RESUMO
Invasive lobular carcinoma (ILC) is the second most prevalent histologic subtype of invasive breast cancer. Here, we comprehensively profiled 817 breast tumors, including 127 ILC, 490 ductal (IDC), and 88 mixed IDC/ILC. Besides E-cadherin loss, the best known ILC genetic hallmark, we identified mutations targeting PTEN, TBX3, and FOXA1 as ILC enriched features. PTEN loss associated with increased AKT phosphorylation, which was highest in ILC among all breast cancer subtypes. Spatially clustered FOXA1 mutations correlated with increased FOXA1 expression and activity. Conversely, GATA3 mutations and high expression characterized luminal A IDC, suggesting differential modulation of ER activity in ILC and IDC. Proliferation and immune-related signatures determined three ILC transcriptional subtypes associated with survival differences. Mixed IDC/ILC cases were molecularly classified as ILC-like and IDC-like revealing no true hybrid features. This multidimensional molecular atlas sheds new light on the genetic bases of ILC and provides potential clinical options.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Antígenos CD , Neoplasias da Mama/metabolismo , Caderinas/química , Caderinas/genética , Caderinas/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/metabolismo , Feminino , Fator 3-alfa Nuclear de Hepatócito/química , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Modelos Moleculares , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Oncogênica v-akt/metabolismo , TranscriptomaRESUMO
Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.
Assuntos
Genótipo , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Neoplasias/genética , Neoplasias/patologia , Transcriptoma/genética , Animais , Antígenos CD34/metabolismo , Calreticulina/genética , Linhagem Celular , Proliferação de Células , Células Clonais/classificação , Células Clonais/metabolismo , Células Clonais/patologia , Endorribonucleases/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Modelos Moleculares , Transtornos Mieloproliferativos/classificação , NF-kappa B/metabolismo , Neoplasias/classificação , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Mielofibrose Primária/genética , Mielofibrose Primária/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Resposta a Proteínas não Dobradas/genéticaRESUMO
Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer1. Chronic lymphocytic leukaemia (CLL) is a highly informative model for cancer evolution as it undergoes substantial genetic diversification and evolution after therapy2,3. The CLL epigenome is also an important disease-defining feature4,5, and growing populations of cells in CLL diversify by stochastic changes in DNA methylation known as epimutations6. However, previous studies using bulk sequencing methods to analyse the patterns of DNA methylation were unable to determine whether epimutations affect CLL populations homogeneously. Here, to measure the epimutation rate at single-cell resolution, we applied multiplexed single-cell reduced-representation bisulfite sequencing to B cells from healthy donors and patients with CLL. We observed that the common clonal origin of CLL results in a consistently increased epimutation rate, with low variability in the cell-to-cell epimutation rate. By contrast, variable epimutation rates across healthy B cells reflect diverse evolutionary ages across the trajectory of B cell differentiation, consistent with epimutations serving as a molecular clock. Heritable epimutation information allowed us to reconstruct lineages at high-resolution with single-cell data, and to apply this directly to patient samples. The CLL lineage tree shape revealed earlier branching and longer branch lengths than in normal B cells, reflecting rapid drift after the initial malignant transformation and a greater proliferative history. Integration of single-cell bisulfite sequencing analysis with single-cell transcriptomes and genotyping confirmed that genetic subclones mapped to distinct clades, as inferred solely on the basis of epimutation information. Finally, to examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells that were preferentially expelled from the lymph node after treatment, marked by distinct transcriptional profiles. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of CLL and its evolution with therapy.
Assuntos
Linhagem da Célula , Epigênese Genética , Evolução Molecular , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Sequência de Bases , Relógios Biológicos , Linhagem da Célula/genética , Metilação de DNA , Epigenoma/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Taxa de Mutação , Análise de Sequência de RNA , Análise de Célula Única , Transcrição GênicaRESUMO
Transcription and pre-mRNA splicing are key steps in the control of gene expression and mutations in genes regulating each of these processes are common in leukaemia1,2. Despite the frequent overlap of mutations affecting epigenetic regulation and splicing in leukaemia, how these processes influence one another to promote leukaemogenesis is not understood and, to our knowledge, there is no functional evidence that mutations in RNA splicing factors initiate leukaemia. Here, through analyses of transcriptomes from 982 patients with acute myeloid leukaemia, we identified frequent overlap of mutations in IDH2 and SRSF2 that together promote leukaemogenesis through coordinated effects on the epigenome and RNA splicing. Whereas mutations in either IDH2 or SRSF2 imparted distinct splicing changes, co-expression of mutant IDH2 altered the splicing effects of mutant SRSF2 and resulted in more profound splicing changes than either mutation alone. Consistent with this, co-expression of mutant IDH2 and SRSF2 resulted in lethal myelodysplasia with proliferative features in vivo and enhanced self-renewal in a manner not observed with either mutation alone. IDH2 and SRSF2 double-mutant cells exhibited aberrant splicing and reduced expression of INTS3, a member of the integrator complex3, concordant with increased stalling of RNA polymerase II (RNAPII). Aberrant INTS3 splicing contributed to leukaemogenesis in concert with mutant IDH2 and was dependent on mutant SRSF2 binding to cis elements in INTS3 mRNA and increased DNA methylation of INTS3. These data identify a pathogenic crosstalk between altered epigenetic state and splicing in a subset of leukaemias, provide functional evidence that mutations in splicing factors drive myeloid malignancy development, and identify spliceosomal changes as a mediator of IDH2-mutant leukaemogenesis.
Assuntos
Processamento Alternativo/genética , Carcinogênese/genética , Epigênese Genética , Leucemia Mieloide Aguda/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Proteínas de Ligação a DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Isocitrato Desidrogenase/genética , Masculino , Mutação/genética , RNA Polimerase II/metabolismo , Fatores de Processamento de Serina-Arginina/genética , TranscriptomaRESUMO
The pathophysiology of neurodegenerative diseases is poorly understood and there are few therapeutic options. Neurodegenerative diseases are characterized by progressive neuronal dysfunction and loss, and chronic glial activation. Whether microglial activation, which is generally viewed as a secondary process, is harmful or protective in neurodegeneration remains unclear. Late-onset neurodegenerative disease observed in patients with histiocytoses, which are clonal myeloid diseases associated with somatic mutations in the RAS-MEK-ERK pathway such as BRAF(V600E), suggests a possible role of somatic mutations in myeloid cells in neurodegeneration. Yet the expression of BRAF(V600E) in the haematopoietic stem cell lineage causes leukaemic and tumoural diseases but not neurodegenerative disease. Microglia belong to a lineage of adult tissue-resident myeloid cells that develop during organogenesis from yolk-sac erythro-myeloid progenitors (EMPs) distinct from haematopoietic stem cells. We therefore hypothesized that a somatic BRAF(V600E) mutation in the EMP lineage may cause neurodegeneration. Here we show that mosaic expression of BRAF(V600E) in mouse EMPs results in clonal expansion of tissue-resident macrophages and a severe late-onset neurodegenerative disorder. This is associated with accumulation of ERK-activated amoeboid microglia in mice, and is also observed in human patients with histiocytoses. In the mouse model, neurobehavioural signs, astrogliosis, deposition of amyloid precursor protein, synaptic loss and neuronal death were driven by ERK-activated microglia and were preventable by BRAF inhibition. These results identify the fetal precursors of tissue-resident macrophages as a potential cell-of-origin for histiocytoses and demonstrate that a somatic mutation in the EMP lineage in mice can drive late-onset neurodegeneration. Moreover, these data identify activation of the MAP kinase pathway in microglia as a cause of neurodegeneration and this offers opportunities for therapeutic intervention aimed at the prevention of neuronal death in neurodegenerative diseases.
Assuntos
Células Precursoras Eritroides/patologia , Sistema de Sinalização das MAP Quinases , Mutação , Células Progenitoras Mieloides/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Células Clonais/enzimologia , Células Clonais/metabolismo , Células Clonais/patologia , Modelos Animais de Doenças , Células Precursoras Eritroides/enzimologia , Células Precursoras Eritroides/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Histiocitose/enzimologia , Histiocitose/genética , Histiocitose/metabolismo , Histiocitose/patologia , Humanos , Macrófagos/enzimologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Microglia/enzimologia , Microglia/metabolismo , Microglia/patologia , Mosaicismo , Células Progenitoras Mieloides/enzimologia , Células Progenitoras Mieloides/metabolismo , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismoRESUMO
BACKGROUND: Acute myeloid leukemia (AML) with initial hyperleukocytosis is associated with high early mortality and a poor prognosis. The aims of this study were to delineate the underlying molecular landscape in the largest cytogenetic risk group, cytogenetically normal acute myeloid leukemia (CN-AML), and to assess the prognostic relevance of recurrent mutations in the context of hyperleukocytosis and clinical risk factors. METHODS: The authors performed a targeted sequencing of 49 recurrently mutated genes in 56 patients with newly diagnosed CN-AML and initial hyperleukocytosis of ≥100 G/L treated in the AMLCG99 study. The median number of mutated genes per patient was 5. The most common mutations occurred in FLT3 (73%), NPM1 (75%), and TET2 (45%). RESULTS: The predominant pathways affected by mutations were signaling (84% of patients), epigenetic modifiers (75% of patients), and nuclear transport (NPM1; 75%) of patients. AML with hyperleukocytosis was enriched for molecular subtypes that negatively affected the prognosis, including a high percentage of patients presenting with co-occurring mutations in signaling and epigenetic modifiers such as FLT3 internal tandem duplications and TET2 mutations. CONCLUSIONS: Despite these unique molecular features, clinical risk factors, including high white blood count, hemoglobin level, and lactate dehydrogenase level at baseline, remained the predictors for overall survival and relapse-free survival in hyperleukocytotic CN-AML.
Assuntos
Leucemia Mieloide Aguda , Proteínas Nucleares , Humanos , Proteínas Nucleares/genética , Nucleofosmina , Leucemia Mieloide Aguda/terapia , Mutação , Prognóstico , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Duodenal-type follicular lymphoma (DTFL) is a rare and highly indolent follicular lymphoma (FL) variant. It is morphologically and immunophenotypically indistinguishable from typical FL, characterized by restricted involvement of intestinal mucosa, and lacks extraintestinal manifestations. The molecular determinants of this distinct clinical behavior are largely unknown. Thirty-eight diagnostic biopsies from patients with DTFL were evaluated. The 10-year overall survival rate was 100% in clinically evaluable patients (n = 19). We compared the targeted mutation profile of DTFL (n = 31), limited-stage typical FL (LSTFL; n = 17), and advanced-stage typical FL (ASTFL; n = 241). The mutation frequencies of recurrently mutated genes, including CREBBP, TNFRSF14/HVEM, and EZH2 were not significantly different. However, KMT2D was less commonly mutated in DTFL (52%) and LSTFL (24%) as compared with ASTFL (79%). In ASTFL, 41% of KMT2D-mutated cases harbored multiple mutations in KMT2D, as compared with only 12% in LSTFL (P = .019) and 0% in DTFL (P < .0001). Whole exome and targeted sequencing of DTFL revealed high mutation frequencies of EEF1A1 (35%) and HVCN1 (22%). We compared the immune microenvironment gene expression signatures of DTFL (n = 8) and LSTFL (n = 7). DTFL clearly separated from LSTFL by unsupervised, hierarchical clustering of 147 chemokines and cytokines and was enriched for a chronic inflammation signature. In conclusion, the mutational landscape of DTFL is highly related to typical FL. The lower frequency of multiple mutations in KMT2D in DTFL and LSTFL indicates an increasing selection pressure for complete KMT2D loss in ASTFL pathogenesis. The highly dissimilar immune microenvironment of DTFL suggests a central role in the biology of this disease.
Assuntos
Biomarcadores Tumorais/genética , Proteínas de Ligação a DNA/genética , Neoplasias Duodenais/imunologia , Inflamação/imunologia , Linfoma Folicular/imunologia , Mutação , Proteínas de Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Citocinas/metabolismo , Análise Mutacional de DNA , Neoplasias Duodenais/genética , Neoplasias Duodenais/patologia , Exoma , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/patologia , Linfoma Folicular/genética , Linfoma Folicular/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Microambiente Tumoral , Adulto JovemRESUMO
BACKGROUND: Follicular lymphoma is a clinically and genetically heterogeneous disease, but the prognostic value of somatic mutations has not been systematically assessed. We aimed to improve risk stratification of patients receiving first-line immunochemotherapy by integrating gene mutations into a prognostic model. METHODS: We did DNA deep sequencing to retrospectively analyse the mutation status of 74 genes in 151 follicular lymphoma biopsy specimens that were obtained from patients within 1 year before beginning immunochemotherapy consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). These patients were recruited between May 4, 2000, and Oct 20, 2010, as part of a phase 3 trial (GLSG2000). Eligible patients had symptomatic, advanced stage follicular lymphoma and were previously untreated. The primary endpoints were failure-free survival (defined as less than a partial remission at the end of induction, relapse, progression, or death) and overall survival calculated from date of treatment initiation. Median follow-up was 7·7 years (IQR 5·5-9·3). Mutations and clinical factors were incorporated into a risk model for failure-free survival using multivariable L1-penalised Cox regression. We validated the risk model in an independent population-based cohort of 107 patients with symptomatic follicular lymphoma considered ineligible for curative irradiation. Pretreatment biopsies were taken between Feb 24, 2004, and Nov 24, 2009, within 1 year before beginning first-line immunochemotherapy consisting of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP). Median follow-up was 6·7 years (IQR 5·7-7·6). FINDINGS: We established a clinicogenetic risk model (termed m7-FLIPI) that included the mutation status of seven genes (EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11), the Follicular Lymphoma International Prognostic Index (FLIPI), and Eastern Cooperative Oncology Group (ECOG) performance status. In the training cohort, m7-FLIPI defined a high-risk group (28%, 43/151) with 5-year failure-free survival of 38·29% (95% CI 25·31-57·95) versus 77·21% (95% CI 69·21-86·14) for the low-risk group (hazard ratio [HR] 4·14, 95% CI 2·47-6·93; p<0·0001; bootstrap-corrected HR 2·02), and outperformed a prognostic model of only gene mutations (HR 3·76, 95% CI 2·10-6·74; p<0·0001; bootstrap-corrected HR 1·57). The positive predictive value and negative predictive value for 5-year failure-free survival were 64% and 78%, respectively, with a C-index of 0·80 (95% CI 0·71-0·89). In the validation cohort, m7-FLIPI again defined a high-risk group (22%, 24/107) with 5-year failure-free survival of 25·00% (95% CI 12·50-49·99) versus 68·24% (58·84-79·15) in the low-risk group (HR 3·58, 95% CI 2·00-6·42; p<0.0001). The positive predictive value for 5-year failure-free survival was 72% and 68% for negative predictive value, with a C-index of 0·79 (95% CI 0·69-0·89). In the validation cohort, risk stratification by m7-FLIPI outperformed FLIPI alone (HR 2·18, 95% CI 1·21-3·92), and FLIPI combined with ECOG performance status (HR 2·03, 95% CI 1·12-3·67). INTERPRETATION: Integration of the mutational status of seven genes with clinical risk factors improves prognostication for patients with follicular lymphoma receiving first-line immunochemotherapy and is a promising approach to identify the subset at highest risk of treatment failure. FUNDING: Deutsche Krebshilfe, Terry Fox Research Institute.
Assuntos
Anticorpos Monoclonais Murinos/administração & dosagem , Imunoterapia , Linfoma Folicular/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Adulto , Idoso , Anticorpos Monoclonais Murinos/imunologia , Ciclofosfamida/administração & dosagem , Intervalo Livre de Doença , Doxorrubicina , Feminino , Humanos , Linfoma Folicular/genética , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Prednisona/administração & dosagem , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Resultado do Tratamento , Vincristina/administração & dosagemAssuntos
Linfoma Folicular/genética , Linfoma Folicular/terapia , Mutação , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Feminino , Humanos , Linfoma Folicular/mortalidade , Masculino , Pessoa de Meia-Idade , Prednisona/uso terapêutico , Rituximab , Análise de Sobrevida , Resultado do Tratamento , Vincristina/uso terapêutico , Adulto JovemRESUMO
Single-agent bortezomib, a potent, selective, and reversible inhibitor of the 26S proteasome, has demonstrated clinical efficacy in relapsed and refractory mantle cell lymphoma (MCL). Objective response is achieved in up to 45% of the MCL patients; however, complete remission rates are low and duration of response proved to be relatively short. These limitations may be overcome by combining proteasome inhibition with conventional chemotherapy. Rational combination treatment and schedules require profound knowledge of underlying molecular mechanisms. Here we show that single-agent bortezomib treatment of MCL cell lines leads to G2/M arrest and induction of apoptosis accompanied by downregulation of EIF4E and CCND1 mRNA but upregulation of p15(INK4B) and p21 mRNA. We further present synergistic efficacy of bortezomib combined with cytarabine in MCL cell lines. Interestingly this sequence-dependent synergistic effect was seen almost exclusively in combination with AraC, indicating that pretreatment with cytarabine, followed by proteasome inhibition, may be the preferred approach.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Ácidos Borônicos/administração & dosagem , Ácidos Borônicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linfoma de Célula do Manto/tratamento farmacológico , Pirazinas/administração & dosagem , Pirazinas/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Bortezomib , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Esquema de Medicação , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/farmacologia , Inibidores de ProteassomaRESUMO
Only a fraction of patients with cancer respond to immune checkpoint blockade (ICB) treatment, but current decision-making procedures have limited accuracy. In this study, we developed a machine learning model to predict ICB response by integrating genomic, molecular, demographic and clinical data from a comprehensively curated cohort (MSK-IMPACT) with 1,479 patients treated with ICB across 16 different cancer types. In a retrospective analysis, the model achieved high sensitivity and specificity in predicting clinical response to immunotherapy and predicted both overall survival and progression-free survival in the test data across different cancer types. Our model significantly outperformed predictions based on tumor mutational burden, which was recently approved by the U.S. Food and Drug Administration for this purpose1. Additionally, the model provides quantitative assessments of the model features that are most salient for the predictions. We anticipate that this approach will substantially improve clinical decision-making in immunotherapy and inform future interventions.
Assuntos
Inibidores de Checkpoint Imunológico , Neoplasias , Biomarcadores Tumorais/genética , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Estudos RetrospectivosRESUMO
We investigated the role of PRMT5 in myeloproliferative neoplasm (MPN) pathogenesis and aimed to elucidate key PRMT5 targets contributing to MPN maintenance. PRMT5 is overexpressed in primary MPN cells, and PRMT5 inhibition potently reduced MPN cell proliferation ex vivo. PRMT5 inhibition was efficacious at reversing elevated hematocrit, leukocytosis, and splenomegaly in a model of JAK2V617F+ polycythemia vera and leukocyte and platelet counts, hepatosplenomegaly, and fibrosis in the MPLW515L model of myelofibrosis. Dual targeting of JAK and PRMT5 was superior to JAK or PRMT5 inhibitor monotherapy, further decreasing elevated counts and extramedullary hematopoiesis in vivo. PRMT5 inhibition reduced expression of E2F targets and altered the methylation status of E2F1 leading to attenuated DNA damage repair, cell-cycle arrest, and increased apoptosis. Our data link PRMT5 to E2F1 regulatory function and MPN cell survival and provide a strong mechanistic rationale for clinical trials of PRMT5 inhibitors in MPN. SIGNIFICANCE: Expression of PRMT5 and E2F targets is increased in JAK2V617F+ MPN. Pharmacologic inhibition of PRMT5 alters the methylation status of E2F1 and shows efficacy in JAK2V617F/MPLW515L MPN models and primary samples. PRMT5 represents a potential novel therapeutic target for MPN, which is now being clinically evaluated.This article is highlighted in the In This Issue feature, p. 1611.
Assuntos
Fator de Transcrição E2F1/metabolismo , Redes Reguladoras de Genes/genética , Janus Quinase 2/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Humanos , Metilação , Mutação , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4+ T cells in vitro. Tumors with hyperactive CTSS showed increased CD4+ T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.
Assuntos
Apresentação de Antígeno/imunologia , Catepsinas/metabolismo , Linfoma Folicular/metabolismo , Microambiente Tumoral/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Citocinas/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Terapia de Imunossupressão , Linfoma Folicular/patologia , CamundongosRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.
Assuntos
Carcinoma Ductal Pancreático/genética , Mutação , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Splicing de RNA , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/terapia , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Terapêutica com RNAi/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Although gene expression following bortezomib treatment has been previously explored, direct effects of bortezomib-induced proteasome inhibition on protein level has not been analyzed so far. Using 2-D PAGE in five mantle cell lymphoma cell lines, we screened for cellular protein level alterations following treatment with 25 nM bortezomib for up to 4 h. Using MS, we identified 38 of the 41 most prominent reliably detected protein spots. Twenty-one were affected in all cell lines, whereas the remaining 20 protein spots were exclusively altered in sensitive cell lines. Western blot analysis was performed for 17 of the 38 identified proteins and 70.6% of the observed protein level alterations in 2-D gels was verified. All cell lines exhibited alterations of the cellular protein levels of heat shock-induced protein species (HSPA9, HSP7C, HSPA5, HSPD1), whereas sensitive cell lines also displayed altered cellular protein levels of energy metabolism (ATP5B, AK5, TPI1, ENO-1, ALDOC, GAPDH), RNA and transcriptional regulation (HNRPL, SFRS12) and cell division (NEBL, ACTB, SMC1A, C20orf23) as well as tumor suppressor genes (ENO-1, FH). These proteins clustered in a tight interaction network centered on the major cellular checkpoints TP53. The results were confirmed in primary mantle cell lymphoma, thus confirming the critical role of these candidate proteins of proteasome inhibition.
Assuntos
Ácidos Borônicos/farmacologia , Eletroforese em Gel Bidimensional , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linfoma de Célula do Manto , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Western Blotting , Bortezomib , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Chaperona BiP do Retículo Endoplasmático , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Mapeamento de Interação de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The proteasome inhibitor bortezomib exhibits antitumor activity in many malignancies including mantle cell lymphoma (MCL). Unfortunately, many patients fail to respond to treatment or become refractory. Hyperthermia is an effective chemosensitizer that in combination with some chemotherapeutic agents has shown clinical activity in phase II and III studies. The aim of this study was to use MCL cell lines to investigate the potential benefit of combining clinically relevant doses of bortezomib with two different thermal doses (41.8 degrees C/120 min and 44 degrees C/30 min) that mimic the heterogeneity of the temperature distributions achieved within tumors during hyperthermia. Treated tumor cells were assessed for proliferation using the WST-1 assay and for apoptosis by annexin V staining, while heat shock protein (HSP) levels were determined following western blot analysis. Our results demonstrated that MCL cell lines that are sensitive to bortezomib are also thermosensitive and have low basal expression of hsp27, whereas the bortezomib-resistant MCL cell line strongly expresses hsp27 and is thermoresistant. Interestingly, pre-treatment of MCL cell lines with heat at the two different thermal doses, and the transient elevation of hsp27 and hsp70, do not impair their primary sensitivity to bortezomib. Finally, we show that the concurrent treatment of heat and bortezomib results in additive killing in MCL cell lines.In conclusion, these results suggest that the application of bortezomib, under thermal conditions, in mantle cell lymphoma cells may be beneficial and warrants further investigation.
Assuntos
Ácidos Borônicos/uso terapêutico , Hipertermia Induzida , Linfoma de Célula do Manto/terapia , Pirazinas/uso terapêutico , Apoptose/efeitos dos fármacos , Bortezomib , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Humanos , Linfoma de Célula do Manto/tratamento farmacológicoRESUMO
We tested whether a single nucleotide polymorphism (SNP) that affects splicing of CD33 predicted response to treatment in adults with acute myeloid leukemia (AML) who received the novel CD33 antibody-drug conjugate SGN-CD33A. This genotype, for the CD33 splice site SNP rs12459419, was not associated with clinical response (30% CR/CRi in both groups), event-free survival, or overall survival.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Genótipo , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/imunologia , Leucemia Mieloide Aguda/imunologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Splicing de RNA , Taxa de Sobrevida , Resultado do TratamentoRESUMO
The Follicular Lymphoma (FL) International Prognostic Index (FLIPI) and FLIPI-2 are well-described clinical risk models. Age >60 years at diagnosis is a risk factor in both scores. Recently, we showed that older age is not associated with higher risk of disease progression or inferior treatment efficacy. Instead, shorter survival of older patients results mainly from an increased risk of nonrelapse deaths. This questions the value of age as a meaningful component of scores intended to predict disease-specific survival. The newly proposed PRIMA-prognostic index (PRIMA-PI) only includes ß2-microglobulin levels and bone marrow infiltration as risk factors. Here, we independently validate the PRIMA-PI in a clinical trial cohort of 475 patients with advanced FL who uniformly received cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, prednisone, and rituximab (R-CHOP) as frontline therapy. The PRIMA-PI separated 3 similar sized risk cohorts with 5-year progression-free survival (PFS) rates of 74%, 59%, and 39%, respectively (P < .0001). Furthermore, we compare the PRIMA-PI with the FLIPI and FLIPI-2. We demonstrate that the PRIMA-PI has the highest specificity to identify high-risk patients (80% for 5-year PFS) because of its superior risk stratification in patients >60 years (73% vs 33% [FLIPI] and 47% [FLIPI-2] for 5-year PFS). Thus, the PRIMA-PI is a promising clinical tool to stringently identify patients at highest risk of poor outcome after frontline R-CHOP for advanced FL, and is particularly useful in patients with older age. Further validation in non-R-CHOP treated cohorts is needed.
Assuntos
Fatores Etários , Linfoma Folicular/diagnóstico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Medula Óssea/patologia , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Humanos , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/mortalidade , Pessoa de Meia-Idade , Prednisona/uso terapêutico , Prognóstico , Intervalo Livre de Progressão , Medição de Risco , Fatores de Risco , Rituximab/uso terapêutico , Vincristina/uso terapêutico , Microglobulina beta-2/análiseRESUMO
RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We systematically interrogated RBP dependencies in human cancers using a comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining RNA splicing and AML survival. Genetic or pharmacologic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. The effects of RBM39 loss on splicing further resulted in preferential lethality of spliceosomal mutant AML, providing a strategy for treatment of AML bearing RBP splicing mutations.