RESUMEN
RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.
Asunto(s)
Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Ratones , Línea Celular Tumoral , Proteínas Proto-Oncogénicas p21(ras)/genética , Femenino , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Guanosina Trifosfato/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutación , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , MasculinoRESUMEN
Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the interactions between oncogenes and tumor suppressor genes that shape this landscape remain poorly resolved and cannot be revealed by human cancer genomics alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. We show that the fitness landscape is rugged-the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context-and shows no evidence of diminishing-returns epistasis within variants of the same oncogene. These findings argue against a simple linear signaling relationship amongst these three oncogenes and imply a critical role for off-axis signaling in determining the fitness effects of inactivating tumor suppressors.
Asunto(s)
Neoplasias Pulmonares , Proteínas Proto-Oncogénicas p21(ras) , Ratones , Humanos , Animales , Proteínas Proto-Oncogénicas p21(ras)/genética , Oncogenes/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , MutaciónRESUMEN
The lack of knowledge about the relationship between tumor genotypes and therapeutic responses remains one of the most critical gaps in enabling the effective use of cancer therapies. Here, we couple a multiplexed and quantitative experimental platform with robust statistical methods to enable pharmacogenomic mapping of lung cancer treatment responses in vivo. The complex map of genotype-specific treatment responses uncovered that over 20% of possible interactions show significant resistance or sensitivity. Known and novel interactions were identified, and one of these interactions, the resistance of KEAP1-mutant lung tumors to platinum therapy, was validated using a large patient response data set. These results highlight the broad impact of tumor suppressor genotype on treatment responses and define a strategy to identify the determinants of precision therapies. SIGNIFICANCE: An experimental and analytical framework to generate in vivo pharmacogenomic maps that relate tumor genotypes to therapeutic responses reveals a surprisingly complex map of genotype-specific resistance and sensitivity.
Asunto(s)
Adenocarcinoma del Pulmón/genética , Proteína 1 Asociada A ECH Tipo Kelch/genética , Neoplasias Pulmonares/genética , Farmacogenética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Animales , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Biblioteca de Genes , Genes Supresores de Tumor , Genotipo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Mutación , Metástasis de la NeoplasiaRESUMEN
Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood. Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR-Cas9-based genetic screens in vivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse KrasG12D-driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and a key component of p53-mediated tumor suppression.
Asunto(s)
Proteínas de Unión al ARN/genética , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma/genética , Empalme Alternativo , Animales , Proteínas de Ciclo Celular/metabolismo , Exones , Perfilación de la Expresión Génica/métodos , Genes Supresores de Tumor , Humanos , Neoplasias Hepáticas/genética , Masculino , Ratones , Ratones Endogámicos ICR , Ratones SCID , Interferencia de ARN , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The kinase LKB1 is a critical tumor suppressor in sporadic and familial human cancers, yet the mechanisms by which it suppresses tumor growth remain poorly understood. To investigate the tumor-suppressive capacity of four canonical families of LKB1 substrates in vivo, we used CRISPR/Cas9-mediated combinatorial genome editing in a mouse model of oncogenic KRAS-driven lung adenocarcinoma. We demonstrate that members of the SIK family are critical for constraining tumor development. Histologic and gene-expression similarities between LKB1- and SIK-deficient tumors suggest that SIKs and LKB1 operate within the same axis. Furthermore, a gene-expression signature reflecting SIK deficiency is enriched in LKB1-mutant human lung adenocarcinomas and is regulated by LKB1 in human cancer cell lines. Together, these findings reveal a key LKB1-SIK tumor-suppressive axis and underscore the need to redirect efforts to elucidate the mechanisms through which LKB1 mediates tumor suppression. SIGNIFICANCE: Uncovering the effectors of frequently altered tumor suppressor genes is critical for understanding the fundamental driving forces of cancer growth. Our identification of the SIK family of kinases as effectors of LKB1-mediated tumor suppression will refocus future mechanistic studies and may lead to new avenues for genotype-specific therapeutic interventions.This article is highlighted in the In This Issue feature, p. 1469.
Asunto(s)
Adenocarcinoma del Pulmón/genética , Neoplasias Pulmonares/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Línea Celular Tumoral , Proliferación Celular , Edición Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
The originally published article failed to acknowledge the equal first authorship contribution of I. P. Winters and C. W. Murray. The article has now been corrected online. The editors apologize for this error.
RESUMEN
Large-scale sequencing of human tumours has uncovered a vast array of genomic alterations. Genetically engineered mouse models recapitulate many features of human cancer and have been instrumental in assigning biological meaning to specific cancer-associated alterations. However, their time, cost and labour-intensive nature limits their broad utility; thus, the functional importance of the majority of genomic aberrations in cancer remains unknown. Recent advances have accelerated the functional interrogation of cancer-associated alterations within in vivo models. Specifically, the past few years have seen the emergence of CRISPR-Cas9-based strategies to rapidly generate increasingly complex somatic alterations and the development of multiplexed and quantitative approaches to ascertain gene function in vivo.
Asunto(s)
Sistemas CRISPR-Cas , Genes Relacionados con las Neoplasias , Genómica/métodos , Neoplasias/genética , Animales , Humanos , Ratones , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
The Nkx2-1 transcription factor promotes differentiation of lung epithelial lineages and suppresses malignant progression of lung adenocarcinoma. However, targets of Nkx2-1 that limit tumor growth and progression remain incompletely understood. Here, direct Nkx2-1 targets are identified whose expression correlates with Nkx2-1 activity in human lung adenocarcinoma. Selenium-binding protein 1 (Selenbp1), an Nkx2-1 effector that limits phenotypes associated with lung cancer growth and metastasis, was investigated further. Loss- and gain-of-function approaches demonstrate that Nkx2-1 is required and sufficient for Selenbp1 expression in lung adenocarcinoma cells. Interestingly, Selenbp1 knockdown also reduced Nkx2-1 expression and Selenbp1 stabilized Nkx2-1 protein levels in a heterologous system, suggesting that these genes function in a positive feedback loop. Selenbp1 inhibits clonal growth and migration and suppresses growth of metastases in an in vivo transplant model. Genetic inactivation of Selenbp1, using CRISPR/Cas9, also enhanced primary tumor growth in autochthonous lung adenocarcinoma mouse models. Collectively, these data demonstrate that Selenbp1 is a direct target of Nkx2-1, which inhibits lung adenocarcinoma growth in vivo Implications: Selenbp1 is an important suppressor of lung tumor growth that functions in a positive feedback loop with Nkx2-1, and whose loss is associated with worse patient outcome. Mol Cancer Res; 16(11); 1737-49. ©2018 AACR.
Asunto(s)
Adenocarcinoma del Pulmón/genética , Neoplasias Pulmonares/genética , Proteínas de Unión al Selenio/genética , Factor Nuclear Tiroideo 1/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Animales , Procesos de Crecimiento Celular , Línea Celular Tumoral , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Supresores de Tumor , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Proteínas de Unión al Selenio/biosíntesis , Proteínas de Unión al Selenio/metabolismo , Factor Nuclear Tiroideo 1/metabolismo , TransfecciónRESUMEN
The functional impact of most genomic alterations found in cancer, alone or in combination, remains largely unknown. Here we integrate tumor barcoding, CRISPR/Cas9-mediated genome editing and ultra-deep barcode sequencing to interrogate pairwise combinations of tumor suppressor alterations in autochthonous mouse models of human lung adenocarcinoma. We map the tumor suppressive effects of 31 common lung adenocarcinoma genotypes and identify a landscape of context dependence and differential effect strengths.
Asunto(s)
Adenocarcinoma del Pulmón/genética , Genes Supresores de Tumor , Neoplasias Pulmonares/genética , Animales , Sistemas CRISPR-Cas , Código de Barras del ADN Taxonómico , Eliminación de Gen , Edición Génica , Genes p53 , Aptitud Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Ratones Transgénicos , Proteína de Retinoblastoma/genética , Análisis de Secuencia de ADNRESUMEN
Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded Kras HDR alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.
Asunto(s)
Carcinogénesis/genética , Análisis Mutacional de ADN/métodos , Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Reparación del ADN por Recombinación/genética , Animales , Sistemas CRISPR-Cas/genética , Análisis Costo-Beneficio , Análisis Mutacional de ADN/economía , Estudios de Factibilidad , Femenino , Genómica/economía , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Masculino , Ratones , Mutación , Neoplasias/patología , Reproducibilidad de los ResultadosRESUMEN
Cancer growth is a multistage, stochastic evolutionary process. While cancer genome sequencing has been instrumental in identifying the genomic alterations that occur in human tumors, the consequences of these alterations on tumor growth remain largely unexplored. Conventional genetically engineered mouse models enable the study of tumor growth in vivo, but they are neither readily scalable nor sufficiently quantitative to unravel the magnitude and mode of action of many tumor-suppressor genes. Here, we present a method that integrates tumor barcoding with ultradeep barcode sequencing (Tuba-seq) to interrogate tumor-suppressor function in mouse models of human cancer. Tuba-seq uncovers genotype-dependent distributions of tumor sizes. By combining Tuba-seq with multiplexed CRISPR-Cas9-mediated genome editing, we quantified the effects of 11 tumor-suppressor pathways that are frequently altered in human lung adenocarcinoma. Tuba-seq enables the broad quantification of the function of tumor-suppressor genes with unprecedented resolution, parallelization, and precision.
Asunto(s)
Neoplasias Experimentales/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenocarcinoma/genética , Animales , ADN/genética , ADN/aislamiento & purificación , ADN/metabolismo , Código de Barras del ADN Taxonómico , Femenino , Ingeniería Genética , Humanos , Lentivirus/genética , Pulmón/metabolismo , Neoplasias Pulmonares/genética , Masculino , Ratones , Modelos Genéticos , Plásmidos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.