RESUMEN
Mutational activation of KRAS occurs commonly in lung carcinogenesis and, with the recent U.S. Food and Drug Administration approval of covalent inhibitors of KRASG12C such as sotorasib or adagrasib, KRAS oncoproteins are important pharmacological targets in non-small cell lung cancer (NSCLC). However, not all KRASG12C-driven NSCLCs respond to these inhibitors, and the emergence of drug resistance in those patients who do respond can be rapid and pleiotropic. Hence, based on a backbone of covalent inhibition of KRASG12C, efforts are underway to develop effective combination therapies. Here, we report that the inhibition of KRASG12C signaling increases autophagy in KRASG12C-expressing lung cancer cells. Moreover, the combination of DCC-3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRASG12C-driven lung cancer cell proliferation in vitro and superior tumor control in vivo. Additionally, in genetically engineered mouse models of KRASG12C-driven NSCLC, inhibition of either KRASG12C or ULK1/2 decreases tumor burden and increases mouse survival. Consequently, these data suggest that ULK1/2-mediated autophagy is a pharmacologically actionable cytoprotective stress response to inhibition of KRASG12C in lung cancer.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia , Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares , Proteínas Proto-Oncogénicas p21(ras) , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Ratones , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Modelos Animales de Enfermedad , Autofagia/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Antineoplásicos/farmacología , Pirimidinas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Piperazinas , PiridinasRESUMEN
Mutational activation of KRAS occurs commonly in lung carcinogenesis and, with the recent FDA approval of covalent inhibitors of KRAS G12C such as sotorasib or adagrasib, KRAS oncoproteins are important pharmacological targets in non-small cell lung cancer (NSCLC). However, not all KRAS G12C -driven NSCLCs respond to these inhibitors, and the emergence of drug resistance in those patients that do respond can be rapid and pleiotropic. Hence, based on a backbone of covalent inhibition of KRAS G12C , efforts are underway to develop effective combination therapies. Here we report that inhibition of KRAS G12C signaling increases autophagy in KRAS G12C expressing lung cancer cells. Moreover, the combination of DCC-3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRAS G12C -driven lung cancer cell proliferation in vitro and superior tumor control in vivo . Additionally, in genetically engineered mouse models of KRAS G12C -driven NSCLC, inhibition of either KRAS G12C or ULK1/2 decreases tumor burden and increases mouse survival. Consequently, these data suggest that ULK1/2-mediated autophagy is a pharmacologically actionable cytoprotective stress response to inhibition of KRAS G12C in lung cancer.
RESUMEN
Animal models, particularly genetically engineered mouse models (GEMMs), continue to have a transformative impact on our understanding of the initiation and progression of hematological malignancies and solid tumors. Furthermore, GEMMs have been employed in the design and optimization of potent anticancer therapies. Increasingly, drug responses are assessed in mouse models either prior, or in parallel, to the implementation of precision medical oncology, in which groups of patients with genetically stratified cancers are treated with drugs that target the relevant oncoprotein such that mechanisms of drug sensitivity or resistance may be identified. Subsequently, this has led to the design and preclinical testing of combination therapies designed to forestall the onset of drug resistance. Indeed, mouse models of human lung cancer represent a paradigm for how a wide variety of GEMMs, driven by a variety of oncogenic drivers, have been generated to study initiation, progression, and maintenance of this disease as well as response to drugs. These studies have now expanded beyond targeted therapy to include immunotherapy. We highlight key aspects of the relationship between mouse models and the evolution of therapeutic approaches, including oncogene-targeted therapies, immunotherapies, acquired drug resistance, and ways in which successful antitumor strategies improve on efficiently translating preclinical approaches into successful antitumor strategies in patients.
Asunto(s)
Neoplasias Pulmonares , Animales , Ratones , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Ingeniería Genética , Modelos Animales de EnfermedadRESUMEN
Mutationally activated BRAF is detected in approximately 7% of human lung adenocarcinomas, with BRAFT1799A serving as a predictive biomarker for treatment of patients with FDA-approved inhibitors of BRAFV600E oncoprotein signaling. In genetically engineered mouse (GEM) models, expression of BRAFV600E in the lung epithelium initiates growth of benign lung tumors that, without additional genetic alterations, rarely progress to malignant lung adenocarcinoma. To identify genes that cooperate with BRAFV600E for malignant progression, we used Sleeping Beauty-mediated transposon mutagenesis, which dramatically accelerated the emergence of lethal lung cancers. Among the genes identified was Rbms3, which encodes an RNA-binding protein previously implicated as a putative tumor suppressor. Silencing of RBMS3 via CRISPR/Cas9 gene editing promoted growth of BRAFV600E lung organoids and promoted development of malignant lung cancers with a distinct micropapillary architecture in BRAFV600E and EGFRL858R GEM models. BRAFV600E/RBMS3Null lung tumors displayed elevated expression of Ctnnb1, Ccnd1, Axin2, Lgr5, and c-Myc mRNAs, suggesting that RBMS3 silencing elevates signaling through the WNT/ß-catenin signaling axis. Although RBMS3 silencing rendered BRAFV600E-driven lung tumors resistant to the effects of dabrafenib plus trametinib, the tumors were sensitive to inhibition of porcupine, an acyltransferase of WNT ligands necessary for their secretion. Analysis of The Cancer Genome Atlas patient samples revealed that chromosome 3p24, which encompasses RBMS3, is frequently lost in non-small cell lung cancer and correlates with poor prognosis. Collectively, these data reveal the role of RBMS3 as a lung cancer suppressor and suggest that RBMS3 silencing may contribute to malignant NSCLC progression. SIGNIFICANCE: Loss of RBMS3 cooperates with BRAFV600E to induce lung tumorigenesis, providing a deeper understanding of the molecular mechanisms underlying mutant BRAF-driven lung cancer and potential strategies to more effectively target this disease.
Asunto(s)
Adenocarcinoma del Pulmón , Carcinogénesis , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Proteínas Proto-Oncogénicas B-raf , Proteínas de Unión al ARN , Transactivadores , Animales , Humanos , Ratones , Adenocarcinoma del Pulmón/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Proliferación Celular , Pulmón/patología , Neoplasias Pulmonares/genética , Mutagénesis , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas de Unión al ARN/genética , Transactivadores/metabolismo , Vía de Señalización Wnt , Carcinogénesis/genéticaRESUMEN
This article provides a brief review of the therapeutic opportunity of inhibiting autophagy in pancreatic cancer. The autophagic process, importance of autophagy in pancreatic cancer, relevant clinical trials, and new agents in preclinical and clinical development are discussed.
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Neoplasias Pancreáticas , Autofagia , Humanos , Neoplasias Pancreáticas/tratamiento farmacológicoRESUMEN
PURPOSE: Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma. EXPERIMENTAL DESIGN: We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity. RESULTS: Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death. CONCLUSIONS: These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.
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Cloroquina/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP/genética , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Mutación , Piridonas/farmacología , Pirimidinonas/farmacología , Neoplasias de la Úvea/tratamiento farmacológico , Animales , Antimaláricos/farmacología , Apoptosis , Proliferación Celular , Resistencia a Antineoplásicos , Quimioterapia Combinada , Humanos , Melanoma/genética , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Inhibidores de Proteínas Quinasas/farmacología , Células Tumorales Cultivadas , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
NTRK1 gene fusions are actionable drivers of numerous human malignancies. Here, we show that expression of the TPR-NTRK1 fusion kinase in immortalized mouse pancreatic ductal epithelial (IMPE) (pancreas) or mouse lung epithelial (MLE-12) cells is sufficient to promote rapidly growing tumors in mice. Both tumor models are exquisitely sensitive to targeted inhibition with entrectinib, a tropomyosin-related kinase A (TRKA) inhibitor. Initial regression of NTRK1-driven tumors is driven by induced expression of BIM, such that BIM silencing leads to a diminished response to entrectinib in vivo. However, the emergence of drug-resistant disease limits the long-term durability of responses. Based on the reactivation of RAF>MEK>ERK signaling observed in entrectinib-treated tumors, we show that the combination of entrectinib plus the MEK1/2 inhibitor cobimetinib dramatically forestalls the onset of drug resistance in vivo. Collectively, these data provide a mechanistic rationale for rapid clinical deployment of combined inhibition of TRKA plus MEK1/2 in NTRK1-driven cancers.
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Benzamidas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Indazoles/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Modelos Biológicos , Neoplasias/enzimología , Neoplasias/patología , Inhibidores de Proteínas Quinasas/farmacología , Receptor trkA/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteína 11 Similar a Bcl2/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Células HEK293 , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Masculino , Ratones Endogámicos NOD , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDA) was responsible for ~ 44,000 deaths in the United States in 2018 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable oncoprotein, KRAS1-4. Downstream of KRAS, the RAFâMEKâERK signaling pathway plays a central role in pancreatic carcinogenesis5. However, paradoxically, inhibition of this pathway has provided no clinical benefit to patients with PDA6. Here we show that inhibition of KRASâRAFâMEKâERK signaling elicits autophagy, a process of cellular recycling that protects PDA cells from the cytotoxic effects of KRAS pathway inhibition. Mechanistically, inhibition of MEK1/2 leads to activation of the LKB1âAMPKâULK1 signaling axis, a key regulator of autophagy. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro and promotes regression of xenografted patient-derived PDA tumors in mice. The observed effect of combination trametinib plus chloroquine was not restricted to PDA as other tumors, including patient-derived xenografts (PDX) of NRAS-mutated melanoma and BRAF-mutated colorectal cancer displayed similar responses. Finally, treatment of a patient with PDA with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers.
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Autofagia/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas ras/metabolismo , Animales , Antígeno CA-19-9/metabolismo , Línea Celular Tumoral , Cloroquina/farmacología , Humanos , Ratones SCID , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridonas/farmacología , Pirimidinonas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
In the version of this article initially published, the label over the bottom schematic in Fig. 1a was "pH > 5.0"; it should have been "pH < 5.0". Further, the original article misspelt the surname of Katrin P. Guillen as "Gullien". These errors have been corrected in the print, PDF and HTML versions of the article.
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Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells. We report here that human osteosarcoma (U2OS, MG63, OS15 and SaOS2), breast cancer (MCF7), and primary human fibroblastic cells (HFF) all exhibit OTR not only on the cell membrane, but also in the various nuclear compartments including the nucleolus. Both an OTR-GFP fusion protein and the native OTR appear to be localized to the nucleus as detected by transfection and/or confocal immunofluorescence, respectively. Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus. Western blots indicate that OTR in the nucleus and on the plasma membrane are likely to be the same biochemical and immunological entities. It appears that OTR is first visible in the nucleoli and subsequently disperses within the nucleus into 4-20 spots while some of the OTR diffuses throughout the nucleoplasm. The behaviour and kinetics of OTR-GFP and OTR are different, indicating interference by GFP in both OTR entrance into the nucleus and subsequent relocalization of OTR within the nucleus. There are important differences among the tested cells, such as the requirement of a ligand for transfer of OTR in nuclei. A constitutive internalization of OTR was found only in osteosarcoma cells, while the nuclear localization in all other tested cells was dependent on ligand binding. The amount of OTR-positive material within and in the vicinity of the nucleus increased following a treatment with oxytocin in both constitutive and ligand-dependent type of cells. The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization.
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Núcleo Celular/metabolismo , Oxitocina/metabolismo , Receptores de Oxitocina/metabolismo , Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Femenino , Fibroblastos/metabolismo , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica , Cinética , Ligandos , Microscopía Confocal , Osteosarcoma/patología , Plásmidos , Unión Proteica , Receptores de Oxitocina/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
A continuous cell line, MC3T3-E1 cells, originally derived from murine calvaria bones, loses its osteogenic properties as a result of extended passage number under stress conditions. These aged/stressed MC3T3-S cells, although nontumorigenic, do not display some of the osteogenic properties characteristic of the MC3T3-E1 cells. Altered properties include low expression of alkaline phosphatase, diminished collagen synthesis and inability to form mineralized nodules in vitro. We attempted to reactivate these osteogenic properties by transfections with a pSV2neo plasmid containing the TGFbeta1 gene. During these experiments we found that transfected MC3T3-S cells not only acquired high alkaline phosphatase activity and a potent mineralization potential, but also properties akin to the transformed state, such as ability to grow in soft agar and ability to produce tumors in immunodeficient animals. Further analysis showed that the TGFbeta1 gene is not required and that the changes can be introduced by transfections with pSV2neo alone. In contrast, MC3T3-S cells transfected with pcDNA3 (a plasmid containing only the SV40 origin of replication, early promoter, enhancer and polyadenylation signals) or mock-transfected MC3T3-S cells did not show any transformation traits. The results identify two additional SV40 fragments present in pSV2neo (SV40 virus sequence; Genbank accession number: NC_001669: 4100-4191 and 2668-2774) as functional elements contributing to the transformation of aged/stressed and immortalized osteoblastic cells. These findings are analogous to earlier reports describing the cell modifying potential of pSV2neo. We conclude that stressed and aged MC3T3-S can be transformed by transfection with pSV2neo and that such cells acquire not only the tumorigenic potential but exhibit also some of the osteogenic properties characteristic of the parent MC3T3-E1 cells.