RESUMO
Mitochondria have long been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal-recessive form of early-onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered because no allosteric regulatory sites for PINK1 are known. Here, we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD-related mutant PINK1(G309D) and PINK1(WT). Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.
Assuntos
Mitocôndrias/metabolismo , Doença de Parkinson/patologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Sequência de Aminoácidos , Animais , Apoptose , Axônios/metabolismo , Linhagem Celular , Células Cultivadas , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Cinetina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Fosforilação , Proteínas Quinases/química , Ratos , Alinhamento de Sequência , Ubiquitina-Proteína Ligases/metabolismo , Proteína bcl-X/metabolismoRESUMO
Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.
Assuntos
Sítio Alostérico/efeitos dos fármacos , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/metabolismo , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteína Oncogênica p21(ras)/metabolismo , Regulação Alostérica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , Descoberta de Drogas , Genes ras/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Modelos Moleculares , Proteínas Mutantes/genética , Proteína Oncogênica p21(ras)/genética , Eletricidade Estática , Especificidade por Substrato , Quinases raf/metabolismoRESUMO
Although a variety of genetic alterations have been found across cancer types, the identification and functional characterization of candidate driver genetic lesions in an individual patient and their translation into clinically actionable strategies remain major hurdles. Here, we use whole genome sequencing of a prostate cancer tumor, computational analyses, and experimental validation to identify and predict novel oncogenic activity arising from a point mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor protein. We demonstrate that this mutation (p.A126G) produces an enzymatic gain-of-function in PTEN, shifting its function from a phosphoinositide (PI) 3-phosphatase to a phosphoinositide (PI) 5-phosphatase. Using cellular assays, we demonstrate that this gain-of-function activity shifts cellular phosphoinositide levels, hyperactivates the PI3K/Akt cell proliferation pathway, and exhibits increased cell migration beyond canonical PTEN loss-of-function mutants. These findings suggest that mutationally modified PTEN can actively contribute to well-defined hallmarks of cancer. Lastly, we demonstrate that these effects can be substantially mitigated through chemical PI3K inhibitors. These results demonstrate a new dysfunction paradigm for PTEN cancer biology and suggest a potential framework for the translation of genomic data into actionable clinical strategies for targeted patient therapy.
Assuntos
Genes Supressores de Tumor , Proteínas de Neoplasias/genética , PTEN Fosfo-Hidrolase/genética , Monoéster Fosfórico Hidrolases/genética , Neoplasias da Próstata/genética , Análise de Variância , Animais , Sequência de Bases , Células CHO , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Biologia Computacional/métodos , Cricetinae , Cricetulus , Humanos , Immunoblotting , Masculino , Microscopia de Fluorescência , Anotação de Sequência Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Análise de Sequência de DNARESUMO
Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis-antagonizing transcription factor (AATF)/Che-1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53-driven expression of these pro-apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF-depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho-mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53-proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53-proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53-driven apoptosis and commend this pathway as a target for DNA damage-sensitizing therapeutic regimens.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/fisiologia , Dano ao DNA/fisiologia , Proteínas Repressoras/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Pontos de Checagem do Ciclo Celular , Dano ao DNA/genética , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias do Endométrio/genética , Feminino , Amplificação de Genes , Dosagem de Genes , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos , Cadeias Leves de Miosina/metabolismo , Neuroblastoma/genética , Neuroblastoma/mortalidade , Pressão Osmótica , Fosforilação , Prognóstico , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/genéticaRESUMO
The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkappaB kinase TBK1 was selectively essential in cells that contain mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-kappaB anti-apoptotic signals involving c-Rel and BCL-XL (also known as BCL2L1) that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations indicate that TBK1 and NF-kappaB signalling are essential in KRAS mutant tumours, and establish a general approach for the rational identification of co-dependent pathways in cancer.
Assuntos
Genes ras/genética , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Alelos , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Perfilação da Expressão Gênica , Genes Letais , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-rel/metabolismo , Transdução de Sinais , Proteína bcl-X/metabolismoRESUMO
Small cell lung cancer (SCLC) accounts for about 15% of all lung cancers. The prognosis of SCLC patients is devastating and no biologically targeted therapeutics are active in this tumor type. To develop a framework for development of specific SCLC-targeted drugs we conducted a combined genomic and pharmacological vulnerability screen in SCLC cell lines. We show that SCLC cell lines capture the genomic landscape of primary SCLC tumors and provide genetic predictors for activity of clinically relevant inhibitors by screening 267 compounds across 44 of these cell lines. We show Aurora kinase inhibitors are effective in SCLC cell lines bearing MYC amplification, which occur in 3-7% of SCLC patients. In MYC-amplified SCLC cells Aurora kinase inhibition associates with G2/M-arrest, inactivation of PI3-kinase (PI3K) signaling, and induction of apoptosis. Aurora dependency in SCLC primarily involved Aurora B, required its kinase activity, and was independent of depletion of cytoplasmic levels of MYC. Our study suggests that a fraction of SCLC patients may benefit from therapeutic inhibition of Aurora B. Thus, thorough chemical and genomic exploration of SCLC cell lines may provide starting points for further development of rational targeted therapeutic intervention in this deadly tumor type.
Assuntos
Inibidores Enzimáticos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Apoptose/efeitos dos fármacos , Aurora Quinase B , Aurora Quinases , Benzotiazóis , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Primers do DNA/genética , Diaminas , Citometria de Fluxo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Immunoblotting , Compostos Orgânicos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinolinas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Analog-sensitive (AS) kinase technology is a powerful approach for studying phospho-signaling pathways in diverse organisms and physiological processes. The key feature of this technique is that a kinase-of-interest can be mutated to sensitize it to inhibitor analogs that do not target wild-type (WT) kinases. In theory, this enables specific inhibition of any kinase in cells and in mouse models of human disease. Typically, these inhibitors are identified from a small library of molecules based on the pyrazolopyrimidine (PP) scaffold. However, we recently identified a subset of native human kinases, including the Ephrin A kinase family, that are sensitive to commonly used PP inhibitors. In an effort to develop a bioorthogonal AS-kinase inhibitor and to extend this technique to PP-sensitive kinases, we sought an alternative inhibitor scaffold. Here we report the structure-based design of synthetically tractable, potent, and extremely selective AS-kinase inhibitors based on the natural product staurosporine. We demonstrate that these molecules, termed staralogs, potently target AS kinases in cells, and we employ X-ray crystallography to elucidate their mechanism of efficacy. Finally, we demonstrate that staralogs target AS mutants of PP-sensitive kinases at concentrations where there is little to no inhibition of native human kinases. Thus, staralogs represent a new class of AS-kinase inhibitors and a core component of the chemical genetic tool kit for probing kinase-signaling pathways.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Animais , Sítios de Ligação , Carbazóis/química , Carbazóis/farmacologia , Humanos , Camundongos , Modelos Moleculares , Mutação , Proteínas Quinases/genética , Relação Estrutura-AtividadeRESUMO
The emergence of resistance to targeted therapies restrains their efficacy. The development of rationally guided drug combinations could overcome this currently insurmountable clinical challenge. However, our limited understanding of the trajectories that drive the outgrowth of resistant clones in cancer cell populations precludes design of drug combinations to forestall resistance. Here, we propose an iterative treatment strategy coupled with genomic profiling and genome-wide CRISPR activation screening to systematically extract and define preexisting resistant subpopulations in an EGFR-driven lung cancer cell line. Integrating these modalities identifies several resistance mechanisms, including activation of YAP/TAZ signaling by WWTR1 amplification, and estimates the associated cellular fitness for mathematical population modeling. These observations led to the development of a combination therapy that eradicated resistant clones in large cancer cell line populations by exhausting the spectrum of genomic resistance mechanisms. However, a small fraction of cancer cells was able to enter a reversible nonproliferative state of drug tolerance. This subpopulation exhibited mesenchymal properties, NRF2 target gene expression, and sensitivity to ferroptotic cell death. Exploiting this induced collateral sensitivity by GPX4 inhibition clears drug-tolerant populations and leads to tumor cell eradication. Overall, this experimental in vitro data and theoretical modeling demonstrate why targeted mono- and dual therapies will likely fail in sufficiently large cancer cell populations to limit long-term efficacy. Our approach is not tied to a particular driver mechanism and can be used to systematically assess and ideally exhaust the resistance landscape for different cancer types to rationally design combination therapies. SIGNIFICANCE: Unraveling the trajectories of preexisting resistant and drug-tolerant persister cells facilitates the rational design of multidrug combination or sequential therapies, presenting an approach to explore for treating EGFR-mutant lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Transdução de Sinais , Receptores ErbB/metabolismo , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , MutaçãoRESUMO
OBJECTIVES: Resistance to MET inhibition occurs inevitably in MET-dependent non-small cell lung cancer and the underlying mechanisms are insufficiently understood. We describe resistance mechanisms in patients with MET exon 14 skipping mutation (METΔex14), MET amplification, and MET fusion and report treatment outcomes after switching therapy from type I to type II MET inhibitors. MATERIALS AND METHODS: Pre- and post-treatment biopsies were analysed by NGS (next generation sequencing), digital droplet PCR (polymerase chain reaction), and FISH (fluorescense in situ hybridization). A patient-derived xenograft model was generated in one case. RESULTS: Of 26 patients with MET tyrosine kinase inhibitor treatment, eight had paired pre- and post-treatment biopsies (Three with MET amplification, three with METΔex14, two with MET fusions (KIF5B-MET and PRKAR2B-MET).) In six patients, mechanisms of resistance were detected, whereas in two cases, the cause of resistance remained unclear. We found off-target resistance mechanisms in four cases with KRAS mutations and HER2 amplifications appearing. Two patients exhibited second-site MET mutations (p.D1246N and p. Y1248H). Three patients received type I and type II MET tyrosine kinase inhibitors sequentially. In two cases, further progressive disease was seen hereafter. The patient with KIF5B-MET fusion received three different MET inhibitors and showed long-lasting stable disease and a repeated response after switching therapy, respectively. CONCLUSION: Resistance to MET inhibition is heterogeneous with on- and off-target mechanisms occurring regardless of the initial MET aberration. Switching therapy between different types of kinase inhibitors can lead to repeated responses in cases with second-site mutations. Controlled clinical trials in this setting with larger patient numbers are needed, as evidence to date is limited to preclinical data and case series.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Proto-Oncogênicas c-met/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , MutaçãoRESUMO
In cancer, genetically activated proto-oncogenes often induce "upstream" dependency on the activity of the mutant oncoprotein. Therapeutic inhibition of these activated oncoproteins can induce massive apoptosis of tumor cells, leading to sometimes dramatic tumor regressions in patients. The PI3K and MAPK signaling pathways are central regulators of oncogenic transformation and tumor maintenance. We hypothesized that upstream dependency engages either one of these pathways preferentially to induce "downstream" dependency. Therefore, we analyzed whether downstream pathway dependency segregates by genetic aberrations upstream in lung cancer cell lines. Here, we show by systematically linking drug response to genomic aberrations in non-small-cell lung cancer, as well as in cell lines of other tumor types and in a series of in vivo cancer models, that tumors with genetically activated receptor tyrosine kinases depend on PI3K signaling, whereas tumors with mutations in the RAS/RAF axis depend on MAPK signaling. However, efficacy of downstream pathway inhibition was limited by release of negative feedback loops on the reciprocal pathway. By contrast, combined blockade of both pathways was able to overcome the reciprocal pathway activation induced by inhibitor-mediated release of negative feedback loops and resulted in a significant increase in apoptosis and tumor shrinkage. Thus, by using a systematic chemo-genomics approach, we identify genetic lesions connected to PI3K and MAPK pathway activation and provide a rationale for combined inhibition of both pathways. Our findings may have implications for patient stratification in clinical trials.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Genótipo , Humanos , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
NRG1 fusions are recurrent somatic genome alterations occurring across several tumor types, including invasive mucinous lung adenocarcinomas and pancreatic ductal adenocarcinomas and are potentially actionable genetic alterations in these cancers. We initially discovered CD74-NRG1 as the first NRG1 fusion in lung adenocarcinomas, and many additional fusion partners have since been identified. Here, we present the first CD74-NRG1 transgenic mouse model and provide evidence that ubiquitous expression of the CD74-NRG1 fusion protein in vivo leads to tumor development at high frequency. Furthermore, we show that ERBB2:ERBB3 heterodimerization is a mechanistic event in transformation by CD74-NRG1 binding physically to ERBB3 and that CD74-NRG1-expressing cells proliferate independent of supplemented NRG1 ligand. Thus, NRG1 gene fusions are recurrent driver oncogenes that cause oncogene dependency. Consistent with these findings, patients with NRG1 fusion-positive cancers respond to therapy targeting the ERBB2:ERBB3 receptors.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Animais , Carcinogênese/genética , Humanos , Camundongos , Neuregulina-1/genética , Oncogenes , Receptor ErbB-2/genética , Receptor ErbB-3/genéticaRESUMO
Despite the clinical efficacy of epidermal growth factor receptor (EGFR) inhibitors, a subset of patients with non-small cell lung cancer displays insertion mutations in exon20 in EGFR and Her2 with limited treatment options. Here, we present the development and characterization of the novel covalent inhibitors LDC8201 and LDC0496 based on a 1H-pyrrolo[2,3-b]pyridine scaffold. They exhibited intense inhibitory potency toward EGFR and Her2 exon20 insertion mutations as well as selectivity over wild type EGFR and within the kinome. Complex crystal structures with the inhibitors and biochemical and cellular on-target activity document their favorable binding characteristics. Ultimately, we observed tumor shrinkage in mice engrafted with patient-derived EGFR-H773_V774insNPH mutant cells during treatment with LDC8201. Together, these results highlight the potential of covalent pyrrolopyridines as inhibitors to target exon20 insertion mutations.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutagênese Insercional , Mutação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
NUT carcinoma is a rare, aggressive cancer defined by rearrangements of the NUTM1 gene. No routinely effective treatments of NUT carcinoma exist, despite harboring a targetable oncoprotein, most commonly BRD4-NUT. The vast majority of cases are fatal. Poor awareness of the disease is a major obstacle to progress in the treatment of NUT carcinoma. While the incidence likely exceeds that of Ewing sarcoma, and BRD4-NUT heralded the bromodomain and extra-terminal domain (BET) inhibitor class of selective epigenetic modulators, NUT carcinoma is incorrectly perceived as "impossibly rare," and therefore receives comparatively little private or governmental funding or prioritization by pharma. To raise awareness, propagate scientific knowledge, and initiate a consensus on standard and targeted treatment of NUT carcinoma, we held the First International Symposium on NUT Carcinoma on March 3, 2021. This virtual event had more than eighty attendees from the Americas, Europe, Asia, and Australia. Patients with NUT carcinoma and family members were represented and shared perspectives. Broadly, the four areas discussed by experts in the field included (1) the biology of NUT carcinoma; (2) standard approaches to the treatment of NUT carcinoma; (3) results of clinical trials using BET inhibitors; and (4) future directions, including novel BET bromodomain inhibitors, combinatorial approaches, and immunotherapy. It was concluded that standard chemotherapeutic approaches and first-generation BET bromodomain inhibitors, the latter complicated by a narrow therapeutic window, are only modestly effective in a minority of cases. Nonetheless, emerging second-generation targeted inhibitors, novel rational synergistic combinations, and the incorporation of immuno-oncology approaches hold promise to improve the prognosis of this disease.
Assuntos
Carcinoma , Sarcoma de Ewing , Carcinoma/genética , Proteínas de Ciclo Celular , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: The L1 cell adhesion molecule (L1CAM) is potentially involved in epithelial-mesenchymal transition (EMT). EMT marker expression is of prognostic significance in non-small cell lung cancer (NSCLC). The relevance of L1CAM for NSCLC is unclear. We investigated the protein expression of L1CAM in a cohort of NSCLC patients. L1CAM protein expression was correlated with clinico-pathological parameters including survival and markers of epithelial-mesenchymal transition. RESULTS: L1CAM protein expression was found in 25% of squamous cell carcinomas and 24% of adenocarcinomas and correlated with blood vessel invasion and metastasis (p < 0.05). L1CAM was an independent predictor of survival in a multivariate analysis including pT, pN, and pM category, and tumor differentiation grade. L1CAM expression positively correlated with vimentin, beta-catenin, and slug, but inversely with E-cadherin (all p-values < 0.05). E-cadherin expression was higher in the tumor center than in the tumor periphery, whereas L1CAM and vimentin were expressed at the tumor-stroma interface. In L1CAM-negative A549 cells the L1CAM expression was upregulated and matrigel invasion was increased after stimulation with TGF-beta1. In L1CAM-positive SK-LU-1 and SK-LC-LL cells matrigel invasion was decreased after L1CAM siRNA knockdown. CONCLUSIONS: A subset of NSCLCs with vessel tropism and increased metastasis aberrantly expresses L1CAM. L1CAM is a novel prognostic marker for NSCLCs that is upregulated by EMT induction and appears to be instrumental for enhanced cell invasion.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Invasividade Neoplásica , Molécula L1 de Adesão de Célula Nervosa/genética , Prognóstico , Interferência de RNARESUMO
Here we present the synthesis and biological activity of a series of 7-substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitriles as inhibitors of myosin light chain kinase (MLCK) and the epidermal growth factor receptor kinase (EGFR). The inhibitory effect of these molecules was found to be dependent on the nature of the substituents at the 7-position of the isoquinoline scaffold.
Assuntos
Receptores ErbB/antagonistas & inibidores , Isoquinolinas/farmacologia , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/metabolismo , Humanos , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
EGFR mutations account for the majority of druggable targets in lung adenocarcinoma. Over the past decades the optimization of EGFR inhibitors revolutionized the treatment options for patients suffering from this disease. The pace of this development was largely dictated by the inevitable emergence of resistance mutations during drug treatment. As a result, a rapid understanding of the structural and molecular biology of the individual mutations is the key for the development of next-generation inhibitors. Currently, the field faces an unprecedented number of combinations of activating mutations with distinct resistance mutations in parallel to the approval of osimertinib as a first-line drug for EGFR-mutant lung cancer. In this review, we present a survey of the diverse landscape of EGFR resistance mechanisms with a focus on new insights into on-target EGFR kinase mutations. We discuss array of mutations, their structural effects on the EGFR kinase domain as well as the most promising strategies to overcome the individual resistance profiles found in lung cancer patients.
Assuntos
Adenocarcinoma de Pulmão/genética , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/genética , Mutação , Adenocarcinoma de Pulmão/tratamento farmacológico , Receptores ErbB/química , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Loss of TP53 and RB1 in treatment-naïve small cell lung cancer (SCLC) suggests selective pressure to inactivate cell death pathways prior to therapy. Yet, which of these pathways remain available in treatment-naïve SCLC is unknown. Here, through systemic analysis of cell death pathway availability in treatment-naïve SCLC, we identify non-neuroendocrine (NE) SCLC to be vulnerable to ferroptosis through subtype-specific lipidome remodeling. While NE SCLC is ferroptosis resistant, it acquires selective addiction to the TRX anti-oxidant pathway. In experimental settings of non-NE/NE intratumoral heterogeneity, non-NE or NE populations are selectively depleted by ferroptosis or TRX pathway inhibition, respectively. Preventing subtype plasticity observed under single pathway targeting, combined treatment kills established non-NE and NE tumors in xenografts, genetically engineered mouse models of SCLC and patient-derived cells, and identifies a patient subset with drastically improved overall survival. These findings reveal cell death pathway mining as a means to identify rational combination therapies for SCLC.
Assuntos
Ferroptose , Tumores Neuroendócrinos/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Animais , Antioxidantes/metabolismo , Apoptose , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos Nus , Modelos Biológicos , Necroptose , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeos/metabolismo , Prognóstico , Tiorredoxinas/metabolismoRESUMO
Activation of MAPK signaling via BRAF mutations may limit the activity of EGFR inhibitors in EGFR-mutant lung cancer patients. However, the impact of BRAF mutations on the selection and fitness of emerging resistant clones during anti-EGFR therapy remains elusive. We tracked the evolution of subclonal mutations by whole-exome sequencing and performed clonal analyses of individual metastases during therapy. Complementary functional analyses of polyclonal EGFR-mutant cell pools showed a dose-dependent enrichment of BRAFV600E and a loss of EGFR inhibitor susceptibility. The clones remain stable and become vulnerable to combined EGFR, RAF, and MEK inhibition. Moreover, only osimertinib/trametinib combination treatment, but not monotherapy with either of these drugs, leads to robust tumor shrinkage in EGFR-driven xenograft models harboring BRAFV600E mutations. These data provide insights into the dynamics of clonal evolution of EGFR-mutant tumors and the therapeutic implications of BRAF co-mutations that may facilitate the development of treatment strategies to improve the prognosis of these patients.
RESUMO
Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8+ T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients.
Assuntos
Proteína DEAD-box 58/metabolismo , Imunidade Inata , Inflamação/patologia , Sistema de Sinalização das MAP Quinases , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores Imunológicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citocinas/metabolismo , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Fator Regulador 1 de Interferon/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Oncogenes , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Treatment of patients with solid tumors and KRAS mutations remains disappointing. One option is the combined inhibition of pathways involved in RAF-MEK-ERK and PI3K-AKT-mTOR. METHODS: Patients with relapsed solid tumors were treated with escalating doses of everolimus (E) 2.5-10.0 mg/d in a 14-day run-in phase followed by combination therapy with sorafenib (S) 800 mg/d from day 15. KRAS mutational status was assessed retrospectively in the escalation phase. Extension phase included KRAS-mutated non-small-cell lung cancer (NSCLC) only. Pharmacokinetic analyses were accompanied by pharmacodynamics assessment of E by FDG-PET. Efficacy was assessed by CT scans every 6 weeks of combination. RESULTS: Of 31 evaluable patients, 15 had KRAS mutation, 4 patients were negative for KRAS mutation, and the KRAS status remained unknown in 12 patients. Dose-limiting toxicity (DLT) was not reached. The maximum tolerated dose (MTD) was defined as 7.5 mg/d E + 800 mg/d S due to toxicities at previous dose level (10 mg/d E + 800 mg/d S) including leucopenia/thrombopenia III° and pneumonia III° occurring after the DLT interval. The metabolic response rate in FDG-PET was 17% on day 5 and 20% on day 14. No patient reached partial response in CT scan. Median progression free survival (PFS) and overall survival (OS) were 3.25 and 5.85 months, respectively. CONCLUSIONS: Treatment of patients with relapsed solid tumors with 7.5 mg/d E and 800 mg/d S is safe and feasible. Early metabolic response in FDG-PET was not confirmed in CT scan several weeks later. The combination of S and E is obviously not sufficient to induce durable responses in patients with KRAS-mutant solid tumors.