por 28 dias. Dos 26 pacientes, 23 (88%) encontravam-se em progressão de doença, e 24 (92%) estavam sendo submetidos ao segundo TMO. O seguimento mediano global foi de 436 dias (3-1397 dias), e o seguimento mediano dos pacientes vivos ao término deste estudo foi de 640 dias (135-1397 dias). São apresentados também, sem qualquer finalidade comparativa, os resultados de 47 pacientes que no mesmo período e na mesma unidade, foram submetidos ao TMO com CPP após um regime de condicionamento mieloablativo. Nos pacientes que receberam TMO com regime de condicionamento não-mieloablativo, a toxicidade não-hematológica foi branda e a recuperação rápida. Infecção bacteriana foi observada em 11 pacientes (44%). O tempo mediano com contagem de neutrófilos menor que 500 células por ml de sangue foi de 6 dias, e apenas 3 pacientes (12%) atingiram contagem plaquetária abaixo de 20.000. Quimerismo hematopoiético completo foi atingido em 25 pacientes (95%), e infusão de linfócitos do doador foi necessária em 6 indivíduos, sendo em 3 devido ao estado de quimerismo misto, e em 3 devido à progressão de doença. A doença do enxerto-contra-hospedeiro (DECH) aguda graus II ou maior ocorreu em 9 pacientes (37%) e a DECH crônica extensa ocorreu em 15 (60%). Houve 12 óbitos (46%), 7 relacionados a complicações do procedimento e 5 devido a progressão de doença. A sobrevida global, sobrevida livre de progressão e tempo para progressão nestes pacientes foram respectivamente 39% (95% IC, 15-63%), 33% (95% IC, 11-55%) e 64% (95% IC, 32-96%), considerando-se um tempo de seguimento mediano de 436 dias, com variação de 3 a 1397. O TMO com regime de condicionamento pré-transplante não-mieloablativo mostrou-se factível e seguro, com redução da toxicidade e mortalidade relacionadas ao procedimento, mesmo para pacientes com neoplasias hematológicas de alto risco / Abstract: Background and Objectives. High mortality rate after allogeneic hematopoietic stem cell transplantation (HSCT) with myeloablative conditioning prompted us to offer HSCT with non-myeloablative conditioning (NST). Design and Methods. Between July, 2001 and October, 2005, 26 patients [acute myeloid leukemia, n=1; chronic myeloid leukemia, n=1; Hodgkin¿s lymphoma, n=4; aggressive non-Hodgkin¿s lymphoma n=6; multiple myeloma (MM), n=14], received peripheral NST from HLA-identical siblings. Conditioning regimen consisted of fludarabine plus 2 Gy of TBI, and GVHD prophylaxis with cyclosporine-A plus micofenolate mofetil. Eighty-eight percent were in progression of disease (PD) and 92% were undergoing their second HSCT. Results. Non-hematologic toxicities were mild and transient. Bacterial infection occurred in 44%. Median time to granulocyte recovery was six days and only 12% achieved platelets count lower than 20 x 109/L. Full donor chimerism was established in 95%. Donor lymphocyte infusion was required in 6 patients, 3 because of mixed chimerism and 3 because of PD. A-GVHD grade II or higher was observed in 37%, and extensive c-GVHD in 60%. There were 12 deaths (46%), 7 transplant related mortality and 5 related to PD. OS, PFS and time to progression (TTP) were respectively 39% (95% CI, 15-63%), 33% (95% CI, 11-55%) and 64% (95% CI, 32-96%) for the whole group. MM patients had OS, PFS and TTP of 27% (CI 95%, 0-67%), 42% (95% CI, 8-76%) and 86% (95% CI, 60-100%), respectively. Interpretation and Conclusion. This approach showed feasible and safe with reduction in transplant related toxicities, even for these high risk hematopoietic malignancies / Doutorado / Clinica Medica / Doutor em Clínica Médica

Les mécanismes épigénétiques jouent un rôle essentiel dans la régulation de l’expression des gènes. L’enzyme du complexe répresseur Polycomb II EZH2, capable de triméthyler la lysine 27 de l’histone H3 (H3K27me3) est impliquée dans la régulation de nombreux processus normaux, tels que le développement et la différenciation cellulaire. Les plasmocytes jouent un rôle majeur dans la réponse immunitaire humorale. La différenciation des lymphocytes B en plasmocytes (PCD) est finement régulée par un réseau de facteurs de transcription impliqué de l’induction et le maintien de l’identité de ces deux types cellulaires. Par ailleurs, peu de mécanismes épigénétiques ont été décrits dans la PCD. En utilisant un modèle in vitro de PCD développé dans notre laboratoire, nous avons mis en évidence une surexpression d’EZH2 dans le stade préplasmablaste (prePB) de la PCD. Grâce à l’analyse globale des séquences d’ADN associées à EZH2 et H3K27me3 dans ce type cellulaire, nous avons montré que l’enzyme était capable de réprimer l’expression de gènes impliqués dans différentes fonctions des lymphocytes B et des plasmocytes. EZH2 est également capable de se fixer sur le promoteur de gènes actifs dans les prePB, impliqués dans la régulation de la prolifération de ces cellules. En outre, nous avons montré que l’inhibition chimique d’EZH2 dans notre modèle résultent en une dérégulation transcriptionnelle associée à une accélération du processus de différenciation. Nos résultats suggèrent qu’EZH2 est impliqué dans le maintien de l’état transitoire, immature et prolifératif des prePBs via la régulation de gènes, dépendante et indépendante de H3K27me3, favorisant l’amplification des cellules à défaut de leur différenciation en plasmocytes. Des anomalies de séquence ou de l’expression d’EZH2 ont été mis en évidence de nombreux cancers hématologiques et solides. Le myélome multiple (MM) est une hémopathie caractérisée par l’accumulation de plasmocytes tumoraux dans la moelle osseuse. Nos travaux ont notamment permis d’identifier une surexpression des membres du complexe Polycomb Repressive Complex 2 (PRC2) dans les cellules de MM, en association avec leur prolifération. Afin de comprendre le rôle de PRC2 dans le MM, nous avons utilisé un inhibiteur de l’activité méthyltransférase d’EZH2 (EPZ-6438). L’effet de l’inhibiteur d’EZH2 sur la prolifération et la survie des cellules de MM est très hétérogène. En effet, les cellules sensibles présentent un arrêt du cycle cellulaire et entrent en apoptose. De manière intéressante, la résistance des cellules de MM à l’inhibiteur d’EZH2 pourrait être médiée induite par la méthylation des promoteurs des gènes cibles de PRC2. Nous avons ainsi établi un score (EZ-score), basé sur l’expression des gènes, permettant d’identifier des patients de mauvais pronostic pouvant bénéficier d’un traitement avec un inhibiteur d’EZH2. Nous avons également mis en évidence un effet synergique de EPZ-6438 et du Lenalidomide, un agent immuno-modulateurs utilisé en traitement conventionnelle du MM. Cette inhibition de la croissance cellulaire est notamment due à l’induction de l’expression de facteurs de transcription, spécifiques des lymphocytes B, et des suppresseurs de tumeurs en association avec la répression de l’expression de MYC. Aussi, un prétraitement avec l’inhibiteur d’EZH2 permet de surmonter la résistance des cellules tumorales au Lenalidomide. Ces données suggèrent que le ciblage de PRC2 pourrait avoir un intérêt thérapeutique chez les patients caractérisés par un mauvais pronostic et un fort EZ-score. Ainsi, l’inhibiteur d’EZH2 pourrait également permettre de resensibiliser les patients aux chimiothérapies basées sur des agents immuno-modulateurs. / Epigenetic mechanisms play an essential role in gene expression regulation. EZH2, the catalytic sub-unit of PRC2, is able to trimethylate the lysine 27 of histone H3 (H3K27me3) and is involved in the regulation of numerous normal processes, such as development and cell differentiation. Plasma cells (PCs) play a major role in the defense of the host organism against pathogens, by producing antigen-specific antibodies. B cell differentiation into PC is mediated by a fine-tuned regulatory network of cell specific transcription factors involved in B and plasma cell identity. Although numerous key actors involved in plasma cell differentiation (PCD) have been described, most of the epigenetic mechanisms associated with this process are yet to be unveiled. Using an in vitro model of PCD developed in our laboratory, we showed that EZH2 is upregulated in the preplasmablast stage (prePB) of the PCD. By analyzing DNA sequences associated with EZH2 and H3K27me3 in this cell type, we highlighted that EZH2 is recruited to and represses through H3K27me3 a subset of genes involved in B cell and plasma cell identity. Interestingly, in prePBs and PBs, EZH2 was also found to be recruited to H3K27me3-free promoters of transcriptionally active genes known to regulate cell proliferation and DNA repair. Inhibition of EZH2 catalytic activity resulted in B to PC transcriptional changes associated with PC maturation induction together with higher immunoglobulin secretion. Altogether, our data suggests that EZH2 is involved in the maintenance of prePBs/PBs transitory immature proliferative state through H3K27me3-dependent and independent gene regulation supporting their amplification. Moreover, while EZH2 overexpression was previously shown to inhibit PCD in mice, this study highlights for the first time that EZH2 inhibition can accelerate normal human PCD by prematurely inducing a plasma cell transcriptional program.EZH2 mutations or abnormal expression were shown to be involved in numerous hematological malignancies and solid tumors. Multiple Myeloma (MM) is a malignant plasma cell disease with a poor survival, characterized by the accumulation of myeloma cells (MMCs) within the bone marrow. We identified a significant upregulation of the Polycomb Repressive Complex 2 (PRC2) core genes in MM cells in association with proliferation. We used EPZ-6438, a specific small molecule inhibitor of EZH2 methyltransferase activity, to evaluate its effects on MM cells phenotype and gene expression profile. PRC2 targeting results in cell growth inhibition due to cell cycle arrest and apoptosis together with Polycomb, DNA methylation, TP53 and RB1 target genes induction. EZH2 inhibitor induced toxicity was heterogeneous in human myeloma cell lines and primary MM cells from patients. Interestingly, we found that MM cell resistance to EZH2 inhibitor could be mediated by DNA methylation of PRC2 target genes. We established a gene expression-based EZ-score allowing to identify poor prognosis patients that could benefit from EZH2 inhibitor treatment. We also demonstrated a synergistic effect of EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment, through the activation of B cell transcription factors and tumor suppressor gene expression in concert with MYC repression. Moreover, EPZ-6438 pre-treatment was able to overcome MM cells resistance to lenalidomide. These data suggest that PRC2 targeting could have a therapeutic interest in MM patients characterized by high-risk EZ-score values, reactivating B cell transcription factors and tumor suppressor genes. EZH2 inhibitor could also re-sensitize MM patients to chemotherapies based on immunomodulatory agents.

Multiple Myeloma (MM) is the second most common hematological malignancy, and although patient outcomes have significantly improved since the introduction of autologous stem cell transplantation (ASCT) and novel pharmacological agents such as immunomodulators (IMID), proteasome inhibitors (PI), and monoclonal antibodies (mAb), the disease remains incurable. The pathological complexity of MM results from accumulating mutations in clonal populations of malignant B-cells, which are cytogenetically heterogenous and selectively sensitive to different therapeutic agents. Drug regimens therefore include a diverse combination of therapeutics designed to target specific pathways to inhibit cell proliferation. Recent advances in genomic analytics and novel pharmacological agents potentially allow for more targeted treatments which improve patient outcomes and frequency of remission with minimal adverse effects. Only recently have studies began to correlate an increased understanding of the many subtypes of MM with optimal treatment regimens, and practices such as “Direct to Drug” screening can give clinicians a look at a patient’s likely response to a combination of drugs. By incorporating emerging pharmaceutical agents into studies based on patient characteristics, the management of MM is making incremental strides towards a more targeted treatment paradigm.

No description available.