RESUMO
Li et al. (2020) elucidate the resistance mechanisms to small-molecule inhibitors targeting the G2/M cell cycle checkpoint kinase, CHK1, in a variety of non-small cell lung cancer cell lines using CRISPR-mediated genetic approaches and identify biomarkers of response.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Fosforilação , Proteínas Quinases/metabolismoRESUMO
Malignant transformation of cells typically involves several genetic lesions, whose combined activity gives rise to cancer1. Here we analyse 1,148 patient-derived B-cell leukaemia (B-ALL) samples, and find that individual mutations do not promote leukaemogenesis unless they converge on one single oncogenic pathway that is characteristic of the differentiation stage of transformed B cells. Mutations that are not aligned with this central oncogenic driver activate divergent pathways and subvert transformation. Oncogenic lesions in B-ALL frequently mimic signalling through cytokine receptors at the pro-B-cell stage (via activation of the signal-transduction protein STAT5)2-4 or pre-B-cell receptors in more mature cells (via activation of the protein kinase ERK)5-8. STAT5- and ERK-activating lesions are found frequently, but occur together in only around 3% of cases (P = 2.2 × 10-16). Single-cell mutation and phospho-protein analyses reveal the segregation of oncogenic STAT5 and ERK activation to competing clones. STAT5 and ERK engage opposing biochemical and transcriptional programs that are orchestrated by the transcription factors MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway comes at the expense of the principal oncogenic driver and reverses transformation. Conversely, deletion of divergent pathway components accelerates leukaemogenesis. Thus, persistence of divergent signalling pathways represents a powerful barrier to transformation, while convergence on one principal driver defines a central event in leukaemia initiation. Pharmacological reactivation of suppressed divergent circuits synergizes strongly with inhibition of the principal oncogenic driver. Hence, reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to enhance treatment responses.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Transformação Celular Neoplásica , Leucemia de Células B/metabolismo , Leucemia de Células B/patologia , Transdução de Sinais , Animais , Linfócitos B/patologia , Linhagem Celular Tumoral , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT5/metabolismoRESUMO
Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis.
Assuntos
Transformação Celular Viral , Infecções por Vírus Epstein-Barr/patologia , Herpesvirus Humano 4/patogenicidade , Linfoma de Células B/patologia , Plasmócitos/patologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Fibroblastos , Herpesvirus Humano 4/metabolismo , Humanos , Linfoma de Células B/virologia , Camundongos , Camundongos Knockout , Plasmócitos/virologia , Cultura Primária de Células , Transativadores/genética , Transativadores/metabolismo , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/metabolismoRESUMO
Hereditary Cancer makes up around 5-10% of all cancers. It is important to diagnose hereditary cancer in a timely fashion, as not only do patients require long-term care from a young age, but their relatives also require management. The main approach to capture at-risk relatives is cascade testing. It involves genetic testing of relatives of the first detected carrier of a pathogenic variant in a family i.e. the proband. The current standard of care for cascade testing is a patient-mediated approach. Probands are then advised to inform and encourage family members to undergo genetic testing. In Singapore, cascade testing is inefficient, around 10-15%, lower than the 30% global average. Here, we describe the cascade testing process and its effort to increase testing in Singapore. Precision Health Research, Singapore (PRECISE), was set up to coordinate Singapore's National Precision Medicine strategy and has awarded five clinical implementation pilots, with one of them seeking to identify strategies for how cascade testing for hereditary cancer can be increased in a safe and cost-efficient manner. Achieving this will be done through addressing barriers such as cost, manpower shortages, exploring a digital channel for contacting at-risk relatives, and getting a deeper insight into why genetic testing gets declined. If successful, it will likely result in care pathways that are a cost-effective public health intervention for identifying individuals at risk. Surveillance and management of those unaffected at-risk individuals, if caught early, will result in improved patient outcomes, and further reduce the healthcare burden for the economy.
Assuntos
Predisposição Genética para Doença , Testes Genéticos , Genômica , Humanos , Singapura , Testes Genéticos/métodos , Genômica/métodos , Neoplasias/genética , Neoplasias/diagnóstico , Medicina de Precisão/métodos , Política de Saúde , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/diagnósticoRESUMO
Molecular subtypes of Small Cell Lung Cancer (SCLC) have been described based on differential expression of transcription factors (TFs) ASCL1, NEUROD1, POU2F3 and immune-related genes. We previously reported an additional subtype based on expression of the neurogenic TF ATOH1 within our SCLC Circulating tumour cell-Derived eXplant (CDX) model biobank. Here we show that ATOH1 protein was detected in 7/81 preclinical models and 16/102 clinical samples of SCLC. In CDX models, ATOH1 directly regulated neurogenesis and differentiation programs consistent with roles in normal tissues. In ex vivo cultures of ATOH1-positive CDX, ATOH1 was required for cell survival. In vivo, ATOH1 depletion slowed tumour growth and suppressed liver metastasis. Our data validate ATOH1 as a bona fide oncogenic driver of SCLC with tumour cell survival and pro-metastatic functions. Further investigation to explore ATOH1 driven vulnerabilities for targeted treatment with predictive biomarkers is warranted.
RESUMO
INTRODUCTION: Although targeted therapies have revolutionized the therapeutic landscape of lung adenocarcinomas (LUADs), disease progression on single-agent targeted therapy against known oncogenic drivers is common, and therapeutic options after disease progression are limited. In patients with MDM2 amplification (MDM2amp) and a concurrent oncogenic driver alteration, we hypothesized that targeting of the tumor-suppressor pathway (by means of restoration of p53 using MDM2 inhibition) and simultaneous targeting of co-occurring MAPK oncogenic pathway might represent a more durably effective therapeutic strategy. METHODS: We evaluated genomic next-generation sequencing data using the Memorial Sloan Kettering Cancer Center-Integrated Mutation Profiling of Actionable Cancer Targets platform to nominate potential targets for combination therapy in LUAD. We investigated the small molecule MDM2 inhibitor milademetan in cell lines and patient-derived xenografts of LUAD with a known driver alteration and MDM2amp. RESULTS: Of 10,587 patient samples from 7121 patients with LUAD profiled by next-generation sequencing, 6% (410 of 7121) harbored MDM2amp. MDM2amp was significantly enriched among tumors with driver alterations in METex14 (36%, p < 0.001), EGFR (8%, p < 0.001), RET (12%, p < 0.01), and ALK (10%, p < 0.01). The combination of milademetan and the MEK inhibitor trametinib was synergistic in growth inhibition of ECLC5-GLx (TRIM33-RET/MDM2amp), LUAD12c (METex14/KRASG12S/MDM2amp), SW1573 (KRASG12C, TP53 wild type), and A549 (KRASG12S) cells and in increasing expression of proapoptotic proteins PUMA and BIM. Treatment of ECLC5-GLx and LUAD12c with single-agent milademetan increased ERK phosphorylation, consistent with previous data on ERK activation with MDM2 inhibition. This ERK activation was effectively suppressed by concomitant administration of trametinib. In contrast, ERK phosphorylation induced by milademetan was not suppressed by concurrent RET inhibition using selpercatinib (in ECLC5-GLx) or MET inhibition using capmatinib (in LUAD12c). In vivo, combination milademetan and trametinib was more effective than either agent alone in ECLC5-GLx, LX-285 (EGFRex19del/MDM2amp), L13BS1 (METex14/MDM2amp), and A549 (KRASG12S, TP53 wild type). CONCLUSIONS: Combined MDM2/MEK inhibition was found to have efficacy across multiple patient-derived LUAD models harboring MDM2amp and concurrent oncogenic drivers. This combination, potentially applicable to LUADs with a wide variety of oncogenic driver mutations and kinase fusions activating the MAPK pathway, has evident clinical implications and will be investigated as part of a planned phase 1/2 clinical trial.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Mutação , Progressão da Doença , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Proteínas Proto-Oncogênicas c-mdm2/genética , Fatores de Transcrição/genéticaRESUMO
Multiple myeloma (MM) shows constitutive activation of canonical and noncanonical nuclear factor κB (NF-κB) signaling via genetic mutations or tumor microenvironment (TME) stimulations. A subset of MM cell lines showed dependency for cell growth and survival on the canonical NF-κB transcription factor RELA alone, suggesting a critical role for a RELA-mediated biological program in MM pathogenesis. Here, we determined the RELA-dependent transcriptional program in MM cell lines and found the expression of the cell surface molecules interleukin-27 receptor-α (IL-27Rα) and the adhesion molecule JAM2 to be responsive to RELA at the messenger RNA and protein levels. IL-27Rα and JAM2 were expressed on primary MM cells at higher levels than on healthy long-lived plasma cells (PCs) in the bone marrow. IL-27 activated STAT1, and to a lesser extent STAT3, in MM cell lines and in PCs generated from memory B cells in an IL-21-dependent in vitro PC differentiation assay. Concomitant activity of IL-21 and IL-27 enhanced differentiation into PCs and increased the cell-surface expression of the known STAT target gene CD38. In accordance, a subset of MM cell lines and primary MM cells cultured with IL-27 upregulated CD38 cell-surface expression, a finding with potential implications for enhancing the efficacy of CD38-directed monoclonal antibody therapies by increasing CD38 expression on tumor cells. The elevated expression of IL-27Rα and JAM2 on MM cells compared with that on healthy PCs may be exploited for the development of targeted therapeutic strategies that modulate the interaction of MM cells with the TME.
Assuntos
Interleucina-27 , Mieloma Múltiplo , Humanos , Interleucina-27/metabolismo , Mieloma Múltiplo/genética , NF-kappa B/metabolismo , Receptores de Citocinas/metabolismo , Microambiente Tumoral , Regulação para CimaRESUMO
Access to clinically relevant small cell lung cancer (SCLC) tissue is limited because surgical resection is rare in metastatic SCLC. Patient-derived xenografts (PDX) and circulating tumor cell-derived xenografts (CDX) have emerged as valuable tools to characterize SCLC. Here, we present a resource of 46 extensively annotated PDX/CDX models derived from 33 patients with SCLC. We perform multi-omic analyses, using targeted tumor next-generation sequencing, RNA-sequencing, and immunohistochemistry to deconvolute the mutational landscapes, global expression profiles, and molecular subtypes of these SCLC models. SCLC subtypes characterized by transcriptional regulators, ASCL1, NEUROD1 and POU2F3 are confirmed in this cohort. A subset of SCLC clinical specimens, including matched PDX/CDX and clinical specimen pairs, confirm that the primary features and genomic and proteomic landscapes of the tumors of origin are preserved in the derivative PDX models. This resource provides a powerful system to study SCLC biology.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteômica , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Transcriptoma/genéticaRESUMO
Small cell lung cancer (SCLC) is characterized by morphologic, epigenetic and transcriptomic heterogeneity. Subtypes based upon predominant transcription factor expression have been defined that, in mouse models and cell lines, exhibit potential differential therapeutic vulnerabilities, with epigenetically distinct SCLC subtypes also described. The clinical relevance of these subtypes is unclear, due in part to challenges in obtaining tumor biopsies for reliable profiling. Here we describe a robust workflow for genome-wide DNA methylation profiling applied to both patient-derived models and to patients' circulating cell-free DNA (cfDNA). Tumor-specific methylation patterns were readily detected in cfDNA samples from patients with SCLC and were correlated with survival outcomes. cfDNA methylation also discriminated between the transcription factor SCLC subtypes, a precedent for a liquid biopsy cfDNA-methylation approach to molecularly subtype SCLC. Our data reveal the potential clinical utility of cfDNA methylation profiling as a universally applicable liquid biopsy approach for the sensitive detection, monitoring and molecular subtyping of patients with SCLC.
Assuntos
Ácidos Nucleicos Livres , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Animais , Camundongos , Ácidos Nucleicos Livres/genética , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Epigenoma/genética , Metilação de DNA/genética , Neoplasias Pulmonares/diagnóstico , Fatores de Transcrição/genéticaRESUMO
Small cell lung cancers (SCLCs) have high mutational burden but are relatively unresponsive to immune checkpoint blockade (ICB). Using SCLC models, we demonstrate that inhibition of WEE1, a G2/M checkpoint regulator induced by DNA damage, activates the STING-TBK1-IRF3 pathway, which increases type I interferons (IFN-α and IFN-ß) and pro-inflammatory chemokines (CXCL10 and CCL5), facilitating an immune response via CD8+ cytotoxic T cell infiltration. We further show that WEE1 inhibition concomitantly activates the STAT1 pathway, increasing IFN-γ and PD-L1 expression. Consistent with these findings, combined WEE1 inhibition (AZD1775) and PD-L1 blockade causes remarkable tumor regression, activation of type I and II interferon pathways, and infiltration of cytotoxic T cells in multiple immunocompetent SCLC genetically engineered mouse models, including an aggressive model with stabilized MYC. Our study demonstrates cell-autonomous and immune-stimulating activity of WEE1 inhibition in SCLC models. Combined inhibition of WEE1 plus PD-L1 blockade represents a promising immunotherapeutic approach in SCLC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Antígeno B7-H1 , Proteínas de Ciclo Celular , Neoplasias Pulmonares , Proteínas de Membrana , Proteínas Tirosina Quinases , Fator de Transcrição STAT1 , Carcinoma de Pequenas Células do Pulmão , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT1/metabolismo , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologiaRESUMO
Next-generation sequencing has transformed our knowledge of the genetics of lymphoid malignancies. However, limited experimental systems are available to model the functional effects of these genetic changes and their implications for therapy. The majority of mature B-cell malignancies arise from the germinal center (GC) stage of B-cell differentiation. Here we describe a detailed protocol for the purification and ex vivo expansion of primary, nonmalignant human GC B cells. We present methodology for the high-efficiency transduction of these cells to enable combinatorial expression of putative oncogenes. We also describe alternative approaches for CRISPR-Cas9-mediated deletion of putative tumor suppressors. Mimicking genetic changes commonly found in lymphoid malignancies leads to immortalized growth in vitro, while engraftment into immunodeficient mice generates genetically customized, synthetic models of human lymphoma. The protocol is simple and inexpensive and can be implemented in any laboratory with access to standard cell culture and animal facilities. It can be easily scaled up to enable high-throughput screening and thus provides a versatile platform for the functional interrogation of lymphoma genomic data.
Assuntos
Linfócitos B/metabolismo , Técnicas de Cultura de Células/métodos , Técnicas Genéticas , Centro Germinativo/citologia , Linfócitos B/citologia , Sistemas CRISPR-Cas , Proliferação de Células/genética , Deleção de Genes , Genômica , HumanosRESUMO
Activation of mitogenic signaling pathways is a common oncogenic driver of many solid tumors including lung cancer. Although activating mutations in the mitogen-activated protein kinase (MAPK) pathway are prevalent in non-small cell lung cancers, MAPK pathway activity, counterintuitively, is relatively suppressed in the more aggressively proliferative small cell lung cancer (SCLC). Here, we elucidate the role of the MAPK pathway and how it interacts with other signaling pathways in SCLC. We find that the most common SCLC subtype, SCLC-A associated with high expression of ASCL1, is selectively sensitive to MAPK activation in vitro and in vivo through induction of cell-cycle arrest and senescence. We show strong upregulation of ERK negative feedback regulators and STAT signaling upon MAPK activation in SCLC-A lines. These findings provide insight into the complexity of signaling networks in SCLC and suggest subtype-specific mitogenic vulnerabilities.
RESUMO
Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf-Mycl-driven mouse model of SCLC. RLF-MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF-MYCL genetically engineered mouse model displayed gene expression similarities with human RLF-MYCL SCLC. Together, our studies support RLF-MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. SIGNIFICANCE: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF-MYCL gene fusion by developing a Rlf-Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis. This article is highlighted in the In This Issue feature, p. 2945.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Fusão Gênica , Genes myc , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Proto-Oncogênicas c-myc , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Proteínas de Ligação a TelômerosRESUMO
Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models.
Assuntos
Linfócitos B/patologia , Linfoma Difuso de Grandes Células B/genética , Cultura Primária de Células/métodos , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/genética , Técnicas de Cocultura/métodos , Vetores Genéticos/genética , Centro Germinativo/citologia , Ensaios de Triagem em Larga Escala , Humanos , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Gradação de Tumores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-myc/genética , Retroviridae/genética , Transdução Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Studying mechanisms of malignant transformation of human pre-B cells, we found that acute activation of oncogenes induced immediate cell death in the vast majority of cells. Few surviving pre-B cell clones had acquired permissiveness to oncogenic signaling by strong activation of negative feedback regulation of Erk signaling. Studying negative feedback regulation of Erk in genetic experiments at three different levels, we found that Spry2, Dusp6, and Etv5 were essential for oncogenic transformation in mouse models for pre-B acute lymphoblastic leukemia (ALL). Interestingly, a small molecule inhibitor of DUSP6 selectively induced cell death in patient-derived pre-B ALL cells and overcame conventional mechanisms of drug-resistance.