RESUMO
KRAS inhibitors demonstrate clinical efficacy in pancreatic ductal adenocarcinoma (PDAC); however, resistance is common. Among patients with KRASG12C-mutant PDAC treated with adagrasib or sotorasib, mutations in PIK3CA and KRAS, and amplifications of KRASG12C, MYC, MET, EGFR, and CDK6 emerged at acquired resistance. In PDAC cell lines and organoid models treated with the KRASG12D inhibitor MRTX1133, epithelial-to-mesenchymal transition and PI3K-AKT-mTOR signaling associate with resistance to therapy. MRTX1133 treatment of the KrasLSL-G12D/+;Trp53LSL-R172H/+;p48-Cre (KPC) mouse model yielded deep tumor regressions, but drug resistance ultimately emerged, accompanied by amplifications of Kras, Yap1, Myc, and Cdk6/Abcb1a/b, and co-evolution of drug-resistant transcriptional programs. Moreover, in KPC and PDX models, mesenchymal and basal-like cell states displayed increased response to KRAS inhibition compared to the classical state. Combination treatment with KRASG12D inhibition and chemotherapy significantly improved tumor control in PDAC mouse models. Collectively, these data elucidate co-evolving resistance mechanisms to KRAS inhibition and support multiple combination therapy strategies.
RESUMO
BACKGROUND & AIMS: Transforming growth factor-b (TGFb) plays pleiotropic roles in pancreatic cancer, including promoting metastasis, attenuating CD8 T-cell activation, and enhancing myofibroblast differentiation and deposition of extracellular matrix. However, single-agent TGFb inhibition has shown limited efficacy against pancreatic cancer in mice or humans. METHODS: We evaluated the TGFß-blocking antibody NIS793 in combination with gemcitabine/nanoparticle (albumin-bound)-paclitaxel or FOLFIRINOX (folinic acid [FOL], 5-fluorouracil [F], irinotecan [IRI] and oxaliplatin [OX]) in orthotopic pancreatic cancer models. Single-cell RNA sequencing and immunofluorescence were used to evaluate changes in tumor cell state and the tumor microenvironment. RESULTS: Blockade of TGFß with chemotherapy reduced tumor burden in poorly immunogenic pancreatic cancer, without affecting the metastatic rate of cancer cells. Efficacy of combination therapy was not dependent on CD8 T cells, because response to TGFß blockade was preserved in CD8-depleted or recombination activating gene 2 (RAG2-/-) mice. TGFß blockade decreased total α-smooth muscle actin-positive fibroblasts but had minimal effect on fibroblast heterogeneity. Bulk RNA sequencing on tumor cells sorted ex vivo revealed that tumor cells treated with TGFß blockade adopted a classical lineage consistent with enhanced chemosensitivity, and immunofluorescence for cleaved caspase 3 confirmed that TGFß blockade increased chemotherapy-induced cell death in vivo. CONCLUSIONS: TGFß regulates pancreatic cancer cell plasticity between classical and basal cell states. TGFß blockade in orthotropic models of pancreatic cancer enhances sensitivity to chemotherapy by promoting a classical malignant cell state. This study provides scientific rationale for evaluation of NIS793 with FOLFIRINOX or gemcitabine/nanoparticle (albumin-bound) paclitaxel chemotherapy backbone in the clinical setting and supports the concept of manipulating cancer cell plasticity to increase the efficacy of combination therapy regimens.
Assuntos
Antineoplásicos , Neoplasias Pancreáticas , Humanos , Camundongos , Animais , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Fator de Crescimento Transformador beta/metabolismo , Antineoplásicos/uso terapêutico , Gencitabina , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Albuminas , Fatores de Crescimento Transformadores/uso terapêutico , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.
Assuntos
Neoplasias , Quimera de Direcionamento de Proteólise , Camundongos , Animais , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias/tratamento farmacológico , Transdução de Sinais , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/químicaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in > 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell-cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC. SIGNIFICANCE: A high-throughput pipeline using multiplex immunofluorescence in pancreatic cancer reveals striking expression subtype intratumoral heterogeneity with implications for therapy selection and identifies co-expressor cells that may serve as intermediates during subtype switching.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Prognóstico , Fenótipo , RNA , Regulação Neoplásica da Expressão Gênica , ClaudinasRESUMO
Pancreatic ductal adenocarcinomas (PDAC) depend on autophagy for survival; however, the metabolic substrates that autophagy provides to drive PDAC progression are unclear. Ferritin, the cellular iron storage complex, is targeted for lysosomal degradation (ferritinophagy) by the selective autophagy adaptor NCOA4, resulting in release of iron for cellular utilization. Using patient-derived and murine models of PDAC, we demonstrate that ferritinophagy is upregulated in PDAC to sustain iron availability, thereby promoting tumor progression. Quantitative proteomics reveals that ferritinophagy fuels iron-sulfur cluster protein synthesis to support mitochondrial homeostasis. Targeting NCOA4 leads to tumor growth delay and prolonged survival but with the development of compensatory iron acquisition pathways. Finally, enhanced ferritinophagy accelerates PDAC tumorigenesis, and an elevated ferritinophagy expression signature predicts for poor prognosis in patients with PDAC. Together, our data reveal that the maintenance of iron homeostasis is a critical function of PDAC autophagy, and we define NCOA4-mediated ferritinophagy as a therapeutic target in PDAC. SIGNIFICANCE: Autophagy and iron metabolism are metabolic dependencies in PDAC. However, targeted therapies for these pathways are lacking. We identify NCOA4-mediated selective autophagy of ferritin ("ferritinophagy") as upregulated in PDAC. Ferritinophagy supports PDAC iron metabolism and thereby tumor progression and represents a new therapeutic target in PDAC. See related commentary by Jain and Amaravadi, p. 2023. See related article by Ravichandran et al., p. 2198. This article is highlighted in the In This Issue feature, p. 2007.
Assuntos
Carcinoma Ductal Pancreático , Proteínas Ferro-Enxofre , Neoplasias Pancreáticas , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Disponibilidade Biológica , Carcinoma Ductal Pancreático/genética , Ferritinas/genética , Ferritinas/metabolismo , Humanos , Ferro/metabolismo , Ferro/farmacologia , Proteínas Ferro-Enxofre/metabolismo , Camundongos , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Neoplasias Pancreáticas/genética , Enxofre/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias PancreáticasRESUMO
The mechanisms underlying metabolic adaptation of pancreatic ductal adenocarcinoma (PDA) cells to pharmacologic inhibition of RAS-MAPK signaling are largely unknown. Using transcriptome and chromatin immunoprecipitation profiling of PDA cells treated with the MEK inhibitor (MEKi) trametinib, we identify transcriptional antagonism between c-MYC and the master transcription factors for lysosome gene expression, the MiT/TFE proteins. Under baseline conditions, c-MYC and MiT/TFE factors compete for binding to lysosome gene promoters to fine-tune gene expression. Treatment of PDA cells or patient organoids with MEKi leads to c-MYC downregulation and increased MiT/TFE-dependent lysosome biogenesis. Quantitative proteomics of immunopurified lysosomes uncovered reliance on ferritinophagy, the selective degradation of the iron storage complex ferritin, in MEKi-treated cells. Ferritinophagy promotes mitochondrial iron-sulfur cluster protein synthesis and enhanced mitochondrial respiration. Accordingly, suppressing iron utilization sensitizes PDA cells to MEKi, highlighting a critical and targetable reliance on lysosome-dependent iron supply during adaptation to KRAS-MAPK inhibition. SIGNIFICANCE: Reduced c-MYC levels following MAPK pathway suppression facilitate the upregulation of autophagy and lysosome biogenesis. Increased autophagy-lysosome activity is required for increased ferritinophagy-mediated iron supply, which supports mitochondrial respiration under therapy stress. Disruption of ferritinophagy synergizes with KRAS-MAPK inhibition and blocks PDA growth, thus highlighting a key targetable metabolic dependency. See related commentary by Jain and Amaravadi, p. 2023. See related article by Santana-Codina et al., p. 2180. This article is highlighted in the In This Issue feature, p. 2007.
Assuntos
Carcinoma Ductal Pancreático , Proteínas Ferro-Enxofre , Neoplasias Pancreáticas , Humanos , Disponibilidade Biológica , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Ferro/metabolismo , Ferro/uso terapêutico , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/uso terapêutico , Coativadores de Receptor Nuclear/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Enxofre/metabolismo , Enxofre/uso terapêutico , Fatores de Transcrição/metabolismo , Neoplasias PancreáticasRESUMO
PRMT5 is an essential arginine methyltransferase and a therapeutic target in MTAP-null cancers. PRMT5 uses adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (CLNS1A, RIOK1, and COPR5) and show that it is necessary and sufficient for interaction with PRMT5. We demonstrate that PRMT5 uses modular adaptor proteins containing a common binding motif for substrate recruitment, comparable with other enzyme classes such as kinases and E3 ligases. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosomal complexes. Furthermore, disruption of this site affects Sm spliceosome activity, leading to intron retention. Genetic disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells and is thus a site for development of therapeutic inhibitors of PRMT5.
Assuntos
Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/fisiologia , Animais , Linhagem Celular Tumoral , Citoplasma/metabolismo , Feminino , Células HCT116 , Células HEK293 , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Canais Iônicos/metabolismo , Masculino , Metilação , Camundongos , Camundongos Nus , Proteínas Nucleares/metabolismo , Peptídeos/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Spliceossomos/metabolismoRESUMO
Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-ß/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization. We perform an in vivo screen identifying FOSL1 as both a SMAD4 target and sufficient to drive colonization to the lung. The targeting of these genes early in treatment may provide a therapeutic benefit.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteína Smad4/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Elementos Facilitadores Genéticos/genética , Humanos , Camundongos , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Neoplasias PancreáticasRESUMO
Loss of major histocompatibility complex (MHC) class I and interferon-γ (IFN-γ) sensing are major causes of primary and acquired resistance to checkpoint blockade immunotherapy. Thus, additional treatment options are needed for tumors that lose expression of MHC class I. The cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) regulate classical and alternative nuclear factor κB (NF-κB) signaling. Induction of noncanonical NF-κB signaling with cIAP1/2 antagonists mimics costimulatory signaling, augmenting antitumor immunity. We show that induction of noncanonical NF-κB signaling induces T cell-dependent immune responses, even in ß2-microglobulin (ß2M)-deficient tumors, demonstrating that direct CD8 T cell recognition of tumor cell-expressed MHC class I is not required. Instead, T cell-produced lymphotoxin reprograms both mouse and human macrophages to be tumoricidal. In wild-type mice, but not mice incapable of antigen-specific T cell responses, cIAP1/2 antagonism reduces tumor burden by increasing phagocytosis of live tumor cells. Efficacy is augmented by combination with CD47 blockade. Thus, activation of noncanonical NF-κB stimulates a T cell-macrophage axis that curtails growth of tumors that are resistant to checkpoint blockade because of loss of MHC class I or IFN-γ sensing. These findings provide a potential mechanism for controlling checkpoint blockade refractory tumors.
Assuntos
Reprogramação Celular , Antígenos de Histocompatibilidade Classe I , Imunoterapia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neoplasias/terapia , Fagócitos , Linfócitos T/imunologia , Animais , Humanos , Interferon gama , Macrófagos , Camundongos , NF-kappa B , Neoplasias/imunologia , Transdução de SinaisRESUMO
The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Few therapies target the loss of tumor suppressor genes in cancer. We examine CRISPR-SpCas9 and RNA-interference loss-of-function screens to identify new therapeutic targets associated with genomic loss of tumor suppressor genes. The endosomal sorting complexes required for transport (ESCRT) ATPases VPS4A and VPS4B score as strong synthetic lethal dependencies. VPS4A is essential in cancers harboring loss of VPS4B adjacent to SMAD4 on chromosome 18q and VPS4B is required in tumors with co-deletion of VPS4A and CDH1 (E-cadherin) on chromosome 16q. We demonstrate that more than 30% of cancers selectively require VPS4A or VPS4B. VPS4A suppression in VPS4B-deficient cells selectively leads to ESCRT-III filament accumulation, cytokinesis defects, nuclear deformation, G2/M arrest, apoptosis, and potent tumor regression. CRISPR-SpCas9 screening and integrative genomic analysis reveal other ESCRT members, regulators of abscission, and interferon signaling as modifiers of VPS4A dependency. We describe a compendium of synthetic lethal vulnerabilities and nominate VPS4A and VPS4B as high-priority therapeutic targets for cancers with 18q or 16q loss.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Neoplasias/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Linhagem Celular Tumoral , HumanosRESUMO
Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Movimento Celular , Proteína Duplacortina , Quinases Semelhantes a Duplacortina , Ensaios de Seleção de Medicamentos Antitumorais , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacocinética , Proteômica , Ratos , Relação Estrutura-Atividade , Peixe-Zebra , Neoplasias PancreáticasRESUMO
SRC is a major regulator of many signaling pathways and contributes to cancer development. However, development of a selective SRC inhibitor has been challenging, and FDA-approved SRC inhibitors, dasatinib and bosutinib, are multitargeted kinase inhibitors. Here, we describe our efforts to develop a selective SRC covalent inhibitor by targeting cysteine 277 on the P-loop of SRC. Using a promiscuous covalent kinase inhibitor (CKI) SM1-71 as a starting point, we developed covalent inhibitor 15a, which discriminates SRC from other covalent targets of SM1-71 including TAK1 and FGFR1. As an irreversible covalent inhibitor, compound 15a exhibited sustained inhibition of SRC signaling both in vitro and in vivo. Moreover, 15a exhibited potent antiproliferative effects in nonsmall cell lung cancer cell lines harboring SRC activation, thus providing evidence that this approach may be promising for further drug development efforts.
Assuntos
Anilidas/farmacologia , Cisteína/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Domínio AAA , Sequência de Aminoácidos , Anilidas/síntese química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Pirimidinas/síntese química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Quinases da Família src/químicaRESUMO
Cyclin-dependent kinase 7 (CDK7) is a central regulator of the cell cycle and gene transcription. However, little is known about its impact on genomic instability and cancer immunity. Using a selective CDK7 inhibitor, YKL-5-124, we demonstrated that CDK7 inhibition predominately disrupts cell-cycle progression and induces DNA replication stress and genome instability in small cell lung cancer (SCLC) while simultaneously triggering immune-response signaling. These tumor-intrinsic events provoke a robust immune surveillance program elicited by T cells, which is further enhanced by the addition of immune-checkpoint blockade. Combining YKL-5-124 with anti-PD-1 offers significant survival benefit in multiple highly aggressive murine models of SCLC, providing a rationale for new combination regimens consisting of CDK7 inhibitors and immunotherapies.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Instabilidade Genômica , Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/genética , Animais , Antineoplásicos/farmacologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Quimiocina CXCL9/metabolismo , Dano ao DNA , Feminino , Humanos , Sistema Imunitário , Inflamação , Interferon gama/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Masculino , Camundongos , Testes para Micronúcleos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Pirazóis/farmacologia , Pirróis/farmacologia , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
From the perspective of media protocol control and routing of directional wireless ad hoc sensors networks, neighbor discovery protocol is an important problem to be solved first. In the past period of time, some methods have been studied on neighbor discovery protocol, but they have a common defect of link collision. The collision is caused by mutual interference of multiple transmitting nodes which are in one reception beam of the receiving node. To solve this problem, we propose a neighbor discovery algorithm using a bi-directional carrier sense collision avoidance and multi subchannels based on a scan-based algorithm (BD-SBA). Based on a scan-based algorithm (SBA), bi-directional carrier sense of the BD-SBA algorithm is performed in the first broadcast step which can reduce the collision of broadcasting the scanning request (SREQ) frames. In the second step (the reply step), the mechanism of multiple subchannels and multiple slots is applied to reduce the collision of the scanning response (SRES) frames. From the analysis and simulation, we can see that nodes using proposed algorithm can discover their neighbor nodes in fewer time. Moreover, the proposed algorithm has better performance for different beamwidths and densely distributed scenes. So it has great significance in engineering application.
RESUMO
Allosteric kinase inhibitors offer a potentially complementary therapeutic strategy to ATP-competitive kinase inhibitors due to their distinct sites of target binding. In this study, we identify and study a mutant-selective EGFR allosteric inhibitor, JBJ-04-125-02, which as a single agent can inhibit cell proliferation and EGFRL858R/T790M/C797S signaling in vitro and in vivo. However, increased EGFR dimer formation limits treatment efficacy and leads to drug resistance. Remarkably, osimertinib, an ATP-competitive covalent EGFR inhibitor, uniquely and significantly enhances the binding of JBJ-04-125-02 for mutant EGFR. The combination of osimertinib and JBJ-04-125-02 results in an increase in apoptosis, a more effective inhibition of cellular growth, and an increased efficacy in vitro and in vivo compared with either single agent alone. Collectively, our findings suggest that the combination of a covalent mutant-selective ATP-competitive inhibitor and an allosteric EGFR inhibitor may be an effective therapeutic approach for patients with EGFR-mutant lung cancer. SIGNIFICANCE: The clinical efficacy of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutant lung cancer is limited by acquired drug resistance, thus highlighting the need for alternative strategies to inhibit EGFR. Here, we identify a mutant EGFR allosteric inhibitor that is effective as a single agent and in combination with the EGFR TKI osimertinib.This article is highlighted in the In This Issue feature, p. 813.
Assuntos
Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Benzenoacetamidas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologia , Regulação Alostérica , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alternative splicing (AS) events occur in the majority of human genes. AS in a single gene can give rise to different functions among multiple isoforms. Human ortholog of mammalian enabled (Mena) is a conserved regulator of actin dynamics that plays an important role in metastasis. Mena has been shown to have multiple splice variants in human tumor cells due to AS. However, the mechanism mediated Mena AS has not been elucidated. Here we showed that polypyrimidine tract-binding protein 1 (PTBP1) could modulate Mena AS. First, PTBP1 levels were elevated in metastatic lung cancer cells as well as during epithelial-mesenchymal transition (EMT) process. Then, knockdown of PTBP1 using shRNA inhibited migration and invasion of lung carcinoma cells and decreased the Mena exon11a skipping, whereas overexpression of PTBP1 had the opposite effects. The results of RNA pull-down assays and mutation analyses demonstrated that PTBP1 functionally targeted and physically interacted with polypyrimidine sequences on both upstream intron11 (TTTTCCCCTT) and downstream intron11a (TTTTTTTTTCTTT). In addition, the results of migration and invasion assays as well as detection of filopodia revealed that the effect of PTBP1 was reversed by knockdown of Mena but not Mena11a+. Overexpressed MenaΔ11a also rescued the PTBP1-induced migration and invasion. Taken together, our study provides a novel mechanism that PTBP1 modulates Mena exon11a skipping, and indicates that PTBP1 depends on the level of Mena11a- to promote lung cancer cells migration and invasion. The regulation of Mena AS may be a potential prognostic marker and a promising target for treatment of lung carcinoma.
Assuntos
Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Neoplasias Pulmonares/genética , Proteínas dos Microfilamentos/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Células A549 , Movimento Celular , Transição Epitelial-Mesenquimal , Éxons , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Invasividade Neoplásica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Regulação para CimaRESUMO
Oncogenic KRAS is the key driver of pancreatic ductal adenocarcinoma (PDAC). We previously described a role for KRAS in PDAC tumor maintenance through rewiring of cellular metabolism to support proliferation. Understanding the details of this metabolic reprogramming in human PDAC may provide novel therapeutic opportunities. Here we show that the dependence on oncogenic KRAS correlates with specific metabolic profiles that involve maintenance of nucleotide pools as key mediators of KRAS-dependence. KRAS promotes these effects by activating a MAPK-dependent signaling pathway leading to MYC upregulation and transcription of the non-oxidative pentose phosphate pathway (PPP) gene RPIA, which results in nucleotide biosynthesis. The use of MEK inhibitors recapitulates the KRAS-dependence pattern and the expected metabolic changes. Antagonizing the PPP or pyrimidine biosynthesis inhibits the growth of KRAS-resistant cells. Together, these data reveal differential metabolic rewiring between KRAS-resistant and sensitive cells, and demonstrate that targeting nucleotide metabolism can overcome resistance to KRAS/MEK inhibition.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirimidinas/biossíntese , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Via de Pentose Fosfato/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
A ratiometric probe is described for the fluorometric determination of Cu(II) ions based on their quenching effect on the luminescence of dually-emitting quantum dots (QDs). ZnS QDs were doped with Mn(II) and subsequently modified with mercaptopropionic acid to give the QD probe which consists of a sole fluorophore but has two emission peaks (at 430 and 590 nm under 310 nm excitation, respectively). On addition of Cu(II) ions, the 590 nm band is quenched while the 430 nm band exhibits a little change. The changes in the intensity ratios of the yellow and the purple bands increases linearly in the 0 to 3.0 µM Cu(II) concentration range, and the detection limit reached 14 nM. The QD probe was validated and successfully applied to the determination of Cu(II) in spiked real water samples. Graphical abstract Mn-doped ZnS (ZnS:Mn(II)) quantum dots were synthesized with yellow fluorescence. After the modification of 3-mercaptopropionic acid (MPA), ZnS:Mn(II) was transferred to aqueous phase and became MPA modified Mn-doped ZnS (MPA- ZnS:Mn(II)). The fluorescence was changed to purple upon the addition of copper ions because the yellow band was largely quenched while the purple band only changed a little.