RESUMEN
Lung cancer is a major disease carrying heterogeneous molecular lesions and many of them remain to be analyzed functionally in vivo. Gain-of-function (GOF) SHP2 (PTPN11) mutations have been found in various types of human cancer, including lung cancer. However, the role of activating SHP2 mutants in lung cancer has not been established. We generated transgenic mice containing a doxycycline (Dox)-inducible activating SHP2 mutant (tetO-SHP2(E76K)) and analyzed the role of SHP2(E76K) in lung tumorigenesis in the Clara cell secretory protein (CCSP)-reverse tetracycline transactivator (rtTA)/tetO-SHP2(E76K) bitransgenic mice. SHP2(E76K) activated Erk1/Erk2 (Erk1/2) and Src, and upregulated c-Myc and Mdm2 in the lungs of bitransgenic mice. Atypical adenomatous hyperplasia and small adenomas were observed in CCSP-rtTA/tetO-SHP2(E76K) bitransgenic mice induced with Dox for 2-6 months and progressed to larger adenoma and adenocarcinoma by 9 months. Dox withdrawal from bitransgenic mice bearing magnetic resonance imaging-detectable lung tumors resulted in tumor regression. These results show that the activating SHP2 mutant promotes lung tumorigenesis and that the SHP2 mutant is required for tumor maintenance in this mouse model of non-small cell lung cancer. SHP2(E76K) was associated with Gab1 in the lung of transgenic mice. Elevated pGab1 was observed in the lung of Dox-induced CCSP-rtTA/tetO-SHP2(E76K) mice and in cell lines expressing SHP2(E76K), indicating that the activating SHP2 mutant autoregulates tyrosine phosphorylation of its own docking protein. Gab1 tyrosine phosphorylation is sensitive to inhibition by the Src inhibitor dasatinib in GOF SHP2-mutant-expressing cells, suggesting that Src family kinases are involved in SHP2 mutant-induced Gab1 tyrosine phosphorylation.
Asunto(s)
Adenocarcinoma/patología , Adenoma/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Transformación Celular Neoplásica/patología , Neoplasias Pulmonares/patología , Mutación/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenoma/genética , Adenoma/metabolismo , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Femenino , Humanos , Inmunoprecipitación , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Transgénicos , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
PURPOSE: PARP inhibitors (PARPi) are a novel class of small molecule therapeutics for small cell lung cancer (SCLC). Identification of predictors of response would advance our understanding, and guide clinical application, of this therapeutic strategy. EXPERIMENTAL DESIGN: Efficacy of PARP inhibitors olaparib, rucaparib, and veliparib, as well as etoposide and cisplatin in SCLC cell lines, and gene expression correlates, was analyzed using public datasets. HRD genomic scar scores were calculated from Affymetrix SNP 6.0 arrays. In vitro talazoparib efficacy was measured by cell viability assays. For functional studies, CRISPR/Cas9 and shRNA were used for genomic editing and transcript knockdown, respectively. Protein levels were assessed by immunoblotting and immunohistochemistry (IHC). Quantitative synergy of talazoparib and temozolomide was determined in vitro In vivo efficacy of talazoparib, temozolomide, and the combination was assessed in patient-derived xenograft (PDX) models. RESULTS: We identified SLFN11, but not HRD genomic scars, as a consistent correlate of response to all three PARPi assessed, with loss of SLFN11 conferring resistance to PARPi. We confirmed these findings in vivo across multiple PDX and defined IHC staining for SLFN11 as a predictor of talazoparib response. As temozolomide has activity in SCLC, we investigated combination therapy with talazoparib and found marked synergy in vitro and efficacy in vivo, which did not solely depend on SLFN11 or MGMT status. CONCLUSIONS: SLFN11 is a relevant predictive biomarker of sensitivity to PARP inhibitor monotherapy in SCLC and we identify combinatorial therapy with TMZ as a particularly promising therapeutic strategy that warrants further clinical investigation. Clin Cancer Res; 23(2); 523-35. ©2016 AACR.
Asunto(s)
Sinergismo Farmacológico , Proteínas Nucleares/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Bencimidazoles/administración & dosificación , Línea Celular Tumoral , Cisplatino/administración & dosificación , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Etopósido/administración & dosificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genómica/métodos , Humanos , Indoles/administración & dosificación , Ratones , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Temozolomida , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Small cell lung cancer is initially highly responsive to cisplatin and etoposide but in almost every case becomes rapidly chemoresistant, leading to death within 1 year. We modeled acquired chemoresistance in vivo using a series of patient-derived xenografts to generate paired chemosensitive and chemoresistant cancers. Multiple chemoresistant models demonstrated suppression of SLFN11, a factor implicated in DNA-damage repair deficiency. In vivo silencing of SLFN11 was associated with marked deposition of H3K27me3, a histone modification placed by EZH2, within the gene body of SLFN11, inducing local chromatin condensation and gene silencing. Inclusion of an EZH2 inhibitor with standard cytotoxic therapies prevented emergence of acquired resistance and augmented chemotherapeutic efficacy in both chemosensitive and chemoresistant models of small cell lung cancer.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/fisiología , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Nucleares/fisiología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Animales , Resistencia a Antineoplásicos , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Humanos , Ratones , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Proteína 1 Relacionada con Twist/fisiologíaRESUMEN
Patient-derived xenograft (PDX) mouse models are increasingly used for preclinical therapeutic testing of human cancer. A limitation in molecular and genetic characterization of PDX tumors is the presence of integral murine stroma. This is particularly problematic for genomic sequencing of PDX models. Rapid and dependable approaches for quantitating stromal content and purifying the malignant human component of these tumors are needed. We used a recently developed technique exploiting species-specific polymerase chain reaction (PCR) amplicon length (ssPAL) differences to define the fractional composition of murine and human DNA, which was proportional to the fractional composition of cells in a series of lung cancer PDX lines. We compared four methods of human cancer cell isolation: fluorescence-activated cell sorting (FACS), an immunomagnetic mouse cell depletion (MCD) approach, and two distinct EpCAM-based immunomagnetic positive selection methods. We further analyzed DNA extracted from the resulting enriched human cancer cells by targeted sequencing using a clinically validated multi-gene panel. Stromal content varied widely among tumors of similar histology, but appeared stable over multiple serial tumor passages of an individual model. FACS and MCD were superior to either positive selection approach, especially in cases of high stromal content, and consistently allowed high quality human-specific genomic profiling. ssPAL is a dependable approach to quantitation of murine stromal content, and MCD is a simple, efficient, and high yield approach to human cancer cell isolation for genomic analysis of PDX tumors.
Asunto(s)
Genómica , Neoplasias/genética , Neoplasias/patología , Células del Estroma/metabolismo , Animales , Biología Computacional/métodos , Modelos Animales de Enfermedad , Genómica/métodos , Xenoinjertos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Células del Estroma/patologíaRESUMEN
RET fusions have been found in lung adenocarcinoma, of which KIF5B-RET is the most prevalent. We established inducible KIF5B-RET transgenic mice and KIF5B-RET-dependent cell lines for preclinical modeling of KIF5B-RET-associated lung adenocarcinoma. Doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET transgenic mice developed invasive lung adenocarcinoma with desmoplastic reaction. Tumors regressed upon suppression of KIF5B-RET expression. By culturing KIF5B-RET-dependent BaF3 (B/KR) cells with increasing concentrations of cabozantinib or vandetanib, we identified cabozantinib-resistant RETV804L mutation and vandetanib-resistant-RETG810A mutation. Among cabozantinib, lenvatinib, ponatinib, and vandetanib, ponatinib was identified as the most potent inhibitor against KIF5B-RET and its drug-resistant mutants. Interestingly, the vandetanib-resistant KIF5B-RETG810A mutant displayed gain-of-sensitivity (GOS) to ponatinib and lenvatinib. Treatment of doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET bitransgenic mice with ponatinib effectively induced tumor regression. These results indicate that KIF5B-RET-associated lung tumors are addicted to the fusion oncogene and ponatinib is the most effective inhibitor for targeting KIF5B-RET in lung adenocarcinoma. Moreover, this study finds a novel vandetanib-resistant RETG810A mutation and identifies lenvatinib and ponatinib as the secondary drugs to overcome this vandetanib resistance mechanism. Mol Cancer Ther; 15(10); 2521-9. ©2016 AACR.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma del Pulmón , Animales , Antineoplásicos/farmacología , Biopsia , Línea Celular Tumoral , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Expresión Génica , Orden Génico , Vectores Genéticos/genética , Humanos , Imidazoles/farmacología , Inmunohistoquímica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/farmacología , Transgenes , Microtomografía por Rayos XRESUMEN
Epidermal growth factor receptor (EGFR) mutants drive lung tumorigenesis and are targeted for therapy. However, resistance to EGFR inhibitors has been observed, in which the mutant EGFR remains active. Thus, it is important to uncover mediators of EGFR mutant-driven lung tumors to develop new treatment strategies. The protein tyrosine phosphatase (PTP) Shp2 mediates EGF signaling. Nevertheless, it is unclear if Shp2 is activated by oncogenic EGFR mutants in lung carcinoma or if inhibiting the Shp2 PTP activity can suppress EGFR mutant-induced lung adenocarcinoma. Here, we generated transgenic mice containing a doxycycline (Dox)-inducible PTP-defective Shp2 mutant (tetO-Shp2CSDA). Using the rat Clara cell secretory protein (CCSP)-rtTA-directed transgene expression in the type II lung pneumocytes of transgenic mice, we found that the Gab1-Shp2 pathway was activated by EGFRL858R in the lungs of transgenic mice. Consistently, the Gab1-Shp2 pathway was activated in human lung adenocarcinoma cells containing mutant EGFR. Importantly, Shp2CSDA inhibited EGFRL858R-induced lung adenocarcinoma in transgenic animals. Analysis of lung tissues showed that Shp2CSDA suppressed Gab1 tyrosine phosphorylation and Gab1-Shp2 association, suggesting that Shp2 modulates a positive feedback loop to regulate its own activity. These results show that inhibition of the Shp2 PTP activity impairs mutant EGFR signaling and suppresses EGFRL858R-driven lung adenocarcinoma.
Asunto(s)
Adenocarcinoma/enzimología , Adenocarcinoma/genética , Receptores ErbB/genética , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Línea Celular Tumoral , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Interferon-α2b (IFN-α2b) is used to treat melanoma but there is a need to improve its efficacy. IFN-α2b signaling requires STAT1/STAT2 tyrosine phosphorylation and is subject to negative regulation by phosphatases. In this study, we determined whether inhibition of the protein tyrosine phosphatase Shp2 could enhance IFN-α2b responses in human melanoma cells. Shp2 knockdown increased IFN-α2b-stimulated STAT1 Tyr-701 phosphorylation and ISRE-luciferase activity even though it did not affect STAT2 Tyr-690 phosphorylation in A375 cells. In A375 tumor xenografts, Shp2 knockdown enhanced the anti-melanoma effect of IFN-α2b. Furthermore, the Shp2 inhibitor SPI-112Me increased the IFN-α2b-induced STAT1 activation and anti-proliferative response in A375 and SK-MEL-2 cells. These results demonstrate that inhibition of Shp2 can enhance the anti-melanoma activity of IFN-α2b.