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1.
Mol Cell ; 82(13): 2443-2457.e7, 2022 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-35613620

RESUMEN

RAF protein kinases are effectors of the GTP-bound form of small guanosine triphosphatase RAS and function by phosphorylating MEK. We showed here that the expression of ARAF activated RAS in a kinase-independent manner. Binding of ARAF to RAS displaced the GTPase-activating protein NF1 and antagonized NF1-mediated inhibition of RAS. This reduced ERK-dependent inhibition of RAS and increased RAS-GTP. By this mechanism, ARAF regulated the duration and consequences of RTK-induced RAS activation and supported the RAS output of RTK-dependent tumor cells. In human lung cancers with EGFR mutation, amplification of ARAF was associated with acquired resistance to EGFR inhibitors, which was overcome by combining EGFR inhibitors with an inhibitor of the protein tyrosine phosphatase SHP2 to enhance inhibition of nucleotide exchange and RAS activation.


Asunto(s)
Neurofibromina 1 , Proteínas Proto-Oncogénicas A-raf , Proteínas Activadoras de ras GTPasa , Receptores ErbB/genética , Receptores ErbB/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Neurofibromina 1/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas A-raf/metabolismo , Transducción de Señal , Proteínas Activadoras de ras GTPasa/metabolismo
2.
Angiogenesis ; 20(4): 655-662, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28752390

RESUMEN

Pericytes are essential mural cells distinguished by their association with small caliber blood vessels and the presence of a basement membrane shared with endothelial cells. Pericyte interaction with the endothelium plays an important role in angiogenesis; however, very few tools are currently available that allow for the targeting of pericytes in mouse models, limiting our ability to understand their biology. We have generated a novel mouse line expressing tamoxifen-inducible Cre-recombinase under the control of the platelet-derived growth factor receptor ß promoter: PDGFRß-P2A-CreER T2 . We evaluated the expression of the PDGFRß-P2A-CreER T2 line by crossing it with fluorescent reporter lines and analyzed reporter signal in the angiogenic retina and brain at different time points after tamoxifen administration. Reporter lines showed labeling of NG2+, desmin+, PDGFRß+ perivascular cells in the retina and the brain, indicating successful targeting of pericytes; however, signal from reporter lines was also observed in a small subset of glial cells both in the retina and the brain. We also evaluated recombination in tumors and found efficient recombination in perivascular cells associated with tumor vasculature. As a proof of principle, we used our newly generated driver to delete Notch signaling in perivascular cells and observed a loss of smooth muscle cells in retinal arteries, consistent with previously published studies evaluating Notch3 null mice. We conclude that the PDGFRß-P2A-CreER T2 line is a powerful new tool to target pericytes and will aid the field in gaining a deeper understanding of the role of these cells in physiological and pathological settings.


Asunto(s)
Técnicas Genéticas , Neovascularización Fisiológica , Pericitos/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Femenino , Genes Reporteros , Integrasas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Recombinación Genética/genética , Retina/metabolismo , Tamoxifeno/farmacología , Factores de Tiempo
3.
Cancer Res ; 82(8): 1633-1645, 2022 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-35149586

RESUMEN

In-frame insertions in exon 20 of HER2 are the most common HER2 mutations in patients with non-small cell lung cancer (NSCLC), a disease in which approved EGFR/HER2 tyrosine kinase inhibitors (TKI) display poor efficiency and undesirable side effects due to their strong inhibition of wild-type (WT) EGFR. Here, we report a HER2-selective covalent TKI, JBJ-08-178-01, that targets multiple HER2 activating mutations, including exon 20 insertions as well as amplification. JBJ-08-178-01 displayed strong selectivity toward HER2 mutants over WT EGFR compared with other EGFR/HER2 TKIs. Determination of the crystal structure of HER2 in complex with JBJ-08-178-01 suggests that an interaction between the inhibitor and Ser783 may be responsible for HER2 selectivity. The compound showed strong antitumoral activity in HER2-mutant or amplified cancers in vitro and in vivo. Treatment with JBJ-08-178-01 also led to a reduction in total HER2 by promoting proteasomal degradation of the receptor. Taken together, the dual activity of JBJ-08-178-01 as a selective inhibitor and destabilizer of HER2 represents a combination that may lead to better efficacy and tolerance in patients with NSCLC harboring HER2 genetic alterations or amplification. SIGNIFICANCE: This study describes unique mechanisms of action of a new mutant-selective HER2 kinase inhibitor that reduces both kinase activity and protein levels of HER2 in lung cancer.


Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Exones , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Inhibidores de Proteínas Quinasas/efectos adversos , Receptor ErbB-2/metabolismo
4.
Clin Cancer Res ; 28(8): 1640-1650, 2022 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-35091439

RESUMEN

PURPOSE: Activating missense mutations of KRAS are the most frequent oncogenic driver events in lung adenocarcinoma (LUAD). However, KRAS isoforms are highly heterogeneous, and data on the potential isoform-dependent therapeutic vulnerabilities are still lacking. EXPERIMENTAL DESIGN: We developed an isogenic cell-based platform to compare the oncogenic properties and specific therapeutic actionability of KRAS-mutant isoforms. In parallel, we analyzed clinicopathologic and genomic data from 3,560 patients with non-small cell lung cancer (NSCLC) to survey allele-specific features associated with oncogenic KRAS mutations. RESULTS: In isogenic cell lines expressing different mutant KRAS isoforms, we identified isoform-specific biochemical, biological, and oncogenic properties both in vitro and in vivo. These exclusive features correlated with different therapeutic responses to MEK inhibitors, with KRAS G12C and Q61H mutants being more sensitive compared with other isoforms. In vivo, combined KRAS G12C and MEK inhibition was more effective than either drug alone. Among patients with NSCLCs that underwent comprehensive tumor genomic profiling, STK11 and ATM mutations were significantly enriched among tumors harboring KRAS G12C, G12A, and G12V mutations. KEAP1 mutation was significantly enriched among KRAS G12C and KRAS G13X LUADs. KRAS G13X-mutated tumors had the highest frequency of concurrent STK11 and KEAP1 mutations. Transcriptomic profiling revealed unique patterns of gene expression in each KRAS isoform, compared with KRAS wild-type tumors. CONCLUSIONS: This study demonstrates that KRAS isoforms are highly heterogeneous in terms of concurrent genomic alterations and gene-expression profiles, and that stratification based on KRAS alleles should be considered in the design of future clinical trials.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Mutación , Factor 2 Relacionado con NF-E2/genética , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética
5.
Mol Oncol ; 15(1): 27-42, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32191822

RESUMEN

Small-cell lung cancer (SCLC) occurs infrequently in never/former light smokers. We sought to study this rare clinical subset through next-generation sequencing (NGS) and by characterizing a representative patient-derived model. We performed targeted NGS, as well as comprehensive pathological evaluation, in 11 never/former light smokers with clinically diagnosed SCLC. We established a patient-derived model from one such patient (DFCI168) harboring an NRASQ61K mutation and characterized the sensitivity of this model to MEK and TORC1/2 inhibitors. Despite the clinical diagnosis of SCLC, the majority (8/11) of cases were either of nonpulmonary origin or of mixed histology and included atypical carcinoid (n = 1), mixed non-small-cell lung carcinoma and SCLC (n = 4), unspecified poorly differentiated carcinoma (n = 1), or small-cell carcinoma from different origins (n = 2). RB1 and TP53 mutations were found in four and five cases, respectively. Predicted driver mutations were detected in EGFR (n = 2), NRAS (n = 1), KRAS (n = 1), BRCA1 (n = 1), and ATM (n = 1), and one case harbored a TMPRSS2-ERG fusion. DFCI168 (NRASQ61K ) exhibited marked sensitivity to MEK inhibitors in vitro and in vivo. The combination of MEK and mTORC1/2 inhibitors synergized to prevent compensatory mTOR activation, resulting in prolonged growth inhibition in this model and in three other NRAS mutant lung cancer cell lines. SCLC in never/former light smokers is rare and is potentially a distinct disease entity comprised of oncogenic driver mutation-harboring carcinomas morphologically and/or clinically mimicking SCLC. Comprehensive pathologic review integrated with genomic profiling is critical in refining the diagnosis and in identifying potential therapeutic options.


Asunto(s)
Heterogeneidad Genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Terapia Molecular Dirigida , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Fumadores , Anciano , Animales , Secuencia de Bases , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , GTP Fosfohidrolasas/genética , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteínas de la Membrana/genética , Ratones , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Mutación/genética , Sistemas Neurosecretores/efectos de los fármacos , Sistemas Neurosecretores/patología , Fenotipo , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Carcinoma Pulmonar de Células Pequeñas/diagnóstico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico
6.
Mol Cancer Ther ; 20(4): 641-654, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33536188

RESUMEN

RAS gene mutations are the most frequent oncogenic event in lung cancer. They activate multiple RAS-centric signaling networks among them the MAPK, PI3K, and RB pathways. Within the MAPK pathway, ERK1/2 proteins exert a bottleneck function for transmitting mitogenic signals and activating cytoplasmic and nuclear targets. In view of disappointing antitumor activity and toxicity of continuously applied MEK inhibitors in patients with KRAS-mutant lung cancer, research has recently focused on ERK1/2 proteins as therapeutic targets and on ERK inhibitors for their ability to prevent bypass and feedback pathway activation. Here, we show that intermittent application of the novel and selective ATP-competitive ERK1/2 inhibitor LY3214996 exerts single-agent activity in patient-derived xenograft (PDX) models of RAS-mutant lung cancer. Combination treatments were well tolerated and resulted in synergistic (ERKi plus PI3K/mTORi LY3023414) and additive (ERKi plus CDK4/6i abemaciclib) tumor growth inhibition in PDX models. Future clinical trials are required to investigate if intermittent ERK inhibitor-based treatment schedules can overcome toxicities observed with continuous MEK inhibition and-equally important-to identify biomarkers for patient stratification.


Asunto(s)
Genes ras/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Oncogenes/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/patología , Inhibidores de Proteínas Quinasas/farmacología
7.
Clin Cancer Res ; 27(1): 34-42, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33082208

RESUMEN

PURPOSE: The RET proto-oncogene encodes a receptor tyrosine kinase that is activated by gene fusion in 1%-2% of non-small cell lung cancers (NSCLC) and rarely in other cancer types. Selpercatinib is a highly selective RET kinase inhibitor that has recently been approved by the FDA in lung and thyroid cancers with activating RET gene fusions and mutations. Molecular mechanisms of acquired resistance to selpercatinib are poorly understood. PATIENTS AND METHODS: We studied patients treated on the first-in-human clinical trial of selpercatinib (NCT03157129) who were found to have MET amplification associated with resistance to selpercatinib. We validated MET activation as a targetable mediator of resistance to RET-directed therapy, and combined selpercatinib with the MET/ALK/ROS1 inhibitor crizotinib in a series of single patient protocols (SPP). RESULTS: MET amplification was identified in posttreatment biopsies in 4 patients with RET fusion-positive NSCLC treated with selpercatinib. In at least one case, MET amplification was clearly evident prior to therapy with selpercatinib. We demonstrate that increased MET expression in RET fusion-positive tumor cells causes resistance to selpercatinib, and this can be overcome by combining selpercatinib with crizotinib. Using SPPs, selpercatinib with crizotinib were given together generating anecdotal evidence of clinical activity and tolerability, with one response lasting 10 months. CONCLUSIONS: Through the use of SPPs, we were able to offer combination therapy targeting MET-amplified resistance identified on the first-in-human study of selpercatinib. These data suggest that MET dependence is a recurring and potentially targetable mechanism of resistance to selective RET inhibition in advanced NSCLC.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas , Ensayos Clínicos Fase I como Asunto , Crizotinib/farmacología , Crizotinib/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Amplificación de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Proyectos Piloto , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-ret/genética , Pirazoles/farmacología , Pirazoles/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Resultado del Tratamiento
8.
Sci Transl Med ; 13(609): eabb3738, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34516823

RESUMEN

The clinical efficacy of epidermal growth factor receptor (EGFR)­targeted therapy in EGFR-mutant non­small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor­resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.


Asunto(s)
Receptores ErbB , Neoplasias Pulmonares , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico
9.
J Comp Physiol B ; 188(3): 461-469, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29350264

RESUMEN

Skeletal muscle has diverse mechanical roles during locomotion. In swimming fish, power-producing muscles work in concert with the accessory muscles of the fins which augment and control power transfer to the water. Although fin muscles represent a significant proportion of the locomotor muscle mass, their physiological properties are poorly characterized. To examine the relationship between muscle metabolism and the differing mechanical demands placed on distinct muscle groups, we quantified the aerobic and glycolytic capacities of the myotomal, pectoral and caudal muscles of bluegill sunfish. These were indicated by the activities of citrate synthase and lactate dehydrogenase, rate-limiting enzymes for aerobic respiration and glycolysis, respectively. The well-established roles of slow and fast myotomal muscle types in sustained and transient propulsive movements allows their use as benchmarks to which other muscles can be compared to assess their function. Slow myotomal muscle had the highest CS activity, consistent with meeting the high metabolic and mechanical power demands of body-caudal fin (BCF) swimming at the upper end of the aerobically supported speed range. The largest pectoral adductors and abductors had CS activities lower than the slow myotomal muscle, in line with their role supplying thrust for low-speed, low-power swimming. The metabolic capacities of the caudal muscles were surprisingly low and inconsistent with their activity during steady-state BCF swimming at high speeds. This may reflect adaptation to the observed swimming behavior in the field, which typically involved short bouts of BCF-propulsive cycles rather than sustained propulsive activity.


Asunto(s)
Peces/fisiología , Músculo Esquelético/fisiología , Natación/fisiología , Animales , Citrato (si)-Sintasa/metabolismo , Proteínas de Peces/metabolismo , L-Lactato Deshidrogenasa/metabolismo
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