RESUMO
The neural transcription factor SOX11 is usually highly expressed in typical mantle cell lymphoma (MCL), but it is absent in the more indolent form of MCL. Despite being an important diagnostic marker for this hard-to-treat malignancy, the mechanisms of aberrant SOX11 expression are largely unknown. Herein, we describe 2 modes of SOX11 regulation by the cell-cycle regulator cyclin D1 (CCND1) and the signal transducer and activator of transcription 3 (STAT3). We found that ectopic expression of CCND1 in multiple human MCL cell lines resulted in increased SOX11 transcription, which correlated with increased acetylated histones H3K9 and H3K14 (H3K9/14Ac). Increased H3K9/14Ac and SOX11 expression was also observed after histone deacetylase 1 (HDAC1) or HDAC2 was depleted by RNA interference or inhibited by the HDAC inhibitor vorinostat. Mechanistically, we showed that CCND1 interacted with and sequestered HDAC1 and HDAC2 from the SOX11 locus, leading to SOX11 upregulation. Interestingly, our data revealed a potential inverse relationship between phosphorylated Y705 STAT3 and SOX11 expression in MCL cell lines, primary tumors, and patient-derived xenografts. Functionally, inactivation of STAT3 by inhibiting the upstream Janus kinase (JAK) 1 or JAK2 or by STAT3 knockdown was found to increase SOX11 expression, whereas interleukin-21 (IL-21)-induced STAT3 activation or overexpression of the constitutively active form of STAT3 decreased SOX11 expression. In addition, targeting SOX11 directly by RNA interference or indirectly by IL-21 treatment induced toxicity in SOX11+ MCL cells. Collectively, we demonstrate the involvement of CCND1 and STAT3 in the regulation of SOX11 expression, providing new insights and therapeutic implications in MCL.
Assuntos
Ciclina D1/metabolismo , Linfoma de Célula do Manto/genética , Fatores de Transcrição SOXC/genética , Fator de Transcrição STAT3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Loci Gênicos , Células HEK293 , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Humanos , Interleucinas/farmacologia , Fosfotirosina/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Fatores de Transcrição SOXC/metabolismo , Regulação para Cima/genéticaRESUMO
Knowledge of programmed death ligand 1 (PD-L1) expression and its regulation in B-cell lymphoma cells is limited. Investigating mechanisms that control PD-L1 expression in B-cell lymphoma cells might identify biomarkers that predict the efficacy of immunotherapy with anti-programmed death-1/PD-L1 antibodies. In addition, identification of mechanisms that regulate PD-L1 may identify molecules that can be targeted to improve the clinical efficacy of immune checkpoint inhibitors. In this study, we used proteomic approaches and patient-derived B-cell lymphoma cell lines to investigate mechanisms that regulate PD-L1 expression. We found that PD-L1 expression, particularly in nongerminal center B cell-derived diffuse large B-cell lymphoma (DLBCL), is controlled and regulated by several interactive signaling pathways, including the B-cell receptor (BCR) and JAK2/STAT3 signaling pathways. We found that that BCR-mediated NFATc1 activation upregulates IL-10 chemokine expression in PD-L1+ B-cell lymphoma cells. Released IL-10 activates the JAK2/STAT3 pathway, leading to STAT3-induced PD-L1 expression. IL-10 antagonist antibody abrogates IL-10/STAT3 signaling and PD-L1 protein expression. We also found that BCR pathway inhibition by BTK inhibitors (ibrutinib, acalabrutinib, and BGB-3111) blocks NFATc1 and STAT3 activation, thereby inhibiting IL-10 and PD-L1 expression. Finally, we validated the PD-L1 signaling network in 2 primary DLBCL cohorts consisting of 428 and 350 cases and showed significant correlations among IL-10, STAT3, and PD-L1. Thus, our findings reveal a complex signaling network regulating PD-L1 expression in B-cell lymphoma cells and suggest that PD-L1 expression can be modulated by small molecule inhibitors to potentiate immunotherapies.
Assuntos
Antígeno B7-H1/biossíntese , Regulação Leucêmica da Expressão Gênica/fisiologia , Linfoma Difuso de Grandes Células B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/fisiologia , Humanos , Interleucina-10/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Fatores de Transcrição NFATC/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Toll-like receptor 4 (TLR4) play a key role in activating the innate immune system during pathogen recognition. In the pathogenesis of multiple sclerosis (MS), activated TLR4 together with myeloid differentiation primary response gene 88 (MyD88) produce an inflammatory microenvironment that promotes the differentiation of microglia into the M1 phenotype, who plays a key role in the pathogenesis of MS. Interleukin-1 receptor-associated kinase (IRAK)-M is specifically expressed in microglia in central nervous system (CNS) and act as a negative regulator of TLR4-MyD88 signaling pathway. Moreover, previous studies have shown that IRAK-M promotes the differentiation of type 2 microglia; however, its role in MS has not been explored. In the present study, we demonstrated that IRAK-M expression is elevated during EAE, and IRAK-M-/- mice significantly accelerated course and increased severity of disease, accompanied by a visible increase of the M1 microglia infiltrated. In conclusion, these data indicates that IRAK-M significantly improves EAE onset through down-regulation of the TLR4-MyD88 signaling pathway, which finally leads to differentiation of M2 phenotype in the microglia. Our study suggests that IRAK-M may be a potential therapeutic target for the treatment of MS.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Microglia/citologia , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Diferenciação Celular/imunologia , Dependovirus/genética , Regulação para Baixo , Encefalomielite Autoimune Experimental/imunologia , Feminino , Quinases Associadas a Receptores de Interleucina-1/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/imunologia , Células Th1/imunologia , Células Th17/imunologiaRESUMO
AKT signaling is important for proliferation and survival of tumor cells. The clinical significance of AKT activation in diffuse large B-cell lymphoma (DLBCL) is not well analyzed. Here, we assessed expression of phosphorylated AKT (p-AKT) in 522 DLBCL patients. We found that high levels of p-AKT nuclear expression, observed in 24.3% of the study cohort, were associated with significantly worse progression-free survival and Myc and Bcl-2 overexpression. However, multivariate analysis indicated that AKT hyperactivation was not an independent factor. miRNA profiling analysis demonstrated that 63 miRNAs directly or indirectly related to the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin pathway were differentially expressed between DLBCLs with high and low p-AKT nuclear expression. We further targeted AKT signaling using a highly selective AKT inhibitor MK-2206 in 26 representative DLBCL cell lines and delineated signaling alterations using a reverse-phase protein array. MK-2206 treatment inhibited lymphoma cell viability, and MK-2206 sensitivity correlated with AKT activation status in DLBCL cells. On MK-2206 treatment, p-AKT levels and downstream targets of AKT signaling were significantly decreased, likely because of the decreased feedback repression; Rictor and phosphatidylinositol 3-kinase expression and other compensatory pathways were also induced. This study demonstrates the clinical and therapeutic implications of AKT hyperactivation in DLBCL and suggests that AKT inhibitors need to be combined with other targeted agents for DLBCL to achieve optimal clinical efficacy.
Assuntos
Linfoma Difuso de Grandes Células B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/mortalidade , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Taxa de SobrevidaRESUMO
Aberrant nuclear factor κB (NF-κB) signaling has been found to be of particular importance in diffuse, large B-cell lymphoma (DLBCL) cell survival and proliferation. Although the canonical NF-κB signaling pathway has been studied in some detail, activation of the alternative NF-κB pathway in DLBCL is not well characterized. Important insights into the regulation of the alternative NF-κB pathway in B lymphocytes has recently revealed the regulatory importance of the survival kinase NIK (NF-κB-inducing kinase) in genetically engineered murine models. Our studies demonstrate that both the canonical and alternative NF-κB pathways are constitutively activated in DLBCL. We also demonstrate that NIK kinase aberrantly accumulates in DLBCL cells due to constitutive activation of B-cell activation factor (BAFF)-R (BR3) through interaction with autochthonous B-lymphocyte stimulator (BLyS) ligand in DLBCL cells. Activation of BR3 in DLBCL induces recruitment and degradation of tumor necrosis factor receptor-associated factor 3, which results in NIK kinase accumulation, IκBα phosphorylation, and NF-κB p100 processing, thereby resulting in continuous activation of both NF-κB pathways in DLBCL cells, leading to autonomous lymphoma cell growth and survival. These results further elucidate mechanisms involved in abnormal NF-κB activation in DLBCL, and should contribute to better future therapeutic approaches for patients with DLBCL.
Assuntos
Receptor do Fator Ativador de Células B/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Receptor do Fator Ativador de Células B/antagonistas & inibidores , Receptor do Fator Ativador de Células B/genética , Proteína 3 com Repetições IAP de Baculovírus , Western Blotting , Núcleo Celular/metabolismo , Proliferação de Células , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Técnicas Imunoenzimáticas , Imunoprecipitação , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator 2 Associado a Receptor de TNF/antagonistas & inibidores , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/antagonistas & inibidores , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Análise Serial de Tecidos , Ubiquitina-Proteína Ligases , Quinase Induzida por NF-kappaBRESUMO
High grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH) represents an uncommon B-cell lymphoma (BCL) with aggressive clinical courses and poor prognosis. Despite revolutionary therapeutic advances in BCL, there has been limited treatment progress in HGBCL-DH, thus necessitating additional therapeutic strategies for HGBCL-DH. This study demonstrated that the BET antagonist INCB057643 synergized with the XPO1 inhibitors (selinexor and eltanexor) to decrease cell viability and increase cell apoptosis in HGBCL-DH cells with or without TP53 mutations. As anticipated, the combined treatment of INCB057643 with selinexor slowed tumor growth and reduced the tumor burden in TP53-mutated HGBCL-DH xenografts. Mechanistically, MYC functional inhibition was a potential molecular mechanism underlying the synergy of the combined INCB057643 and selinexor treatment in HGBCL-DH cells independent of TP53 mutation status. In TP53 mutated HGBCL-DH cells, inducing DNA damage and impairing the DNA damage response (DDR) were involved in the therapeutic interaction of the combined regimen. In TP53 wild-type cells, the molecular mechanism was linked with upregulation of p53 levels and activation of its targeted pathways, rather than dysregulation of the DDR. Collectively, we might provide a potential promising combination therapy regimen for the management of HGBCL-DH. Clinical evaluations are warranted to confirm this conclusion.
Assuntos
Linfoma de Células B , Linfoma Difuso de Grandes Células B , Humanos , Regulação para Baixo , Linfoma de Células B/genética , Linfoma Difuso de Grandes Células B/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
The nuclear factor of activated T cells (NFAT) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for cell growth and survival in hematopoietic cells. Recent experimental evidence has implicated the calcineurin/NFAT signaling pathway in the pathogenesis of various malignancies, including diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanism(s) underlying NFATc1 regulation of genes controlling lymphoma cell growth and survival is still unclear. In this study, we demonstrate that the transcription factor NFATc1 regulates gene expression in DLBCL cells through a chromatin remodeling mechanism that involves recruitment of the SWItch/Sucrose NonFermentable chromatin remodeling complex ATPase enzyme SMARCA4 (also known as Brahma-related gene 1) to NFATc1 targeted gene promoters. The NFATc1/Brahma-related gene 1 complex induces promoter DNase I hypersensitive sites and recruits other transcription factors to the active chromatin site to regulate gene transcription. Targeting NFATc1 with specific small hairpin RNA inhibits DNase I hypersensitive site formation and down-regulates target gene expression. Our data support a novel epigenetic control mechanism for the transcriptional regulation of growth and survival genes by NFATc1 in the pathophysiology of DLBCL and suggests that targeting NFATc1 could potentially have therapeutic value.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Epigênese Genética , Linfoma Difuso de Grandes Células B/genética , Fatores de Transcrição NFATC/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , DNA Helicases/metabolismo , Primers do DNA/genética , Desoxirribonuclease I , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Modelos Biológicos , Fatores de Transcrição NFATC/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Chronic lymphocytic leukemia (CLL) involves a profound humoral immune defect and tumor-specific humoral tolerance that directly contribute to disease morbidity and mortality. CD154 gene therapy can reverse this immune defect, but attempts to do this pharmacologically have been unsuccessful. The immune-modulatory agent lenalidomide shows clinical activity in CLL, but its mechanism is poorly understood. Here, we demonstrate that lenalidomide induces expression of functional CD154 antigen on CLL cells both in vitro and in vivo. This occurs via enhanced CD154 transcription mediated by a Nuclear Factor of Activated T cells c1 (NFATc1)/Nuclear Factor-kappaB (NF-kappaB) complex and also through phosphoinositide-3 (PI3)-kinase pathway-dependent stabilization of CD154 mRNA. Importantly, CD154-positive CLL cells up-regulate BID, DR5, and p73, become sensitized to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, and promote costimulatory activation of normal B cells to produce antibodies. In CLL patients receiving lenalidomide, similar evidence of CD154 activation is observed including BID, DR5, and p73 induction and also development of anti-ROR1 tumor-directed antibodies. Our data demonstrate that lenalidomide promotes CD154 expression on CLL cells with subsequent activation phenotype, and may therefore reverse the humoral immune defect observed in this disease. This study is registered at http://clinicaltrials.gov as NCT00466895.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antineoplásicos/farmacologia , Linfócitos B/efeitos dos fármacos , Ligante de CD40/biossíntese , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Neoplasias/biossíntese , Fosfatidilinositol 3-Quinases/fisiologia , Estabilidade de RNA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Talidomida/análogos & derivados , Adjuvantes Imunológicos/uso terapêutico , Idoso , Antineoplásicos/uso terapêutico , Linfócitos B/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/biossíntese , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Ligante de CD40/genética , Ligante de CD40/fisiologia , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Lenalidomida , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Fatores de Transcrição NFATC/fisiologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Talidomida/farmacologia , Talidomida/uso terapêutico , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genéticaRESUMO
FOXM1 is a transcription factor that controls cell cycle regulation, cell proliferation, and differentiation. Overexpression of FOXM1 has been implicated in various cancer types. However, the activation status and functional significance of FOXM1 in diffuse large B cell lymphoma (DLBCL) have not been well investigated. Using proteomic approaches, we discovered that the protein expression levels of FOXM1 and PLK1 were positively correlated in DLBCL cell lines and primary DLBCL. Expression levels of FOXM1 and PLK1 mRNAs were also significantly higher in DLBCL than in normal human B cells and could predict poor prognosis of DLBCL, particularly in patients with germinal center B cell-like (GCB) DLBCL. Furthermore, proteomic studies defined a FOXM1-PLK1 signature that consisted of proteins upstream and downstream of that axis involved in the p38-MAPK-AKT pathway, cell cycle, and DNA damage/repair. Further studies demonstrated a mechanistic function of the FOXM1/PLK1 axis in connection with the DNA damage response pathways regulating the S/G2 checkpoint of the cell cycle. Therapeutic targeting of FOXM1/PLK1 using a FOXM1 or PLK1 inhibitor, as well as other clinically relevant small-molecule inhibitors targeting ATR-CHK1, was highly effective in DLBCL in vitro models. These findings are instrumental for lymphoma drug discovery aiming at the FOXM1/PLK1/ATR/CHK1 axis.
RESUMO
BLyS and its major receptor BAFF-R have been shown to be critical for development and homeostasis of normal B lymphocytes, and for cell growth and survival of neoplastic B lymphocytes, but the biologic mechanisms of this ligand/receptor-derived intracellular signaling pathway(s) have not been completely defined. We have discovered that the BAFF-R protein was present in the cell nucleus, in addition to its integral presence in the plasma membrane and cytoplasm, in both normal and neoplastic B cells. BAFF-R interacted with histone H3 and IKKbeta in the cell nucleus, enhancing histone H3 phosphorylation through IKKbeta. Nuclear BAFF-R was also associated with NF-kappaB/c-Rel and bound to NF-kappaB targeted promoters including BLyS, CD154, Bcl-xL, IL-8, and Bfl-1/A1, promoting the transcription of these genes. These observations suggested that in addition to activating NF-kappaB pathways in the plasma membrane, BAFF-R also promotes normal B-cell and B-cell non-Hodgkin lymphoma (NHL-B) survival and proliferation by functioning as a transcriptional regulator through a chromatin remodeling mechanism(s) and NF-kappaB association. Our studies provide an expanded conceptual view of the BAFF-R signaling, which should contribute a better understanding of the physiologic mechanisms involved in normal B-cell survival and growth, as well as in the pathophysiology of aggressive B-cell malignancies and autoimmune diseases.
Assuntos
Receptor do Fator Ativador de Células B/fisiologia , Núcleo Celular/metabolismo , Proliferação de Células , Sobrevivência Celular/fisiologia , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Genes rel , Histonas/metabolismo , Humanos , Quinase I-kappa B/genética , Linfócitos/metabolismo , Linfoma de Células B/metabolismo , Mutagênese Sítio-Dirigida , NF-kappa B/genética , Fosforilação , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-rel/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações SubcelularesRESUMO
High-grade B-cell lymphoma with concurrent MYC and BCL2 rearrangements (HGBL-DHL) is a rare, aggressive mature B-cell malignancy with a high likelihood of treatment failure following front-line immunochemotherapies. Patients with HGBL-DHL who develop a relapsed or refractory disease have little effective therapeutic strategies and show very poor clinical outcomes, thus calling for development of novel therapies for this specific patient population. In this study, we investigated the preclinical anti-lymphoma efficacies and potential mechanism of action of a novel treatment approach, combining the BCL2 inhibitor venetoclax with CS2164, a new orally active multitarget inhibitor, in HGBL-DHL models. This combination therapy exhibited a robust synergistic cytotoxicity against HGBL-DHL cells, evidenced by cooperatively inducing loss of cell viability and promoting cell apoptosis. Moreover, coadministration of CS2164 and venetoclax resulted in significant superior suppression of HGBL-DHL cell growth and remarkably abrogated tumor burden in a HGBL-DHL-xenografted mouse model. The synergistic lethality of CS2164 and venetoclax in HGBL-DHL cells was associated with induction of DNA damage and impairment of DNA repair ability. Of importance, the combined treatment almost abolished the expression of both BCL2 and MYC, two hallmark proteins of HGBL-DHL, and substantially blunted the activity of PI3K/AKT/mTOR signaling cascade. In addition, MCL1 and BCL-XL, two well-characterized contributors for venetoclax resistance, were significantly lessened in the presence of CS2164 and venetoclax, thus leading to the accumulation of proapoptotic proteins BAX and PUMA and then initiating the intrinsic apoptosis pathway. Taken together, these findings suggest that the regimen of CS2164 and venetoclax is highly effective to eliminate HGBL-DHL cells in the preclinical setting, warranting further clinical investigations of this regimen for the treatment of unfavorable HGBL-DHL patients.
RESUMO
Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities (MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2-targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL-MYC/BCL2-DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC/BCL2/TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. IMPLICATIONS: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg.
Assuntos
Perfilação da Expressão Gênica/métodos , Linfoma Difuso de Grandes Células B/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Linhagem Celular Tumoral , Humanos , Linfoma Difuso de Grandes Células B/patologiaRESUMO
'Cancer stem cells' or 'tumour initiating cells' in B-cell non-Hodgkin lymphomas have not been demonstrated, although some studies focused on other cancer types suggest that such populations exist and represent tumour cells resistant to therapy and involved in relapse. These cells may also represent a putative neoplastic 'cell of origin' in lymphomas, but there is little substantive data to support this suggestion. Using cell lines derived from a recently established murine IL-14alphax c-Myc double transgenic/mantle cell lymphoma-blastoid variant model, heretofore referred to as DTG cell lines, we identified a subset of cells within the side population (SP) with features of 'tumour-initiating cells'. These features include higher expression of ABCG2 and BCL-2, longer telomere length, greater self-renewal ability and higher in vitro clonogenic and in vivo tumorigenic capacities compared with non-SP. In addition, in vitro viability studies demonstrated that the non-SP lymphoma subpopulation has a limited lifespan in comparison with the SP fraction. Syngenic transplant studies showed that non-SP derived tumours, in comparison to the SP-derived tumours, exhibit greater necrosis/apoptosis and less systemic dissemination capability. In conclusion, our data support the interpretation that the DTG SP fraction contains a cell population highly capable of tumour maintenance and systemic dissemination and lends support to the concept that 'tumour-initiating cells' occur in lymphomas.
Assuntos
Linfoma de Célula do Manto/patologia , Células-Tronco Neoplásicas/patologia , ADP-Ribosil Ciclase 1/metabolismo , Aneuploidia , Animais , Antígenos CD34/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Células Clonais , Modelos Animais de Doenças , Linfoma de Célula do Manto/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fase S , Frações Subcelulares/metabolismo , Telômero/metabolismoRESUMO
The XPO1 inhibitor selinexor was recently approved in relapsed/refractory DLBCL patients but only demonstrated modest anti-DLBCL efficacy, prompting us to investigate the prognostic effect of XPO1 in DLBCL patients and the rational combination therapies in high-risk DLBCL. High XPO1 expression (XPO1high) showed significant adverse prognostic impact in 544 studied DLBCL patients, especially in those with BCL2 overexpression. Therapeutic study in 30 DLBCL cell lines with various molecular and genetic background found robust cytotoxicity of selinexor, especially in cells with BCL2-rearranged (BCL2-R+) DLBCL or high-grade B-cell lymphoma with MYC/BCL2 double-hit (HGBCL-DH). However, expression of mutant (Mut) p53 significantly reduced the cytotoxicity of selinexor in overall cell lines and the BCL2-R and HGBCL-DH subsets, consistent with the favorable impact of XPO1high observed in Mut-p53-expressing patients. The therapeutic effect of selinexor in HGBCL-DH cells was significantly enhanced when combined with a BET inhibitor INCB057643, overcoming the drug resistance in Mut-p53-expressing cells. Collectively, these data suggest that XPO1 worsens the survival of DLBCL patients with unfavorable prognostic factors such as BCL2 overexpression and double-hit, in line with the higher efficacy of selinexor demonstrated in BCL2-R+ DLBCL and HGBCL-DH cell lines. Expression of Mut-p53 confers resistance to selinexor treatment, which can be overcome by combined INCB057643 treatment in HGBCL-DH cells. This study provides insight into the XPO1 significance and selinexor efficacy in DLBCL, important for developing combination therapy for relapsed/refractory DLBCL and HGBCL-DH.
Assuntos
Antineoplásicos/uso terapêutico , Hidrazinas/uso terapêutico , Carioferinas/antagonistas & inibidores , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/uso terapêutico , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Carioferinas/genética , Linfoma Difuso de Grandes Células B/diagnóstico , Prognóstico , Receptores Citoplasmáticos e Nucleares/genética , Proteína Supressora de Tumor p53/genética , Proteína Exportina 1RESUMO
"Double-hit lymphomas with MYC and BCL2 translocations can be effectively treated by combined targeting of the driver oncogenes".
RESUMO
Double-hit lymphoma (DHL) is among the most aggressive and chemoresistant lymphoma subtypes. DHLs carry genomic abnormalities in MYC, BCL2, and/or BCL6 oncogenes. Due to the simultaneous overexpression of these driver oncogenes, DHLs are highly resistant to frontline therapies. Most DHLs overexpress both MYC and BCL2 driver oncogenes concurrently. We reasoned that simultaneous suppression of the two driver oncogenes would be more effective in eradicating DHLs than inactivation of single oncogene. XPO1 is a receptor for nuclear cytoplasmic transport of protein and RNA species. Recently, XPO1 inhibition was shown to downregulate MYC expression in several cancer cell lines. We therefore examined the role of XPO1 as a therapeutic target in suppressing MYC function and the potential synergistic effects of simultaneous suppression of XPO1 and BCL2 in the treatment of DHL. Here, we demonstrate that XPO1 inhibition abrogates MYC protein expression and induces massive tumor cell apoptosis. Combined use of XPO1 and BCL2 inhibitors is highly effective in eradicating DHL cells in cell culture. Notably, in a mouse model of DHL bearing primary tumor cells derived from lymphoma patients, combined treatment with XPO1 and BCL2 inhibitors blocks tumor progression, prevents brain metastasis, and extends host survival. Thus, our study confirms the simultaneous targeting of MYC and BCL2 driver oncogenes through the combined use of XPO1 and BCL2 inhibitors as a unique approach for the treatment of DHLs.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Carioferinas/antagonistas & inibidores , Linfoma/tratamento farmacológico , Linfoma/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Rearranjo Gênico , Humanos , Carioferinas/genética , Linfoma/patologia , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores Citoplasmáticos e Nucleares/genética , Translocação Genética , Proteína Exportina 1RESUMO
Metabolic reprogramming is linked to cancer cell growth and proliferation, metastasis, and therapeutic resistance in a multitude of cancers. Targeting dysregulated metabolic pathways to overcome resistance, an urgent clinical need in all relapsed/refractory cancers, remains difficult. Through genomic analyses of clinical specimens, we show that metabolic reprogramming toward oxidative phosphorylation (OXPHOS) and glutaminolysis is associated with therapeutic resistance to the Bruton's tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma (MCL), a B cell lymphoma subtype with poor clinical outcomes. Inhibition of OXPHOS with a clinically applicable small molecule, IACS-010759, which targets complex I of the mitochondrial electron transport chain, results in marked growth inhibition in vitro and in vivo in ibrutinib-resistant patient-derived cancer models. This work suggests that targeting metabolic pathways to subvert therapeutic resistance is a clinically viable approach to treat highly refractory malignancies.
Assuntos
Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/metabolismo , Terapia de Alvo Molecular , Fosforilação Oxidativa , Adenina/análogos & derivados , Animais , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Linfoma de Célula do Manto/genética , Camundongos , Mutação/genética , Fosforilação Oxidativa/efeitos dos fármacos , Piperidinas , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/genética , Sequenciamento do ExomaRESUMO
PD-1/L1 and CTLA-4 blockade immunotherapies have been approved for 13 types of cancers and are being studied in diffuse large B-cell lymphoma (DLBCL), the most common aggressive B-cell lymphoma. However, whether both PD-1 and CTLA-4 checkpoints are active and clinically significant in DLBCL is unknown. Whether PD-1 ligands expressed by tumor cells or by the microenvironment of DLBCL are critical for the PD-1 immune checkpoint is unclear. We performed immunophenotypic profiling for 405 patients with de novo DLBCL using a MultiOmyx immunofluorescence platform and simultaneously quantitated expression/coexpression of 13 immune markers to identify prognostic determinants. In both training and validation cohorts, results demonstrated a central role of the tumor immune microenvironment, and when its functionality was impaired by deficiency in tumor-infiltrating T cells and/or natural killer cells, high PD-1 expression (but not CTLA-4) on CD8+ T cells, or PD-L1 expression on T cells and macrophages, patients had significantly poorer survival after rituximab-CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) immunochemotherapy. In contrast, tumor-cell PD-L2 expression was associated with superior survival, as well as PD-L1+CD20+ cells proximal (indicates interaction) to PD-1 + CD8+ T cells in patients with low PD-1 + percentage of CD8+ T cells. Gene-expression profiling results suggested the reversibility of T-cell exhaustion in PD-1+/PD-L1+ patients with unfavorable prognosis and implication of LILRA/B, IDO1, CHI3L1, and SOD2 upregulation in the microenvironment dysfunction with PD-L1 expression. This study comprehensively characterized the DLBCL immune landscape, deciphered the differential roles of various checkpoint components in rituximab-CHOP resistance in DLBCL patients, and suggests targets for PD-1/PD-L1 blockade and combination immunotherapies.
Assuntos
Antígeno B7-H1/imunologia , Antígeno CTLA-4/imunologia , Linfoma Difuso de Grandes Células B/imunologia , Receptor de Morte Celular Programada 1/imunologia , Microambiente Tumoral/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Células Matadoras Naturais/imunologia , Pessoa de Meia-Idade , Fenótipo , Prednisona/uso terapêutico , Prognóstico , Rituximab/uso terapêutico , Linfócitos T/imunologia , Vincristina/uso terapêuticoRESUMO
Macrophages (MPs) are heterogeneous, multifunctional, myeloid-derived leukocytes that are part of the innate immune system, playing wide-ranging critical roles in basic biological activities, including maintenance of tissue homeostasis involving clearance of microbial pathogens. Tumor-associated MPs (TAMs) are MPs with defined specific M2 phenotypes now known to play central roles in the pathophysiology of a wide spectrum of malignant neoplasms. Also, TAMs are often intrinsic cellular components of the essential tumor microenvironment (TME). In concert with lymphoid-lineage B and T cells at various developmental stages, TAMs can mediate enhanced tumor progression, often leading to poor clinical prognosis, at least partly through secretion of chemokines, cytokines, and various active proteases shown to stimulate tumor growth, angiogenesis, metastasis, and immunosuppression. Researchers recently showed that TAMs express certain key checkpoint-associated proteins [e.g., programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1)] that appear to be involved in T-cell activation and that these proteins are targets of other specific checkpoint-blocking immunotherapies (anti-PD-1/PD-L1) currently part of new therapeutic paradigms for chemotherapy-resistant neoplasms. Although much is known about the wide spectrum and flexibility of MPs under many normal and neoplastic conditions, relatively little is known about the increasingly important interactions between MPs and B-lymphoid cells, particularly in the TME in patients with aggressive B-cell non-Hodgkin lymphoma (NHL-B). Normal and neoplastic lymphoid and myeloid cell/MP lineages appear to share many primitive cellular characteristics as well as transcriptional factor interactions in human and animal ontogenic studies. Such cells are capable of ectopic transcription factor-induced lineage reprogramming or transdifferentiation from early myeloid/monocytic lineages to later induce B-cell lymphomagenesis in experimental in vivo murine systems. Close cellular interactions between endogenous clonal neoplastic B cells and related aberrant myeloid precursor cells/MPs appear to be important interactive components of aggressive NHL-B that we discuss herein in the larger context of the putative role of B-cell/MP cellular lineage interactions involved in NHL-B pathophysiology during ensuing lymphoma development.
RESUMO
Purpose: B-cell lymphoma-2 (BCL-2), an antiapoptotic protein often dysregulated in B-cell lymphomas, promotes cell survival and provides protection from stress. A recent phase I first-in-human study of the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma showed an overall response rate of 44%. These promising clinical results prompted our examination of the biological effects and mechanism of action underlying venetoclax activity in aggressive B-cell lymphoma, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL).Experimental Design: MCL and DLBCL cell lines, primary patient samples, and in vivo patient-derived xenograft (PDX) models were utilized to examine venetoclax efficacy. Furthermore, the mechanisms underlying venetoclax response and the development of venetoclax resistance were evaluated using proteomics analysis and Western blotting.Results: Potential biomarkers linked to venetoclax activity and targeted combination therapies that can augment venetoclax response were identified. We demonstrate that DLBCL and MCL cell lines, primary patient samples, and PDX mouse models expressing high BCL-2 levels are extremely sensitive to venetoclax treatment. Proteomics studies showed that venetoclax substantially alters the expression levels and phosphorylation status of key proteins involved in cellular processes, including the DNA damage response, cell metabolism, cell growth/survival, and apoptosis. Short- and long-term exposure to venetoclax inhibited PTEN expression, leading to enhanced AKT pathway activation and concomitant susceptibility to PI3K/AKT inhibition. Intrinsic venetoclax-resistant cells possess high AKT activation and are highly sensitive to PI3K/AKT inhibition.Conclusions: These findings demonstrate the on-target effect of venetoclax and offer potential mechanisms to overcome acquired and intrinsic venetoclax resistance through PI3K/AKT inhibition. Clin Cancer Res; 24(16); 3967-80. ©2018 AACR.