RESUMO
A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor ß pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes present three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.
Assuntos
Glaucoma , Disco Óptico , Humanos , Camundongos , Animais , Disco Óptico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Camundongos Endogâmicos C57BL , Glaucoma/genética , Glaucoma/metabolismo , Retina/metabolismo , Nervo Óptico/metabolismo , Pressão Intraocular , Compressão Nervosa , Expressão Gênica , Modelos Animais de DoençasRESUMO
OBJECTIVE: It is difficult to predict which mechanically ventilated patients will ultimately require a tracheostomy which further predisposes them to unnecessary spontaneous breathing trials, additional time on the ventilator, increased costs, and further ventilation-related complications such as subglottic stenosis. In this study, we aimed to develop a machine learning tool to predict which patients need a tracheostomy at the onset of admission to the intensive care unit (ICU). STUDY DESIGN: Retrospective Cohort Study. SETTING: Multicenter Study of 335 Intensive Care Units between 2014 and 2015. METHODS: The eICU Collaborative Research Database (eICU-CRD) was utilized to obtain the patient cohort. Inclusion criteria included: (1) Age >18 years and (2) ICU admission requiring mechanical ventilation. The primary outcome of interest included tracheostomy assessed via a binary classification model. Models included logistic regression (LR), random forest (RF), and Extreme Gradient Boosting (XGBoost). RESULTS: Of 38,508 invasively mechanically ventilated patients, 1605 patients underwent a tracheostomy. The XGBoost, RF, and LR models had fair performances at an AUROC 0.794, 0.780, and 0.775 respectively. Limiting the XGBoost model to 20 features out of 331, a minimal reduction in performance was observed with an AUROC of 0.778. Using Shapley Additive Explanations, the top features were an admission diagnosis of pneumonia or sepsis and comorbidity of chronic respiratory failure. CONCLUSIONS: Our machine learning model accurately predicts the probability that a patient will eventually require a tracheostomy upon ICU admission, and upon prospective validation, we have the potential to institute earlier interventions and reduce the complications of prolonged ventilation.
RESUMO
Aged patients with melanoma (>65 years old) have more aggressive disease relative to young patients (<55 years old) for reasons that are not completely understood. Analysis of the young and aged secretome from human dermal fibroblasts identified >5-fold levels of IGF-binding protein 2 (IGFBP2) in the aged fibroblast secretome. IGFBP2 functionally triggers upregulation of the PI3K-dependent fatty acid biosynthesis program in melanoma cells. Melanoma cells co-cultured with aged dermal fibroblasts have higher levels of lipids relative to those co-cultured with young dermal fibroblasts, which can be lowered by silencing IGFBP2 expression in fibroblasts prior to treating with conditioned media. Conversely, ectopically treating melanoma cells with recombinant IGFBP2 in the presence of conditioned media from young fibroblasts or overexpressing IGFBP2 in melanoma cells promoted lipid synthesis and accumulation in melanoma cells. Treatment of young mice with rIGFBP2 increases tumor growth. Neutralizing IGFBP2 in vitro reduces migration and invasion in melanoma cells, and in vivo studies demonstrate that neutralizing IGFBP2 in syngeneic aged mice reduces tumor growth and metastasis. Our results suggest that aged dermal fibroblasts increase melanoma cell aggressiveness through increased secretion of IGFBP2, stressing the importance of considering age when designing studies and treatment. SIGNIFICANCE: The aged microenvironment drives metastasis in melanoma cells. This study reports that IGFBP2 secretion by aged fibroblasts induces lipid accumulation in melanoma cells, driving an increase in tumor invasiveness. Neutralizing IGFBP2 decreases melanoma tumor growth and metastasis.
Assuntos
Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina , Melanoma , Invasividade Neoplásica , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Humanos , Animais , Melanoma/patologia , Melanoma/metabolismo , Camundongos , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Fibroblastos/patologia , Movimento Celular , Idoso , Pessoa de Meia-Idade , Lipídeos , Metabolismo dos Lipídeos , Fatores Etários , Camundongos Endogâmicos C57BLRESUMO
Patients with metastatic acral lentiginous melanoma (ALM) suffer worse outcomes relative to patients with other forms of cutaneous melanoma (CM), and do not benefit as well to approved melanoma therapies. Identification of cyclin-dependent kinase 4 and 6 (CDK4/6) pathway gene alterations in >60% of ALMs has led to clinical trials of the CDK4/6 inhibitor (CDK4i/6i) palbociclib for ALM; however, median progression free survival with CDK4i/6i treatment was only 2.2 months, suggesting existence of resistance mechanisms. Therapy resistance in ALM remains poorly understood; here we report hyperactivation of MAPK signaling and elevated cyclin D1 expression serve as a mechanism of intrinsic early/adaptive CDK4i/6i resistance. ALM cells that have acquired CDK4i/6i resistance following chronic treatment exposure also exhibit hyperactivation of the MAPK pathway. MEK and/or ERK inhibition increases CDK4i/6i efficacy against therapy naïve and CDK4i/6i-resistant AM cells in xenograft and patient-derived xenograft (PDX) models and promotes a defective DNA repair, cell cycle arrested and apoptotic program. Notably, gene alterations poorly correlate with protein expression of cell cycle proteins in ALM or efficacy of CDK4i/6i, urging additional strategies when stratifying patients for CDK4i/6i trial inclusion. Concurrent targeting of the MAPK pathway and CDK4/6 represents a new approach for patients with metastatic ALM to improve outcomes.
Assuntos
Melanoma , Neoplasias Cutâneas , Animais , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Modelos Animais de Doenças , Ciclo Celular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer.
Assuntos
Neoplasias da Mama , Cromatina , Histona-Lisina N-Metiltransferase , Humanos , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Neoplasias da Mama/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Metilação/efeitos dos fármacos , Linhagem Celular Tumoral , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Receptores de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and in human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and on separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor ß pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.
RESUMO
Resistance to combination BRAF/MEK inhibitor (BRAFi/MEKi) therapy arises in nearly every patient with BRAFV600E/K melanoma, despite promising initial responses. Achieving cures in this expanding BRAFi/MEKi-resistant cohort represents one of the greatest challenges to the field; few experience additional durable benefit from immunotherapy and no alternative therapies exist. To better personalize therapy in cancer patients to address therapy relapse, umbrella trials have been initiated whereby genomic sequencing of a panel of potentially actionable targets guide therapy selection for patients; however, the superior efficacy of such approaches remains to be seen. We here test the robustness of the umbrella trial rationale by analyzing relationships between genomic status of a gene and the downstream consequences at the protein level of related pathway, which find poor relationships between mutations, copy number amplification, and protein level. To profile candidate therapeutic strategies that may offer clinical benefit in the context of acquired BRAFi/MEKi resistance, we established a repository of patient-derived xenograft models from heavily pretreated patients with resistance to BRAFi/MEKi and/or immunotherapy (R-PDX). With these R-PDXs, we executed in vivo compound repurposing screens using 11 FDA-approved agents from an NCI-portfolio with pan-RTK, non-RTK and/or PI3K-mTOR specificity. We identify dasatinib as capable of restoring BRAFi/MEKi antitumor efficacy in ~70% of R-PDX tested. A systems-biology analysis indicates elevated baseline protein expression of canonical drivers of therapy resistance (e.g., AXL, YAP, HSP70, phospho-AKT) as predictive of MAPKi/dasatinib sensitivity. We therefore propose that dasatinib-based MAPKi therapy may restore antitumor efficacy in patients that have relapsed to standard-of-care therapy by broadly targeting proteins critical in melanoma therapy escape. Further, we submit that this experimental PDX paradigm could potentially improve preclinical evaluation of therapeutic modalities and augment our ability to identify biomarker-defined patient subsets that may respond to a given clinical trial.
RESUMO
Patients with metastatic acral lentiginous melanoma (ALM) suffer worse outcomes relative to patients with other forms of cutaneous melanoma (CM), and do not benefit as well to approved melanoma therapies. Identification of cyclin-dependent kinase 4 and 6 (CDK4/6) pathway gene alterations in > 60% of ALMs has led to clinical trials of the CDK4/6 inhibitor (CDK4i/6i) palbociclib for ALM; however, median progression free survival with CDK4i/6i treatment was only 2.2 months, suggesting existence of resistance mechanisms. Therapy resistance in ALM remains poorly understood; here we report hyperactivation of MAPK signaling and elevated cyclin D1 expression are a unified mechanism of both intrinsic and acquired CDK4i/6i resistance. MEK and/or ERK inhibition increases CDK4i/6i efficacy in a patient-derived xenograft (PDX) model of ALM and promotes a defective DNA repair, cell cycle arrested and apoptotic program. Notably, gene alterations poorly correlate with protein expression of cell cycle proteins in ALM or efficacy of CDK4i/6i, urging additional strategies when stratifying patients for CDK4i/6i trial inclusion. Concurrent targeting of the MAPK pathway and CDK4/6 represents a new approach to improve outcomes for patients with advanced ALM.
RESUMO
Though crucial for understanding the function of large biomolecular systems, locating the minimum free energy paths (MFEPs) between their key conformational states is far from trivial due to their high-dimensional nature. Most existing path-searching methods require a static collective variable space as input, encoding intuition or prior knowledge of the transition mechanism. Such information is, however, hardly available a priori and expensive to validate. To alleviate this issue, we have previously introduced a Traveling-salesman based Automated Path Searching method (TAPS) and demonstrated its efficiency on simple peptide systems. Having implemented a parallel version of this method, here we assess the performance of TAPS on three realistic systems (tens to hundreds of residues) in explicit solvents. We show that TAPS successfully located the MFEP for the ground/excited state transition of the T4 lysozyme L99A variant, consistent with previous findings. TAPS also helped identifying the important role of the two polar contacts in directing the loop-in/loop-out transition of the mitogen-activated protein kinase kinase (MEK1), which explained previous mutant experiments. Remarkably, at a minimal cost of 126 ns sampling, TAPS revealed that the Ltn40/Ltn10 transition of lymphotactin needs no complete unfolding/refolding of its ß-sheets and that five polar contacts are sufficient to stabilize the various partially unfolded intermediates along the MFEP. These results present TAPS as a general and promising tool for studying the functional dynamics of complex biomolecular systems.