RESUMO
Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2+ ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.
RESUMO
Capicua (CIC)-rearranged sarcomas are an aggressive subset of undifferentiated round cell sarcomas. CIC::DUX4, the proto-typical CIC fusion oncoprotein is associated with rapid clinical progression and chemotherapy resistance leading to poor clinical outcomes. Recent studies have identified additional CIC fusions (CIC::NUTM1, CIC::FOXO4, and CIC::LEUTX) that largely retain CIC-binding specificity but leverage C-terminal binding partners (NUTM1, FOXO4, and LEUTX) to potentially activate transcriptional programs that drive oncogenesis. Moreover, the recent development of preclinical models to study CIC::DUX4 sarcoma have advanced our understanding of the underlying biological mechanisms and uncovered key dependencies that can be translated into rational therapies. In this review, we will highlight these recent advancements in CIC-rearranged sarcoma biology with a vision for clinical translation to improve patient outcomes.
RESUMO
Rearrangements between genes can yield neomorphic fusions that drive oncogenesis. Fusion oncogenes are made up of fractional segments of the partner genes that comprise them, with each partner potentially contributing some of its own function to the nascent fusion oncoprotein. Clinically, fusion oncoproteins driving one diagnostic entity are typically clustered into a single molecular subset and are often treated a similar fashion. However, knowledge of where specific fusion breakpoints occur in partner genes, and the resulting retention of functional domains in the fusion, is an important determinant of fusion oncoprotein activity and may differ between patients. This study investigates this phenomena through the example of CIC::DUX4, a fusion between the transcriptional repressor capicua (CIC) and the double homeobox 4 gene (DUX4), which drives an aggressive subset of undifferentiated round cell sarcoma. Using a harmonized dataset of over 100 patient fusion breakpoints from the literature, we show that most bona fide CIC::DUX4 fusions retain the C1 domain, which is known to contribute to DNA binding by wild type CIC. Mechanistically, deletion or mutation of the C1 domain reduces, but does not eliminate, activation of CIC target genes by CIC::DUX4. We also find that expression of C1-deleted CIC::DUX4 is capable of exerting intermediate transformation-related phenotypes compared with those imparted by full-length CIC::DUX4, but was not sufficient for tumorigenesis in a subcutaneous mouse model. In summary, our results suggest a supercharging role for the C1 domain in the activity of CIC::DUX4.
RESUMO
CIC-DUX4 is a rare and understudied transcription factor fusion oncoprotein. CIC-DUX4 co-opts native gene targets to drive a lethal form of human sarcoma. The molecular underpinnings that lead to oncogenic reprograming and CIC-DUX4 sarcomagenesis remain largely undefined. Through an integrative ChIP and RNA-Seq analysis using patient-derived CIC-DUX4 cells, we define CIC-DUX4 mediated chromatin states and function. We show that CIC-DUX4 primarily localizes to proximal and distal cis-regulatory elements where it associates with active histone marks. Our findings nominate key signaling pathways and molecular targets that enable CIC-DUX4 to mediate tumor cell survival. Collectively, our data demonstrate how the CIC-DUX4 fusion oncoprotein impacts chromatin state and transcriptional responses to drive an oncogenic program in undifferentiated sarcoma. Significance: CIC-DUX4 sarcoma is a rare and lethal sarcoma that affects children, adolescent young adults, and adults. CIC-DUX4 sarcoma is associated with rapid metastatic dissemination and relative insensitivity to chemotherapy. There are no current standard-of-care therapies for CIC-DUX4 sarcoma leading to universally poor outcomes for patients. Through a deep mechanistic understanding of how the CIC-DUX4 fusion oncoprotein reprograms chromatin state and function, we aim to improve outcomes for CIC-DUX4 patients.
RESUMO
Human prostate cancer can result from chromosomal rearrangements that lead to aberrant ETS gene expression. The mechanisms that lead to fusion-independent ETS factor upregulation and prostate oncogenesis remain relatively unknown. Here, we show that two neighboring transcription factors, Capicua (CIC) and ETS2 repressor factor (ERF), which are co-deleted in human prostate tumors can drive prostate oncogenesis. Concurrent CIC and ERF loss commonly occur through focal genomic deletions at chromosome 19q13.2. Mechanistically, CIC and ERF co-bind the proximal regulatory element and mutually repress the ETS transcription factor, ETV1. Targeting ETV1 in CIC and ERF-deficient prostate cancer limits tumor growth. Thus, we have uncovered a fusion-independent mode of ETS transcriptional activation defined by concurrent loss of CIC and ERF.
Assuntos
Proteínas de Ligação a DNA , Próstata , Neoplasias da Próstata , Proteínas Repressoras , Fatores de Transcrição , Humanos , Masculino , Carcinogênese , Proteínas de Ligação a DNA/genética , Próstata/patologia , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Deleção de GenesRESUMO
Inactivation of Capicua (CIC) or upregulation of yes-associated protein 1, YAP1, leads to broad RAS-RAF-MEK-ERK inhibitor resistance and tumor progression in multiple human cancers. Despite these shared malignant phenotypes, it remains unclear whether CIC and YAP1 are mechanistically linked. Here, we show that the ERK-regulated transcription factor CIC can directly repress YAP1 expression through non-consensus GGAAGGAA DNA-binding motifs in a proximal YAP1 regulatory element. Through binding at GGAA repeats, CIC regulates YAP1 transcriptional output in both normal and human cancer cells. Silencing YAP1 in CIC-deficient cells restores MAPK inhibitor sensitivity and suppresses tumor growth. Thus, we uncover a molecular link between the MAPK-ERK effector CIC and YAP1 in human cells and established YAP inhibition as a strategy to target CIC-deficient cancers.
Assuntos
Neoplasias , Proteínas Repressoras , Carcinogênese/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines, we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells down-regulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors.
Assuntos
Glicólise , Neoplasias Pulmonares , Linhagem Celular Tumoral , Glicólise/genética , Humanos , Neoplasias Pulmonares/patologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
There are more than 70 distinct sarcomas, and this diversity complicates the development of precision-based therapeutics for these cancers. Prospective comprehensive genomic profiling could overcome this challenge by providing insight into sarcomas' molecular drivers. Through targeted panel sequencing of 7494 sarcomas representing 44 histologies, we identify highly recurrent and type-specific alterations that aid in diagnosis and treatment decisions. Sequencing could lead to refinement or reassignment of 10.5% of diagnoses. Nearly one-third of patients (31.7%) harbor potentially actionable alterations, including a significant proportion (2.6%) with kinase gene rearrangements; 3.9% have a tumor mutational burden ≥10 mut/Mb. We describe low frequencies of microsatellite instability (<0.3%) and a high degree of genome-wide loss of heterozygosity (15%) across sarcomas, which are not readily explained by homologous recombination deficiency (observed in 2.5% of cases). In a clinically annotated subset of 118 patients, we validate actionable genetic events as therapeutic targets. Collectively, our findings reveal the genetic landscape of human sarcomas, which may inform future development of therapeutics and improve clinical outcomes for patients with these rare cancers.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Sarcoma , Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Genômica , Humanos , Mutação , Estudos Prospectivos , Sarcoma/diagnóstico , Sarcoma/genética , Sarcoma/terapiaRESUMO
Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.
Assuntos
Fator X , Neoplasias Pulmonares , Apoptose/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Fator X/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Fatores de Processamento de RNA , RNA Mensageiro/genética , Motivos de Ligação ao RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
CIC-DUX4 rearrangements define an aggressive and chemotherapy-insensitive subset of undifferentiated sarcomas. The CIC-DUX4 fusion drives oncogenesis through direct transcriptional upregulation of cell cycle and DNA replication genes. Notably, CIC-DUX4-mediated CCNE1 upregulation compromises the G1/S transition to confer a dependence on the G2/M cell cycle checkpoint. Through an integrative transcriptional and kinase activity screen using patient-derived specimens, we now show that CIC-DUX4 sarcomas depend on the G2/M checkpoint regulator WEE1 as part of an adaptive survival mechanism. Specifically, CIC-DUX4 sarcomas depended on WEE1 activity to limit DNA damage and unscheduled mitotic entry. Consequently, genetic or pharmacologic WEE1 inhibition in vitro and in vivo led to rapid DNA damage-associated apoptotic induction of patient-derived CIC-DUX4 sarcomas. Thus, we identified WEE1 as a vulnerability targetable by therapeutic intervention in CIC-DUX4 sarcomas.
Assuntos
Proteínas de Ciclo Celular , Proteínas Tirosina Quinases , Sarcoma de Células Pequenas , Neoplasias de Tecidos Moles , Proteínas de Ciclo Celular/genética , Rearranjo Gênico , Humanos , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Sarcoma de Células Pequenas/genética , Neoplasias de Tecidos Moles/genéticaRESUMO
BACKGROUND: Soft-tissue sarcomas (STS) are a rare group of mesenchymal malignancies that account for approximately 1% of adult human cancer. Undifferentiated pleomorphic sarcoma (UPS) is one of the most common subtypes of adult STS. Clinical stratification of UPS patients has not evolved for decades and continues to rely on tumor-centric metrics including tumor size and depth. Our understanding of how the tumor microenvironment correlates to these clinicopathologic parameters remains limited. METHODS: Here, we performed single-cell flow cytometric immune-based profiling of 15 freshly resected UPS tumors and integrated this analysis with clinical, histopathologic, and outcomes data using both a prospective and retrospective cohort of UPS patients. RESULTS: We uncovered a correlation between physiologic and anatomic properties of UPS tumors and the composition of immune cells in the tumor microenvironment. Specifically, we identified an inverse correlation between tumor-infiltrating CD8 + T cells and UPS tumor size; and a positive correlation between tumor-infiltrating CD8 + T cells and overall survival. Moreover, we demonstrate an association between anatomical location (deep or superficial) and frequency of CD4 + PD1hi infiltrating T cells in UPS tumors. CONCLUSIONS: Our study provides an immune-based analysis of the tumor microenvironment in UPS patients and describes the different composition of tumor infiltrating lymphocytes based on size and tumor depth.
Assuntos
Sarcoma/fisiopatologia , Neoplasias de Tecidos Moles/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Retrospectivos , Microambiente TumoralRESUMO
Detailed phylogenies of tumor populations can recount the history and chronology of critical events during cancer progression, such as metastatic dissemination. We applied a Cas9-based, single-cell lineage tracer to study the rates, routes, and drivers of metastasis in a lung cancer xenograft mouse model. We report deeply resolved phylogenies for tens of thousands of cancer cells traced over months of growth and dissemination. This revealed stark heterogeneity in metastatic capacity, arising from preexisting and heritable differences in gene expression. We demonstrate that these identified genes can drive invasiveness and uncovered an unanticipated suppressive role for KRT17 We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale.
Assuntos
Neoplasias Pulmonares/patologia , Metástase Neoplásica , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Linhagem da Célula , Células Clonais , Regulação Neoplásica da Expressão Gênica , Humanos , Queratina-17/genética , Neoplasias Pulmonares/genética , Camundongos , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Inoculação de Neoplasia , Transplante de Neoplasias , Fenótipo , RNA-Seq , Análise de Célula Única , Transcriptoma , Transplante HeterólogoRESUMO
Capicua (CIC) is a highly conserved transcriptional repressor that is differentially regulated through mitogen-activated protein kinase (MAPK) signaling or genetic alteration across human cancer. CIC contributes to tumor progression and metastasis through direct transcriptional control of effector target genes. Recent findings indicate that CIC dysregulation is mechanistically linked and restricted to specific cancer subtypes, yet convergence on key downstream transcriptional nodes are critical for CIC-regulated oncogenesis across these cancers. In this review, we focus on how differential regulation of CIC through functional and genetic mechanisms contributes to subtype-specific cancer phenotypes and we propose new therapeutic strategies to effectively target CIC-altered cancers.
Assuntos
Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Proteínas Repressoras/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genéticaRESUMO
Lung cancer mortality largely results from metastasis. Despite curative surgery many patients with early-stage non-small cell lung cancer ultimately succumb to metastatic relapse. Current risk reduction strategies based on cytotoxic chemotherapy and radiation have only modest activity. Against this background, we functionally screened for novel metastasis modulators using a barcoded shRNA library and an orthotopic lung cancer model. We identified aryl hydrocarbon receptor (AHR), a sensor of xenobiotic chemicals and transcription factor, as suppressor of lung cancer metastasis. Knockdown of endogenous AHR induces epithelial-mesenchymal transition signatures, increases invasiveness of lung cancer cells in vitro and metastasis formation in vivo. Low intratumoral AHR expression associates with inferior outcome of patients with resected lung adenocarcinomas. Mechanistically, AHR triggers ATF4 signaling and represses matrix metalloproteinase activity, both counteracting metastatic programs. These findings link the xenobiotic defense system with control of lung cancer progression. AHR-regulated pathways are promising targets for innovative anti-metastatic strategies.
RESUMO
Transcription factor fusions (TFFs) are present in â¼30% of soft-tissue sarcomas. TFFs are not readily "druggable" in a direct pharmacologic manner and thus have proven difficult to target in the clinic. A prime example is the CIC-DUX4 oncoprotein, which fuses Capicua (CIC) to the double homeobox 4 gene, DUX4. CIC-DUX4 sarcoma is a highly aggressive and lethal subtype of small round cell sarcoma found predominantly in adolescents and young adults. To identify new therapeutic targets in CIC-DUX4 sarcoma, we performed chromatin immunoprecipitation sequencing analysis using patient-derived CIC-DUX4 cells. We uncovered multiple CIC-DUX4 targets that negatively regulate MAPK-ERK signaling. Mechanistically, CIC-DUX4 transcriptionally up-regulates these negative regulators of MAPK to dampen ERK activity, leading to sustained CIC-DUX4 expression. Genetic and pharmacologic MAPK-ERK activation through DUSP6 inhibition leads to CIC-DUX4 degradation and apoptotic induction. Collectively, we reveal a mechanism-based approach to therapeutically degrade the CIC-DUX4 oncoprotein and provide a precision-based strategy to combat this lethal cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Sarcoma/metabolismo , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Feminino , Genes Homeobox , Humanos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos SCID , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Oncogênicas/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Repressoras/genética , Sarcoma/genética , Sarcoma de Ewing/genética , Sarcoma de Células Pequenas/genética , Fatores de Transcrição/genética , Translocação Genética/genéticaRESUMO
Transcription factor fusion genes create oncoproteins that drive oncogenesis and represent challenging therapeutic targets. Understanding the molecular targets by which such fusion oncoproteins promote malignancy offers an approach to develop rational treatment strategies to improve clinical outcomes. Capicua-double homeobox 4 (CIC-DUX4) is a transcription factor fusion oncoprotein that defines certain undifferentiated round cell sarcomas with high metastatic propensity and poor clinical outcomes. The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this aggressive malignancy remain largely unknown. We demonstrated that increased expression of ETS variant 4 (ETV4) and cyclin E1 (CCNE1) occurs via neomorphic, direct effects of CIC-DUX4 and drives tumor metastasis and survival, respectively. We uncovered a molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-DUX4-expressing tumors sensitive to inhibition of the CCNE-CDK2 complex, suggesting a therapeutic strategy for CIC-DUX4-expressing tumors. Our findings highlight a paradigm of functional diversification of transcriptional repertoires controlled by a genetically aberrant transcriptional regulator, with therapeutic implications.
Assuntos
Carcinogênese/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Sarcoma/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Feminino , Humanos , Camundongos , Camundongos SCID , Metástase Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Sarcoma/genética , Sarcoma/patologia , Transcrição GênicaRESUMO
Metastasis is the leading cause of death in people with lung cancer, yet the molecular effectors underlying tumor dissemination remain poorly defined. Through the development of an in vivo spontaneous lung cancer metastasis model, we show that the developmentally regulated transcriptional repressor Capicua (CIC) suppresses invasion and metastasis. Inactivation of CIC relieves repression of its effector ETV4, driving ETV4-mediated upregulation of MMP24, which is necessary and sufficient for metastasis. Loss of CIC, or an increase in levels of its effectors ETV4 and MMP24, is a biomarker of tumor progression and worse outcomes in people with lung and/or gastric cancer. Our findings reveal CIC as a conserved metastasis suppressor, highlighting new anti-metastatic strategies that could potentially improve patient outcomes.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Carcinoma Pulmonar de Células não Pequenas/secundário , Neoplasias Pulmonares/patologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas E1A de Adenovirus/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/genética , Camundongos , Camundongos SCID , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Tumorais CultivadasRESUMO
Oncogenic activation of protein kinase BRAF drives tumor growth by promoting mitogen-activated protein kinase (MAPK) pathway signaling. Because oncogenic mutations in BRAF occur in â¼2-7% of lung adenocarcinoma (LA), BRAF-mutant LA is the most frequent cause of BRAF-mutant cancer mortality worldwide. Whereas most tumor types harbor predominantly the BRAFV600E-mutant allele, the spectrum of BRAF mutations in LA includes BRAFV600E (â¼60% of cases) and non-V600E mutant alleles (â¼40% of cases) such as BRAFG469A and BRAFG466V The presence of BRAFV600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAFV600E-mutant patients. Despite promising clinical efficacy, both innate and acquired resistance often result from reactivation of MAPK pathway signaling, thus limiting durable responses to the current BRAF inhibitors. Further, the optimal therapeutic strategy to block non-V600E BRAF-mutant LA remains unclear. Here, we report the efficacy of the Raf proto-oncogene serine/threonine protein kinase (RAF) inhibitor, PLX8394, that evades MAPK pathway reactivation in BRAF-mutant LA models. We show that PLX8394 treatment is effective in both BRAFV600E and certain non-V600 LA models, in vitro and in vivo. PLX8394 was effective against treatment-naive BRAF-mutant LAs and those with acquired vemurafenib resistance caused by an alternatively spliced, truncated BRAFV600E that promotes vemurafenib-insensitive MAPK pathway signaling. We further show that acquired PLX8394 resistance occurs via EGFR-mediated RAS-mTOR signaling and is prevented by upfront combination therapy with PLX8394 and either an EGFR or mTOR inhibitor. Our study provides a biological rationale and potential polytherapy strategy to aid the deployment of PLX8394 in lung cancer patients.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Compostos Heterocíclicos com 2 Anéis/efeitos adversos , Compostos Heterocíclicos com 2 Anéis/farmacocinética , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos com 2 Anéis/uso terapêutico , Humanos , Neoplasias Pulmonares/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos SCID , Inibidores de Proteínas Quinases/farmacologia , Proto-Oncogene Mas , Sulfonamidas/efeitos adversos , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Resultado do TratamentoRESUMO
Improvements in technologies to yield purer circulating tumor cells (CTCs) will enable a broader range of clinical applications. We have previously demonstrated the use of a commercially available cell-adhesion matrix (CAM) assay to capture invasive CTCs (iCTCs). To improve the purity of the isolated iCTCs, here we used fluorescence-activated cell sorting (FACS) in combination with the CAM assay (CAM + FACS). Our results showed an increase of median purity from the CAM assay to CAM + FACS for the spiked-in cell lines and patient samples analyzed from three different metastatic cancer types: castration resistant prostate cancer (mCRPC), non-small cell lung cancer (mNSCLC) and pancreatic ductal adenocarcinoma cancer (mPDAC). Copy number profiles for spiked-in mCRPC cell line and mCRPC patient iCTCs were similar to expected mCRPC profiles and a matched biopsy. A somatic epidermal growth factor receptor (EGFR) mutation specific to mNSCLC was observed in the iCTCs recovered from EGFR(+) mNSCLC cell lines and patient samples. Next-generation sequencing (NGS) of spiked-in pancreatic cancer cell line and mPDAC patient iCTCs showed mPDAC common mutations. CAM + FACS iCTC enrichment enables multiple downstream genomic characterizations across different tumor types.