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1.
Nature ; 608(7923): 609-617, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35948633

RESUMO

Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer1. However, clinical responses to FGFR inhibitors have remained variable1-9, emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening10,11 and tumour modelling in mice12,13, and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1-E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2ΔE18). Functional in vitro and in vivo examination of a compendium of FGFR2ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies.


Assuntos
Éxons , Deleção de Genes , Terapia de Alvo Molecular , Neoplasias , Oncogenes , Inibidores de Proteínas Quinases , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Animais , Éxons/genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Oncogenes/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo
3.
Nature ; 535(7612): 382-7, 2016 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-27443740

RESUMO

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.


Assuntos
Replicação do DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Deleção de Genes , Genes BRCA1 , Genes BRCA2 , Neoplasias/patologia , Proteínas Nucleares/deficiência , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA/biossíntese , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , DNA Helicases/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Feminino , Recombinação Homóloga , Proteína Homóloga a MRE11 , Camundongos , Neoplasias/genética , Proteínas Nucleares/genética , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética
4.
Nucleic Acids Res ; 45(12): 7064-7077, 2017 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-28575524

RESUMO

Insertional mutagenesis using engineered transposons is a potent forward genetic screening technique used to identify cancer genes in mouse model systems. In the analysis of these screens, transposon insertion sites are typically identified by targeted DNA-sequencing and subsequently assigned to predicted target genes using heuristics. As such, these approaches provide no direct evidence that insertions actually affect their predicted targets or how transcripts of these genes are affected. To address this, we developed IM-Fusion, an approach that identifies insertion sites from gene-transposon fusions in standard single- and paired-end RNA-sequencing data. We demonstrate IM-Fusion on two separate transposon screens of 123 mammary tumors and 20 B-cell acute lymphoblastic leukemias, respectively. We show that IM-Fusion accurately identifies transposon insertions and their true target genes. Furthermore, by combining the identified insertion sites with expression quantification, we show that we can determine the effect of a transposon insertion on its target gene(s) and prioritize insertions that have a significant effect on expression. We expect that IM-Fusion will significantly enhance the accuracy of cancer gene discovery in forward genetic screens and provide initial insight into the biological effects of insertions on candidate cancer genes.


Assuntos
Neoplasias da Mama/genética , Elementos de DNA Transponíveis , Leucemia de Células B/genética , Mutagênese Insercional , Proteínas de Neoplasias/genética , Software , Doença Aguda , Animais , Neoplasias da Mama/metabolismo , Mapeamento Cromossômico/métodos , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala , Humanos , Leucemia de Células B/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo
5.
BMC Bioinformatics ; 19(1): 366, 2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-30286710

RESUMO

BACKGROUND: Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analyses of their RNA and DNA profiles are challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin result in false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. However, currently available algorithms are limited and improvements in accuracy and ease of use are necessary. RESULTS: We developed the R-package XenofilteR, which separates mouse from human sequence reads based on the edit-distance between a sequence read and reference genome. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human DNA sequence data. These analyses revealed that XenofilteR removes > 99.9% of sequence reads of mouse origin while retaining human sequences. This allowed for mutation analysis of xenograft samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic tumor mutations. CONCLUSIONS: XenofilteR accurately dissects RNA and DNA sequences from mouse and human origin, thereby outperforming currently available tools. XenofilteR is open source and available at https://github.com/PeeperLab/XenofilteR .


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Computadores , Bases de Dados Genéticas , Humanos , Camundongos
7.
Cell Rep ; 42(5): 112538, 2023 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-37209095

RESUMO

BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis), but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanisms that do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance in vivo, we combine molecular profiling with functional analysis of HR of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent reactivation of BRCA1/2. We observe restoration of HR in 62% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we find that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by PARG loss. Furthermore, combined multi-omics analysis identifies additional genes and pathways potentially involved in modulating PARPi response.


Assuntos
Neoplasias , Neoplasias Ovarianas , Animais , Camundongos , Feminino , Humanos , Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Multiômica , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias/genética , Neoplasias Ovarianas/genética
8.
Nat Commun ; 10(1): 397, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30674894

RESUMO

BRCA1-mutated breast cancer is primarily driven by DNA copy-number alterations (CNAs) containing large numbers of candidate driver genes. Validation of these candidates requires novel approaches for high-throughput in vivo perturbation of gene function. Here we develop genetically engineered mouse models (GEMMs) of BRCA1-deficient breast cancer that permit rapid introduction of putative drivers by either retargeting of GEMM-derived embryonic stem cells, lentivirus-mediated somatic overexpression or in situ CRISPR/Cas9-mediated gene disruption. We use these approaches to validate Myc, Met, Pten and Rb1 as bona fide drivers in BRCA1-associated mammary tumorigenesis. Iterative mouse modeling and comparative oncogenomics analysis show that MYC-overexpression strongly reshapes the CNA landscape of BRCA1-deficient mammary tumors and identify MCL1 as a collaborating driver in these tumors. Moreover, MCL1 inhibition potentiates the in vivo efficacy of PARP inhibition (PARPi), underscoring the therapeutic potential of this combination for treatment of BRCA1-mutated cancer patients with poor response to PARPi monotherapy.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Carcinogênese/genética , Variações do Número de Cópias de DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Mutação , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/genética , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Células-Tronco Embrionárias , Feminino , Redes Reguladoras de Genes , Células HEK293 , Humanos , Neoplasias Mamárias Animais/genética , Camundongos , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transcriptoma , Proteína Supressora de Tumor p53/genética
9.
Cancer Res ; 78(19): 5668-5679, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30115694

RESUMO

In human cancers, FGFR signaling is frequently hyperactivated by deregulation of FGF ligands or by activating mutations in the FGFR receptors such as gene amplifications, point mutations, and gene fusions. As such, FGFR inhibitors are considered an attractive therapeutic strategy for patients with mutations in FGFR family members. We previously identified Fgfr2 as a key driver of invasive lobular carcinoma (ILC) in an in vivo insertional mutagenesis screen using the Sleeping Beauty transposon system. Here we explore whether these FGFR-driven ILCs are sensitive to the FGFR inhibitor AZD4547 and use transposon mutagenesis in these tumors to identify potential mechanisms of resistance to therapy. Combined with RNA sequencing-based analyses of AZD4547-resistant tumors, our in vivo approach identified several known and novel potential resistance mechanisms to FGFR inhibition, most of which converged on reactivation of the canonical MAPK-ERK signaling cascade. Observed resistance mechanisms included mutations in the tyrosine kinase domain of FGFR2, overexpression of MET, inactivation of RASA1, and activation of the drug-efflux transporter ABCG2. ABCG2 and RASA1 were identified only from de novo transposon insertions acquired during AZD4547 treatment, demonstrating that insertional mutagenesis in mice is an effective tool for identifying potential mechanisms of resistance to targeted cancer therapies.Significance: These findings demonstrate that a combined approach of transcriptomics and insertional mutagenesis in vivo is an effective method for identifying potential targets to overcome resistance to therapy in the clinic. Cancer Res; 78(19); 5668-79. ©2018 AACR.


Assuntos
Benzamidas/química , Elementos de DNA Transponíveis , Resistencia a Medicamentos Antineoplásicos , Mutagênese , Piperazinas/química , Pirazóis/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Carcinoma Lobular/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Amplificação de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Mutação , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Análise de Sequência de RNA , Transcriptoma , Proteína p120 Ativadora de GTPase/metabolismo
10.
Cancer Cell ; 33(6): 1078-1093.e12, 2018 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-29894693

RESUMO

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically.


Assuntos
Glicosídeo Hidrolases/genética , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mutações Sintéticas Letais , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Camundongos da Linhagem 129 , Camundongos Knockout , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação/efeitos dos fármacos
11.
Nat Genet ; 49(8): 1219-1230, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28650484

RESUMO

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype and accounts for 8-14% of all cases. Although the majority of human ILCs are characterized by the functional loss of E-cadherin (encoded by CDH1), inactivation of Cdh1 does not predispose mice to develop mammary tumors, implying that mutations in additional genes are required for ILC formation in mice. To identify these genes, we performed an insertional mutagenesis screen using the Sleeping Beauty transposon system in mice with mammary-specific inactivation of Cdh1. These mice developed multiple independent mammary tumors of which the majority resembled human ILC in terms of morphology and gene expression. Recurrent and mutually exclusive transposon insertions were identified in Myh9, Ppp1r12a, Ppp1r12b and Trp53bp2, whose products have been implicated in the regulation of the actin cytoskeleton. Notably, MYH9, PPP1R12B and TP53BP2 were also frequently aberrated in human ILC, highlighting these genes as drivers of a novel oncogenic pathway underlying ILC development.


Assuntos
Neoplasias da Mama/genética , Carcinoma Lobular/genética , Mutagênese Insercional , Animais , Caderinas/genética , Linhagem Celular , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Feminino , Haplótipos , Humanos , Masculino , Camundongos , Cadeias Pesadas de Miosina , Fosfatase de Miosina-de-Cadeia-Leve/genética , Miosina não Muscular Tipo IIA/genética , Transposases/genética , Proteínas Supressoras de Tumor/genética
12.
Cell Rep ; 16(8): 2087-2101, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27524621

RESUMO

Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC.


Assuntos
Caderinas/genética , Carcinoma Lobular/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Neoplasias Mamárias Experimentais/genética , PTEN Fosfo-Hidrolase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Antineoplásicos/farmacologia , Caderinas/deficiência , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/mortalidade , Carcinoma Lobular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Perfilação da Expressão Gênica , Imidazóis/farmacologia , Integrases/genética , Integrases/metabolismo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/mortalidade , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Invasividade Neoplásica , PTEN Fosfo-Hidrolase/deficiência , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Quinolinas/farmacologia , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Análise de Sobrevida , Microambiente Tumoral
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