RESUMO
Cells survive harsh environmental conditions by potently upregulating molecular chaperones such as heat shock proteins (HSPs), particularly the inducible members of the HSP70 family. The life cycle of HSP70 mRNA in the cytoplasm is unique-it is translated during stress when most cellular mRNA translation is repressed and rapidly degraded upon recovery. Contrary to its 5' untranslated region's role in maximizing translation, we discovered that the HSP70 coding sequence (CDS) suppresses its translation via the ribosome quality control (RQC) mechanism. The CDS of the most inducible Saccharomyces cerevisiae HSP70 gene, SSA4, is uniquely enriched with low-frequency codons that promote ribosome stalling during heat stress. Stalled ribosomes are recognized by the RQC components Asc1p and Hel2p and two novel RQC components, the ribosomal proteins Rps28Ap and Rps19Bp. Surprisingly, RQC does not signal SSA4 mRNA degradation via No-Go-Decay. Instead, Asc1p destabilizes SSA4 mRNA during recovery from heat stress by a mechanism independent of ribosome binding and SSA4 codon optimality. Therefore, Asc1p operates in two pathways that converge to regulate the SSA4 mRNA life cycle during stress and recovery. Our research identifies Asc1p as a critical regulator of the stress response and RQC as the mechanism tuning HSP70 synthesis.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Códon/metabolismo , Biossíntese de ProteínasRESUMO
A series of macrocyclic ligands were considered for the chelation of Pb2+: 1,4,7,10-tetrakis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO4S), 1,4,7-tris[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO3S), 1,4,7-tris[2-(methylsulfanyl)ethyl]-10-acetamido-1,4,7,10-tetraazacyclododecane (DO3SAm), 1,7-bis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,10-diacetic acid (DO2A2S), 1,5,9-tris[2-(methylsulfanyl)ethyl]-1,5,9-triazacyclododecane (TACD3S), 1,4,7,10-tetrakis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetrazacyclotridecane (TRI4S), and 1,4,8,11-tetrakis[2-(methylsulfanyl)ethyl]-1,4,8,11-tetrazacyclotetradecane (TE4S). The equilibrium, the acid-mediated dissociation kinetics, and the structural properties of the Pb2+ complexes formed by these chelators were examined by UV-Visible and nuclear magnetic resonance (NMR) spectroscopies, combined with potentiometry and density functional theory (DFT) calculations. The obtained results indicated that DO4S, DO3S, DO3SAm, and DO2A2S were able to efficiently chelate Pb2+ and that the most suitable macrocyclic scaffold for Pb2+ is 1,4,7,10-tetrazacyclododecane. NMR spectroscopy gave insights into the solution structures of the Pb2+ complexes, and 1H-207Pb interactions confirmed the involvement of S and/or O donors in the metal coordination sphere. Highly fluxional solution behavior was discovered when Pb2+ was coordinated to symmetric ligands (i.e., DO4S and DO2A2S) while the introduction of structural asymmetry in DO3S and DO3SAm slowed down the intramolecular dynamics. The ligand ability to chelate [203Pb]Pb2+ under highly dilute reaction conditions was explored through radiolabeling experiments. While DO4S and DO3S possessed modest performance, DO3SAm and DO2A2S demonstrated high complexation efficiency under mild reaction conditions (pH = 7, 5 min reaction time). The [203Pb]Pb2+ complexes' integrity in human serum over 24 h was appreciably good for [203Pb][Pb(DO4S)]2+ (80 ± 5%) and excellent for [203Pb][Pb(DO3SAm)]2+ (93 ± 1%) and [203Pb][Pb(DO2A2S)] (94 ± 1%). These results reveal the promise of DO2A2S and DO3SAm as chelators in cutting-edge theranostic [203/212Pb]Pb2+ radiopharmaceuticals.
Assuntos
Ciclamos , Chumbo , Humanos , Medicina de Precisão , Quelantes/química , LigantesRESUMO
Melanoma progression is generally associated with increased transcriptional activity mediated by the Yes-associated protein (YAP). Mechanical signals from the extracellular matrix are sensed by YAP, which then activates the expression of proliferative genes, promoting melanoma progression and drug resistance. Which extracellular signals induce mechanotransduction, and how this is mediated, is not completely understood. Here, using secretome analyses, we reveal the extracellular accumulation of amyloidogenic proteins, i.e. premelanosome protein (PMEL), in metastatic melanoma, together with proteins that assist amyloid maturation into fibrils. We also confirm the accumulation of amyloid-like aggregates, similar to those detected in Alzheimer disease, in metastatic cell lines, as well as in human melanoma biopsies. Mechanistically, beta-secretase 2 (BACE2) regulates the maturation of these aggregates, which in turn induce YAP activity. We also demonstrate that recombinant PMEL fibrils are sufficient to induce mechanotransduction, triggering YAP signaling. Finally, we demonstrate that BACE inhibition affects cell proliferation and increases drug sensitivity, highlighting the importance of amyloids for melanoma survival, and the use of beta-secretase inhibitors as potential therapeutic approach for metastatic melanoma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Melanoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Amiloidogênicas , Humanos , Mecanotransdução Celular , Melanoma/tratamento farmacológico , Melanoma/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
MET is a master gene controlling a genetic program driving proliferation, apoptosis protection and invasion. The ROR1 pseudokinase acts as a MET substrate. However, its contribution to MET signaling and MET-dependent biological outcomes remains to be elucidated. By structure-function analysis of ROR1 mutants, we show that ROR1 encompasses two major substrate regions: one is located in the proline-rich domain and is directly phosphorylated by MET; the other resides in the pseudokinase domain and is phosphorylated through intermediate activation of SRC. Differential phosphorylation of these two regions dictates the execution of specific responses: phosphorylation of the ROR1 proline-rich domain by MET-but not phosphorylation of the pseudokinase domain by SRC-is necessary and sufficient to control MET-driven proliferation and protection from apoptosis. Differently, both the proline-rich and the pseudokinase domains mediate cell invasion. Consistent with the role of ROR1 in specifying the functional consequences of MET-dependent signals, ROR1 silencing leads to selective attenuation of only some of the signal transduction pathways sustained by MET. These data enlighten the so far elusive function(s) of pseudokinases and identify a mechanism of biological diversification, based on substrate specificity of oncogenic kinases.
Assuntos
Proteínas Proto-Oncogênicas c-met/metabolismo , Pseudogenes/fisiologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Animais , Apoptose , Western Blotting , Células COS , Movimento Celular , Proliferação de Células , Células Cultivadas , Chlorocebus aethiops , Humanos , Imunoprecipitação , Lentivirus/genética , Fosforilação , Plasmídeos/genética , Proteínas Proto-Oncogênicas c-met/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genéticaRESUMO
The complexes formed between Pb2+ and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were reinvestigated in aqueous solutions using a combination of pH potentiometry, UV-vis spectroscopy, and NMR spectroscopy. The thermodynamic data were supported by kinetics assays. Differently protonated complexes, i.e., [PbH3L]+, [PbH2L], [PbHL]-, and [PbL]2-, were detected, and the corresponding stability constants (logß) at T = 298 K and I = 0.1 M NaCl were 33.1 ± 0.2, 32.00 ± 0.06, 29.28 ± 0.06, and 25.3 ± 0.1, respectively. Results differed significantly from those previously reported by Chaves et al. (Talanta1992, 39, 249) and Pippin et al. (Inorg. Chim. Acta1995, 239, 43) in both the speciation and the overall complex stability; the latter in particular was found to be remarkably higher. The work disclosed herein provides revised data on the Pb2+-DOTA complexes, which should be used as a new stability benchmark during the development of lead chelators.
RESUMO
The purpose of this paper is to develop and validate a delta-radiomics score to predict the response of individual colorectal cancer liver metastases (lmCRC) to first-line FOLFOX chemotherapy. Three hundred one lmCRC were manually segmented on both CT performed at baseline and after the first cycle of first-line FOLFOX, and 107 radiomics features were computed by subtracting textural features of CT at baseline from those at timepoint 1 (TP1). LmCRC were classified as nonresponders (R-) if they showed progression of disease (PD), according to RECIST1.1, before 8 months, and as responders (R+), otherwise. After feature selection, we developed a decision tree statistical model trained using all lmCRC coming from one hospital. The final output was a delta-radiomics signature subsequently validated on an external dataset. Sensitivity, specificity, positive (PPV), and negative (NPV) predictive values in correctly classifying individual lesions were assessed on both datasets. Per-lesion sensitivity, specificity, PPV, and NPV were 99%, 94%, 95%, 99%, 85%, 92%, 90%, and 87%, respectively, in the training and validation datasets. The delta-radiomics signature was able to reliably predict R- lmCRC, which were wrongly classified by lesion RECIST as R+ at TP1, (93%, averaging training and validation set, versus 67% of RECIST). The delta-radiomics signature developed in this study can reliably predict the response of individual lmCRC to oxaliplatin-based chemotherapy. Lesions forecasted as poor or nonresponders by the signature could be further investigated, potentially paving the way to lesion-specific therapies.
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The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient and unresponsive to immunotherapy, whereas MMR-deficient (MMRd) tumors often respond to immune-checkpoint blockade. We previously reported that the treatment of colorectal cancer preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB), and sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-methylguanine-DNA-methyltransferase (MGMT)-deficient, MMR-proficient, RAS-mutant mCRC patients received priming therapy with TMZ. Analysis of tissue biopsies and circulating tumor DNA (ctDNA) revealed the emergence of a distinct mutational signature and increased TMB after TMZ treatment. Multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes, and the p.T1219I MSH6 variant was detected in ctDNA and tissue of 94% (16/17) of the cases. A subset of patients whose tumors displayed the MSH6 mutation, the TMZ mutational signature, and increased TMB achieved disease stabilization upon pembrolizumab treatment. SIGNIFICANCE: MMR-proficient mCRCs are unresponsive to immunotherapy. We provide the proof of concept that inactivation of MMR genes can be achieved pharmacologically with TMZ and molecularly monitored in the tissue and blood of patients with mCRC. This strategy deserves additional evaluation in mCRC patients whose tumors are no longer responsive to standard-of-care treatments. See related commentary by Willis and Overman, p. 1612. This article is highlighted in the In This Issue feature, p. 1599.
Assuntos
Neoplasias Encefálicas , Neoplasias Colorretais , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA , Proteínas de Ligação a DNA/genética , Dacarbazina/uso terapêutico , Humanos , Mutação , O(6)-Metilguanina-DNA Metiltransferase/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Temozolomida/farmacologia , Temozolomida/uso terapêuticoRESUMO
Metastasis is facilitated by the formation of a "premetastatic niche," which is fostered by primary tumor-derived factors. Colorectal cancer (CRC) metastasizes mainly to the liver. We show that the premetastatic niche in the liver is induced by bacteria dissemination from primary CRC. We report that tumor-resident bacteria Escherichia coli disrupt the gut vascular barrier (GVB), an anatomical structure controlling bacterial dissemination along the gut-liver axis, depending on the virulence regulator VirF. Upon GVB impairment, bacteria disseminate to the liver, boost the formation of a premetastatic niche, and favor the recruitment of metastatic cells. In training and validation cohorts of CRC patients, we find that the increased levels of PV-1, a marker of impaired GVB, is associated with liver bacteria dissemination and metachronous distant metastases. Thus, PV-1 is a prognostic marker for CRC distant recurrence and vascular impairment, leading to liver metastases.
Assuntos
Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/patologia , Neoplasias Hepáticas/patologia , Metástase Neoplásica/patologia , Recidiva Local de Neoplasia/patologia , Bactérias/isolamento & purificação , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Humanos , Fígado/patologia , Neoplasias Hepáticas/secundárioRESUMO
The long-term efficacy of the Epidermal Growth Factor Receptor (EGFR)-targeted antibody cetuximab in advanced colorectal cancer (CRC) patients is limited by the emergence of drug-resistant (persister) cells. Recent studies in other cancer types have shown that cells surviving initial treatment with targeted agents are often vulnerable to alterations in cell metabolism including oxidative stress. Vitamin C (VitC) is an antioxidant agent which can paradoxically trigger oxidative stress at pharmacological dose. Here we tested the hypothesis that VitC in combination with cetuximab could restrain the emergence of secondary resistance to EGFR blockade in CRC RAS/BRAF wild-type models. We found that addition of VitC to cetuximab impairs the emergence of drug persisters, limits the growth of CRC organoids, and significantly delays acquired resistance in CRC patient-derived xenografts. Mechanistically, proteomic and metabolic flux analysis shows that cetuximab blunts carbohydrate metabolism by blocking glucose uptake and glycolysis, beyond promoting slow but progressive ROS production. In parallel, VitC disrupts iron homeostasis and further increases ROS levels ultimately leading to ferroptosis. Combination of VitC and cetuximab orchestrates a synthetic lethal metabolic cell death program triggered by ATP depletion and oxidative stress, which effectively limits the emergence of acquired resistance to anti-EGFR antibodies. Considering that high-dose VitC is known to be safe in cancer patients, our findings might have clinical impact on CRC patients treated with anti-EGFR therapies.
RESUMO
PURPOSE: Defects in the homologous recombination (HR) repair pathway are of clinical interest due to sensitivity of HR-deficient cells to PARP inhibitors. We were interested in defining PARP vulnerability in patients with metastatic colorectal cancer (mCRC) carrying KRAS and BRAF mutations who display poor prognosis, have limited therapeutic options, and represent an unmet clinical need. EXPERIMENTAL DESIGN: We tested colorectal cancer cell lines, patient-derived organoids (PDO), and patient-derived xenografts (PDX) enriched for KRAS and BRAF mutations for sensitivity to the PARP inhibitor olaparib, and the chemotherapeutic agents oxaliplatin and 5-fluorouracil (5-FU). Genomic profiles and DNA repair proficiency of colorectal cancer models were compared with pharmacologic response. RESULTS: Thirteen of 99 (around 13%) colorectal cancer cell lines were highly sensitive to clinically active concentrations of olaparib and displayed functional deficiency in HR. Response to PARP blockade was positively correlated with sensitivity to oxaliplatin in colorectal cancer cell lines as well as patient-derived organoids. Treatment of PDXs with olaparib impaired tumor growth and maintenance therapy with PARP blockade after initial oxaliplatin response delayed disease progression in mice. CONCLUSIONS: These results indicate that a colorectal cancer subset characterized by poor prognosis and limited therapeutic options is vulnerable to PARP inhibition and suggest that PDO-based drug-screening assays can be used to identify patients with colorectal cancer likely to benefit from olaparib. As patients with mCRC almost invariably receive therapies based on oxaliplatin, "maintenance" treatment with PARP inhibitors warrants further clinical investigation.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Oxaliplatina/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Reparo de DNA por Recombinação , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Patient-derived xenograft (PDX) models accurately recapitulate the tumor of origin in terms of histopathology, genomic landscape, and therapeutic response, but some limitations due to costs associated with their maintenance and restricted amenability for large-scale screenings still exist. To overcome these issues, we established a platform of 2D cell lines (xeno-cell lines, XL), derived from PDXs of colorectal cancer with matched patient germline gDNA available. EXPERIMENTAL DESIGN: Whole-exome and transcriptome sequencing analyses were performed. Biomarkers of response and resistance to anti-HER therapy were annotated. Dependency on the WRN helicase gene was assessed in MSS, MSI-H, and MSI-like XLs using a reverse genetics functional approach. RESULTS: XLs recapitulated the entire spectrum of colorectal cancer transcriptional subtypes. Exome and RNA-seq analyses delineated several molecular biomarkers of response and resistance to EGFR and HER2 blockade. Genotype-driven responses observed in vitro in XLs were confirmed in vivo in the matched PDXs. MSI-H models were dependent upon WRN gene expression, while loss of WRN did not affect MSS XLs growth. Interestingly, one MSS XL with transcriptional MSI-like traits was sensitive to WRN depletion. CONCLUSIONS: The XL platform represents a preclinical tool for functional gene validation and proof-of-concept studies to identify novel druggable vulnerabilities in colorectal cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Instabilidade de Microssatélites , Adulto , Idoso , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Estudos de Coortes , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Feminino , Dosagem de Genes , Humanos , Lapatinib/farmacologia , Lapatinib/uso terapêutico , Masculino , Camundongos , Pessoa de Meia-Idade , Medicina de Precisão , Cultura Primária de Células , RNA-Seq , Reto/patologia , Reto/cirurgia , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Resultado do Tratamento , Helicase da Síndrome de Werner/genética , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Attempts at eradicating metastatic cancers with targeted therapies are limited by the emergence of resistant subclones bearing heterogeneous (epi)genetic changes. We used colorectal cancer (CRC) to test the hypothesis that interfering with an ancestral oncogenic event shared by all the malignant cells (such as WNT pathway alterations) could override heterogeneous mechanisms of acquired drug resistance. Here, we report that in CRC-resistant cell populations, phylogenetic analysis uncovers a complex subclonal architecture, indicating parallel evolution of multiple independent cellular lineages. Functional and pharmacological modulation of WNT signalling induces cell death in CRC preclinical models from patients that relapsed during the treatment, regardless of the drug type or resistance mechanisms. Concomitant blockade of WNT and MAPK signalling restrains the emergence of drug-resistant clones. Reliance upon the WNT-APC pathway is preserved throughout the branched genomic drift associated with emergence of treatment relapse, thus offering the possibility of a common therapeutic strategy to overcome secondary drug resistance.
Assuntos
Neoplasias Colorretais/genética , Deriva Genética , Terapia de Alvo Molecular , Mutação , Animais , Biópsia , Técnicas de Cultura de Células , Linhagem da Célula , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Recidiva Local de Neoplasia , Transplante de Neoplasias , Oncogenes , Filogenia , Transdução de Sinais , Via de Sinalização WntRESUMO
Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Lapatinib/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Receptor ErbB-2/antagonistas & inibidores , Tomografia Computadorizada por Raios X , Trastuzumab/administração & dosagem , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/genética , Adenocarcinoma/secundário , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Classe I de Fosfatidilinositol 3-Quinases/genética , Tomada de Decisão Clínica , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Progressão da Doença , Feminino , Amplificação de Genes , Humanos , Itália , Lapatinib/efeitos adversos , Biópsia Líquida , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Imageamento por Ressonância Magnética , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Valor Preditivo dos Testes , Intervalo Livre de Progressão , Inibidores de Proteínas Quinases/efeitos adversos , Receptor ErbB-2/genética , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Trastuzumab/efeitos adversos , Resultado do Tratamento , Células Tumorais Cultivadas , Proteínas ras/genéticaRESUMO
Background: ALK, ROS1, and NTRK fusions occur in 0.2% to 2.4% of colorectal cancers. Pioneer cases of metastatic colorectal cancer (mCRC) patients bearing rearrangements who benefited from anti-ALK, ROS, and TrkA-B-C therapies have been reported previously. Here we aimed at characterizing the clinical and molecular landscape of ALK, ROS1, and NTRK rearranged mCRC. Methods: Clinical features and molecular characteristics of 27 mCRC patients bearing ALK, ROS1, and NTRK rearranged tumors were compared with those of a cohort of 319 patients not bearing rearrangements by means of Fisher's exact, χ2 test, or Mann-Whitney test as appropriate. Overall survival curves were estimated with the Kaplan-Meier method and compared using the log-rank test. A Cox proportional hazard model was adopted in the multivariable analysis. Deep molecular and immunophenotypic characterizations of rearranged cases, including those described in The Cancer Genome Atlas database, were performed. All statistical tests were two-sided. Results: Closely recalling the "BRAF history," ALK, ROS1, and NTRK rearrangements more frequently occurred in elderly patients (P = .02) with right-sided tumors (P < .001) and node-spreading (P = .03), RAS wild-type (P < .001), and MSI-high (P < .001) cancers. All patients bearing ALK, ROS1, and NTRK fusions had shorter overall survival (15.6 months, 95% confidence interval [CI] = 0.0 to 20.4 months) than negative patients (33.7 months, 95% CI = 28.3 to 42.1 months), both in the univariate (hazard ratio [HR] = 2.17, 95% CI = 1.03 to 4.57, P < .001) and multivariable models (HR = 2.33, 95% CI = 1.10 to 4.95, P = .02). All four evaluable patients with rearrangements showed primary resistance to anti-epidermal growth factor receptor agents. Frequent association with potentially targetable RNF43 mutations was observed in MSI-high rearranged tumors. Conclusions: ALK, ROS1, and NTRK rearrangements define a new rare subtype of mCRC with extremely poor prognosis. Primary tumor site, MSI-high, and RAS and BRAF wild-type status may help to identify patients bearing these alterations. While sensitivity to available treatments is limited, targeted strategies inhibiting ALK, ROS, and TrkA-B-C provided encouraging results.
Assuntos
Quinase do Linfoma Anaplásico/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Rearranjo Gênico , Neoplasias Hepáticas/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptor trkA/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Terapia Combinada , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/patologia , Neoplasias Peritoneais/terapia , Prognóstico , Taxa de Sobrevida , Adulto JovemRESUMO
The anti-epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab are used to treat RAS wild-type colorectal cancers (CRCs), but their efficacy is limited by the emergence of acquired drug resistance. After EGFR blockade, about 20% of CRCs develop mutations in the EGFR extracellular domain (ECD) that impair antibody binding and are associated with clinical relapse. We hypothesized that EGFR ECD-resistant variants could be targeted by the recently developed oligoclonal antibody MM-151 that binds multiple regions of the EGFR ECD. MM-151 inhibits EGFR signaling and cell growth in preclinical models, including patient-derived cells carrying mutant EGFR. Upon MM-151 treatment, EGFR ECD mutations decline in circulating cell-free tumor DNA (ctDNA) of CRC patients who previously developed resistance to EGFR blockade. These data provide molecular rationale for the clinical use of MM-151 in patients who become resistant to cetuximab or panitumumab as a result of EGFR ECD mutations.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Mutação/genética , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Sistema Livre de Células , Cetuximab/farmacologia , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Epitopos/química , Receptores ErbB/química , Células HEK293 , Humanos , Ligantes , Panitumumabe , Domínios Proteicos , Transdução de Sinais/efeitos dos fármacosRESUMO
Blockade of the epidermal growth factor receptor (EGFR) with the monoclonal antibodies cetuximab or panitumumab is effective in a subset of colorectal cancers (CRCs), but the emergence of resistance limits the efficacy of these therapeutic agents. At relapse, the majority of patients develop RAS mutations, while a subset acquires EGFR extracellular domain (ECD) mutations. Here we find that patients who experience greater and longer responses to EGFR blockade preferentially develop EGFR ECD mutations, while RAS mutations emerge more frequently in patients with smaller tumour shrinkage and shorter progression-free survival. In circulating cell-free tumour DNA of patients treated with anti-EGFR antibodies, RAS mutations emerge earlier than EGFR ECD variants. Subclonal RAS but not EGFR ECD mutations are present in CRC samples obtained before exposure to EGFR blockade. These data indicate that clonal evolution of drug-resistant cells is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Genes erbB-1 , Genes ras , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/farmacologia , Evolução Clonal , Neoplasias Colorretais/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
UNLABELLED: Entrectinib is a first-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profiling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1, p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confirmed that either mutation renders the TRKA kinase insensitive to entrectinib. These findings can be immediately exploited to design next-generation TRKA inhibitors. SIGNIFICANCE: We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identification of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the first time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements.
Assuntos
Benzamidas/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Indazóis/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Receptor trkA/genética , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Rearranjo Gênico , Humanos , Camundongos , Mutação , Transplante de Neoplasias , Células Neoplásicas Circulantes/patologia , Receptor trkA/químicaRESUMO
UNLABELLED: How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent therapy is unknown. We studied EGFR blockade in colorectal cancer to assess whether tissue and liquid biopsies can be integrated with radiologic imaging to monitor the impact of individual oncogenic alterations on lesion-specific responses. Biopsy of a patient's progressing liver metastasis following prolonged response to cetuximab revealed a MEK1(K57T) mutation as a novel mechanism of acquired resistance. This lesion regressed upon treatment with panitumumab and the MEK inhibitor trametinib. In circulating tumor DNA (ctDNA), mutant MEK1 levels declined with treatment, but a previously unrecognized KRAS(Q61H) mutation was also identified that increased despite therapy. This same KRAS mutation was later found in a separate nonresponding metastasis. In summary, parallel analyses of tumor biopsies and serial ctDNA monitoring show that lesion-specific radiographic responses to subsequent targeted therapies can be driven by distinct resistance mechanisms arising within separate tumor lesions in the same patient. SIGNIFICANCE: Molecular heterogeneity ensuing from acquired resistance drives lesion-specific responses to subsequent targeted therapies. Analysis of a single-lesion biopsy is inadequate to guide selection of subsequent targeted therapies. ctDNA profiles allow the detection of concomitant resistance mechanisms residing in separate metastases and assessment of the effect of therapies designed to overcome resistance.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas/tratamento farmacológico , MAP Quinase Quinase 1/genética , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular Tumoral , Cetuximab/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA de Neoplasias/sangue , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Terapia de Alvo Molecular , Panitumumabe , Medicina de Precisão , Resultado do TratamentoRESUMO
PURPOSE: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models. EXPERIMENTAL DESIGN: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance. RESULTS: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab. CONCLUSIONS: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor-antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies.
Assuntos
Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Genes erbB-1/genética , Mutação , Antineoplásicos/uso terapêutico , Western Blotting , Linhagem Celular Tumoral , Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Análise Mutacional de DNA , Espaço Extracelular/genética , Citometria de Fluxo , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In clinical practice, targeted therapies are usually administered together with chemotherapeutics. However, little is known whether conventional cytotoxic agents enhance the efficacy of targeted compounds, and whether a possible synergy would be dictated by drug-sensitizing genetic alterations. To explore these issues, we leveraged the design of clinical studies in humans to conduct a multi-arm trial in an 'in-cell' format. Using the MET oncogene as a model target and a panel of genetically characterized cell lines as a reference population, we found that two different chemotherapeutic regimens - cisplatin and 5-fluorouracil - exerted widespread cytotoxic activity that was not further enhanced by MET inhibition with a monovalent anti-MET antibody. From a complementary perspective, targeted MET inhibition was successful in a selected complement of cells harboring MET genomic lesions. In this latter setting, addition of chemotherapy did not provide a therapeutic advantage. Mechanistically, chemotherapeutics did not influence the basal activity of MET in cells with normal MET genomic status nor did they contribute to neutralize MET signals in cells with MET amplification. These data suggest that tumors displaying MET aberrations achieve plateau responses by MET monotherapy and do not receive further benefit by addition of cytotoxic treatments.