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Six new ravidomycin analogs (1-4, 6, and 7) were isolated from Streptomyces sp. Am59 using UV- and LCMS-guided separation based on Global Natural Products Social (GNPS) molecular networking analysis. Furthermore, we isolated fucomycin V (9), which possesses the same chromophore as ravidomycin but features a d-fucopyranose instead of d-ravidosamine. This is the first report of 9 as a natural product. Four new analogs (10-13) of 9 were also isolated. The structures were elucidated by combined spectroscopic and computational methods. We also found an inconsistency with the published [α]D25 of deacetylravidomycin, which is reported to have a (-) sign. Instead, we observed a (+) specific rotation for the reported absolute configuration of deacetylravidomycin (containing d-ravidosamine). We confirmed the positive sign by reisolating deacetylravidomycin from S. ravidus and by deacetylating ravidomycin. Finally, antibacterial, antifungal, and cytotoxicity activities were determined for the compounds. Compared to deacetylravidomycin, the compounds 4-6, 9, 11, and 12 exhibited greater antibacterial selectivity.
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Antineoplásicos , Streptomyces , Streptomyces/química , Aminoglicósidos , Antibacterianos/química , Estructura MolecularRESUMEN
Schwannomas are common, highly morbid and medically untreatable tumors that can arise in patients with germ line as well as somatic mutations in neurofibromatosis type 2 (NF2). These mutations most commonly result in the loss of function of the NF2-encoded protein, Merlin. Little is known about how Merlin functions endogenously as a tumor suppressor and how its loss leads to oncogenic transformation in Schwann cells (SCs). Here, we identify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) as a potential drug target driving NF-κB signaling and Merlin-deficient schwannoma genesis. Using a genomic approach to profile aberrant tumor signaling pathways, we describe multiple upregulated NF-κB signaling elements in human and murine schwannomas, leading us to identify a caspase-cleaved, proteasome-resistant NIK kinase domain fragment that amplifies pathogenic NF-κB signaling. Lentiviral-mediated transduction of this NIK fragment into normal SCs promotes proliferation, survival, and adhesion while inducing schwannoma formation in a novel in vivo orthotopic transplant model. Furthermore, we describe an NF-κB-potentiated hepatocyte growth factor (HGF) to MET proto-oncogene receptor tyrosine kinase (c-Met) autocrine feed-forward loop promoting SC proliferation. These innovative studies identify a novel signaling axis underlying schwannoma formation, revealing new and potentially druggable schwannoma vulnerabilities with future therapeutic potential.
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Neurilemoma/genética , Neurofibromatosis 2/genética , Neurofibromina 2/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Comunicación Autocrina/genética , Carcinogénesis/genética , Caspasa 1/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Humanos , Ratones , Terapia Molecular Dirigida , FN-kappa B/genética , Neurilemoma/complicaciones , Neurilemoma/tratamiento farmacológico , Neurilemoma/patología , Neurofibromatosis 2/complicaciones , Neurofibromatosis 2/tratamiento farmacológico , Neurofibromatosis 2/patología , Complejo de la Endopetidasa Proteasomal/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/genética , Células de Schwann , Transducción de Señal/genética , Quinasa de Factor Nuclear kappa BRESUMEN
Although targeted inhibition of oncogenic kinase drivers has achieved remarkable patient responses in many cancers, the development of resistance has remained a significant challenge. Numerous mechanisms have been identified, including the acquisition of gatekeeper mutations, activating pathway mutations, and copy number loss or gain of the driver or alternate nodes. These changes have prompted the development of kinase inhibitors with increased selectivity, use of second-line therapeutics to overcome primary resistance, and combination treatment to forestall resistance. In addition to genomic resistance mechanisms, adaptive transcriptional and signaling responses seen in tumors are gaining appreciation as alterations that lead to a phenotypic state change-often observed as an epithelial-to-mesenchymal shift or reversion to a cancer stem cell-like phenotype underpinned by remodeling of the epigenetic landscape. This epigenomic modulation driving cell state change is multifaceted and includes modulation of repressive and activating histone modifications, DNA methylation, enhancer remodeling, and noncoding RNA species. Consequently, the combination of kinase inhibitors with drugs targeting components of the transcriptional machinery and histone-modifying enzymes has shown promise in preclinical and clinical studies. Here, we review mechanisms of resistance to kinase inhibition in cancer, with special emphasis on the rewired kinome and transcriptional signaling networks and the potential vulnerabilities that may be exploited to overcome these adaptive signaling changes.
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Epigénesis Genética/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Neoplasias/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
PURPOSE: Plexiform neurofibromas (pNF) develop in children with neurofibromatosis type 1 (NF1) and can be associated with several skeletal comorbidities. Preclinical mouse studies revealed Nf1 deficiency in osteoprogenitor cells disrupts, in a MEK-dependent manner, pyrophosphate (PPi) homeostasis and skeletal mineralization. The etiology of NF-associated skeletal manifestations remains unknown. METHODS: We used mouse models of NF1 neurofibromas to assess bone mineralization of skeletal structures adjacent to tumors. Expression of genes involved in pyrophosphate homeostasis was assessed in mouse and human NF tumors and Schwann cell cultures. We used dual-energy X-ray absorptiometry (DXA) to assess tumor-associated changes in bone mineral density (BMD) in an individual with NF1 following treatment with the MEK inhibitor selumetinib. RESULTS: We detected increased nonmineralized bone surfaces adjacent to tumors in mouse models of NF1 neurofibromas. Expression of Enpp1, a PPi-generating ectophosphatase, and ANKH, a PPi transporter, was increased in mouse and human neurofibroma-derived tissues and Schwann cells, respectively. In one patient, tumor-associated reductions in BMD were partially rescued following therapy with selumetinib. CONCLUSION: Results indicate that NF-associated skeletal pathologies in NF1 are associated with dysregulated pyrophosphate homeostasis in adjacent NF tumors and suggest that treatment of NFs with MEK inhibitors may improve skeletal manifestations of the disease.
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Neurofibroma Plexiforme , Neurofibroma , Neurofibromatosis 1 , Animales , Humanos , Ratones , Neurofibroma Plexiforme/genética , Neurofibromatosis 1/genética , Inhibidores de Proteínas Quinasas , Células de SchwannRESUMEN
Protein binding specificities can be manipulated by redesigning contacts that already exist at an interface or by expanding the interface to allow interactions with residues adjacent to the original binding site. Previously, we developed a strategy, called AnchorDesign, for expanding interfaces around linear binding epitopes. The epitope is embedded in a loop of a scaffold protein, in our case a monobody, and then surrounding residues on the monobody are optimized for binding using directed evolution or computational design. Using this strategy, we have increased binding affinities by >100-fold, but we have not tested whether it can be used to control protein binding specificities. Here, we test whether AnchorDesign can be used to engineer a monobody that binds specifically to the mitogen-activated protein kinase (MAPK) p38α but not to the related MAPKs ERK2 and JNK. To anchor the binding interaction, we used a small (D) docking motif from the mitogen-activated protein kinase kinase (MAP2K) MKK6 that interacts with similar affinity with p38α and ERK2. Our hypothesis was that by embedding the motif in a larger protein that we could expand the interface and create contacts with residues that are not conserved between p38α and ERK2. Molecular modeling was used to inform insertion of the D motif into the monobody, and a combination of phage and yeast display were used to optimize the interface. Binding experiments demonstrate that the engineered monobody binds to the target surface on p38α and does not exhibit detectable binding to ERK2 or JNK.
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Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Péptidos/metabolismo , Ingeniería de Proteínas , Secuencia de Aminoácidos , Sitios de Unión , Células HEK293 , Humanos , Proteína Quinasa 14 Activada por Mitógenos/química , Simulación del Acoplamiento Molecular , Mutagénesis , Biblioteca de Péptidos , Péptidos/química , Péptidos/genética , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Programas InformáticosRESUMEN
Previous work has identified distinct functions for E2F proteins during a cellular proliferative response including a role for E2F1-3 in the activation of transcription at G1/S and a role for E2F4-8 in repressing the same group of E2F1-3 target genes as cells progress through S phase. We now find that E2F7 and E2F8, which are induced by E2F1-3 at G1/S, can form a heterodimer with E2F1 through interactions involving the DNA-binding domains of the two proteins. In vitro DNA interaction assays demonstrate the formation of an E2F1-E2F7 complex, as well as an E2F7-E2F7 complex on adjacent E2F-binding sites. We also show that E2F7 recruits the co-repressor C-terminal-binding protein (CtBP) and that CtBP2 is essential for E2F7 to repress E2F1 transcription. Taken together, these findings suggest a mechanism for the repression of transcription by E2F7.
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Oxidorreductasas de Alcohol/metabolismo , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F7/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética/fisiología , Oxidorreductasas de Alcohol/genética , Línea Celular Tumoral , Proteínas Co-Represoras , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F7/genética , Fase G1/fisiología , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Proteínas del Tejido Nervioso/genética , Multimerización de Proteína/fisiología , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Fase S/fisiologíaRESUMEN
Most cells in solid tumors are exposed to oxygen levels between 0.5% and 5%. We developed an approach that allows collection, processing, and evaluation of cancer and non-cancer cells under physioxia, while preventing exposure to ambient air. This aided comparison of baseline and drug-induced changes in signaling pathways under physioxia and ambient oxygen. Using tumor cells from transgenic models of breast cancer and cells from breast tissues of clinically breast cancer-free women, we demonstrate oxygen-dependent differences in cell preference for epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor beta (PDGFRß) signaling. Physioxia caused PDGFRß-mediated activation of AKT and extracellular regulated kinase (ERK) that reduced sensitivity to EGFR and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) inhibition and maintained PDGFRß+ epithelial-mesenchymal hybrid cells with potential cancer stem cell (CSC) properties. Cells in ambient air displayed differential EGFR activation and were more sensitive to targeted therapies. Our data emphasize the importance of oxygen considerations in preclinical cancer research to identify effective drug targets and develop combination therapy regimens.
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Introduction: Approximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy. Methods: To compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition. Results: RNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3. Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells. Conclusions: The findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.
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Schwann cells are vital to development and maintenance of the peripheral nervous system and their dysfunction has been implicated in a range of neurological and neoplastic disorders, including NF2 -related schwannomatosis. We developed a novel human induced pluripotent stem cell (hiPSC) model to study Schwann cell differentiation in health and disease. We performed transcriptomic, immunofluorescence, and morphological analysis of hiPSC derived Schwann cell precursors (SPCs) and terminally differentiated Schwann cells (SCs) representing distinct stages of development. To validate our findings, we performed integrated, cross-species analyses across multiple external datasets at bulk and single cell resolution. Our hiPSC model of Schwann cell development shared overlapping gene expression signatures with human amniotic mesenchymal stem cell (hAMSCs) derived SCs and in vivo mouse models, but also revealed unique features that may reflect species-specific aspects of Schwann cell biology. Moreover, we identified gene co-expression modules that are dynamically regulated during hiPSC to SC differentiation associated with ear and neural development, cell fate determination, the NF2 gene, and extracellular matrix (ECM) organization. By cross-referencing results between multiple datasets, we identified new genes potentially associated with NF2 expression. Our hiPSC model further provides a tractable platform for studying Schwann cell development in the context of human disease.
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Juvenile myelomonocytic leukemia (JMML) is a hematologic malignancy of young children caused by mutations that increase Ras signaling output. Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment, but patients with relapsed or refractory (advanced) disease have dismal outcomes. This phase II trial evaluated the safety and efficacy of trametinib, an oral MEK1/2 inhibitor, in patients with advanced JMML. Ten infants and children were enrolled, and the objective response rate was 50%. Four patients with refractory disease proceeded to HSCT after receiving trametinib. Three additional patients completed all 12 cycles permitted on study and continue to receive off-protocol trametinib without HSCT. The remaining three patients had progressive disease with two demonstrating molecular evolution by the end of cycle 2. Transcriptomic and proteomic analyses provided novel insights into the mechanisms of response and resistance to trametinib in JMML. ClinicalTrials.gov Identifier: NCT03190915. Significance: Trametinib was safe and effective in young children with relapsed or refractory JMML, a lethal disease with poor survival rates. Seven of 10 patients completed the maximum 12 cycles of therapy or used trametinib as a bridge to HSCT and are alive with a median follow-up of 24 months.
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PURPOSE: Plexiform neurofibromas (PNF) are benign peripheral nerve sheath tumors (PNST) associated with neurofibromatosis type 1 (NF1). Despite similar histologic appearance, these neoplasms exhibit diverse evolutionary trajectories, with a subset progressing to malignant peripheral nerve sheath tumor (MPNST), the leading cause of premature death in individuals with NF1. Malignant transformation of PNF often occurs through the development of atypical neurofibroma (ANF) precursor lesions characterized by distinct histopathologic features and CDKN2A copy-number loss. Although genomic studies have uncovered key driver events promoting tumor progression, the transcriptional changes preceding malignant transformation remain poorly defined. EXPERIMENTAL DESIGN: Here we resolve gene-expression profiles in PNST across the neurofibroma-to-MPNST continuum in NF1 patients and mouse models, revealing early molecular features associated with neurofibroma evolution and transformation. RESULTS: Our findings demonstrate that ANF exhibit enhanced signatures of antigen presentation and immune response, which are suppressed as malignant transformation ensues. MPNST further displayed deregulated survival and mitotic fidelity pathways, and targeting key mediators of these pathways, CENPF and BIRC5, disrupted the growth and viability of human MPNST cell lines and primary murine Nf1-Cdkn2a-mutant Schwann cell precursors. Finally, neurofibromas contiguous with MPNST manifested distinct alterations in core oncogenic and immune surveillance programs, suggesting that early molecular events driving disease progression may precede histopathologic evidence of malignancy. CONCLUSIONS: If validated prospectively in future studies, these signatures may serve as molecular diagnostic tools to augment conventional histopathologic diagnosis by identifying neurofibromas at high risk of undergoing malignant transformation, facilitating risk-adapted care.
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Neoplasias de la Vaina del Nervio , Neurofibroma , Neurofibromatosis 1 , Neurofibrosarcoma , Animales , Humanos , Ratones , Perfilación de la Expresión Génica , Neoplasias de la Vaina del Nervio/genética , Neurofibroma/genética , Neurofibromatosis 1/genética , Neurofibrosarcoma/genéticaRESUMEN
PURPOSE: Plexiform neurofibromas (PNF) are peripheral nerve sheath tumors that cause significant morbidity in persons with neurofibromatosis type 1 (NF1), yet treatment options remain limited. To identify novel therapeutic targets for PNF, we applied an integrated multi-omic approach to quantitatively profile kinome enrichment in a mouse model that has predicted therapeutic responses in clinical trials for NF1-associated PNF with high fidelity. EXPERIMENTAL DESIGN: Utilizing RNA sequencing combined with chemical proteomic profiling of the functionally enriched kinome using multiplexed inhibitor beads coupled with mass spectrometry, we identified molecular signatures predictive of response to CDK4/6 and RAS/MAPK pathway inhibition in PNF. Informed by these results, we evaluated the efficacy of the CDK4/6 inhibitor, abemaciclib, and the ERK1/2 inhibitor, LY3214996, alone and in combination in reducing PNF tumor burden in Nf1flox/flox;PostnCre mice. RESULTS: Converging signatures of CDK4/6 and RAS/MAPK pathway activation were identified within the transcriptome and kinome that were conserved in both murine and human PNF. We observed robust additivity of the CDK4/6 inhibitor, abemaciclib, in combination with the ERK1/2 inhibitor, LY3214996, in murine and human NF1(Nf1) mutant Schwann cells. Consistent with these findings, the combination of abemaciclib (CDK4/6i) and LY3214996 (ERK1/2i) synergized to suppress molecular signatures of MAPK activation and exhibited enhanced antitumor activity in Nf1flox/flox;PostnCre mice in vivo. CONCLUSIONS: These findings provide rationale for the clinical translation of CDK4/6 inhibitors alone and in combination with therapies targeting the RAS/MAPK pathway for the treatment of PNF and other peripheral nerve sheath tumors in persons with NF1.
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Neoplasias de la Vaina del Nervio , Neurofibroma Plexiforme , Neurofibroma , Neurofibromatosis 1 , Humanos , Ratones , Animales , Neurofibroma Plexiforme/etiología , Neurofibroma Plexiforme/genética , Neurofibromatosis 1/tratamiento farmacológico , Neurofibromatosis 1/genética , Sistema de Señalización de MAP Quinasas , Proteómica , Neoplasias de la Vaina del Nervio/tratamiento farmacológico , Neoplasias de la Vaina del Nervio/genética , Inhibidores de Proteínas Quinasas/farmacología , Neurofibroma/complicaciones , Quinasa 4 Dependiente de la Ciclina/genéticaRESUMEN
Dysregulation of PI3K/Akt signaling is a dominant feature in basal-like or triple-negative breast cancers (TNBC). However, the mechanisms regulating this pathway are largely unknown in this subset of aggressive tumors. Here we demonstrate that the transcription factor SOX4 is a key regulator of PI3K signaling in TNBC. Genomic and proteomic analyses coupled with mechanistic studies identified TGFBR2 as a direct transcriptional target of SOX4 and demonstrated that TGFBR2 is required to mediate SOX4-dependent PI3K signaling. We further report that SOX4 and the SWI/SNF ATPase SMARCA4, which are uniformly overexpressed in basal-like tumors, form a previously unreported complex that is required to maintain an open chromatin conformation at the TGFBR2 regulatory regions in order to mediate TGFBR2 expression and PI3K signaling. Collectively, our findings delineate the mechanism by which SOX4 and SMARCA4 cooperatively regulate PI3K/Akt signaling and suggest that this complex may play an essential role in TNBC genesis and/or progression.
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Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.
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Glutamatos/metabolismo , Procesamiento de Imagen Asistido por Computador , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Neuronas/patología , Proteómica , Tauopatías/patología , Proteínas tau/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismo , Piridinas/química , Piridinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.
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Neurofibromatosis type 1 (NF1) plexiform neurofibromas (PNs) are progressive, multicellular neoplasms that cause morbidity and may transform to sarcoma. Treatment of Nf1fl/fl;Postn-Cre mice with cabozantinib, an inhibitor of multiple tyrosine kinases, caused a reduction in PN size and number and differential modulation of kinases in cell lineages that drive PN growth. Based on these findings, the Neurofibromatosis Clinical Trials Consortium conducted a phase II, open-label, nonrandomized Simon two-stage study to assess the safety, efficacy and biologic activity of cabozantinib in patients ≥16 years of age with NF1 and progressive or symptomatic, inoperable PN ( NCT02101736 ). The trial met its primary outcome, defined as ≥25% of patients achieving a partial response (PR, defined as ≥20% reduction in target lesion volume as assessed by magnetic resonance imaging (MRI)) after 12 cycles of therapy. Secondary outcomes included adverse events (AEs), patient-reported outcomes (PROs) assessing pain and quality of life (QOL), pharmacokinetics (PK) and the levels of circulating endothelial cells and cytokines. Eight of 19 evaluable (42%) trial participants achieved a PR. The median change in tumor volume was 15.2% (range, +2.2% to -36.9%), and no patients had disease progression while on treatment. Nine patients required dose reduction or discontinuation of therapy due to AEs; common AEs included gastrointestinal toxicity, hypothyroidism, fatigue and palmar plantar erythrodysesthesia. A total of 11 grade 3 AEs occurred in eight patients. Patients with PR had a significant reduction in tumor pain intensity and pain interference in daily life but no change in global QOL scores. These data indicate that cabozantinib is active in NF1-associated PN, resulting in tumor volume reduction and pain improvement.
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Anilidas/uso terapéutico , Neurofibroma Plexiforme/tratamiento farmacológico , Neurofibromatosis 1/tratamiento farmacológico , Piridinas/uso terapéutico , Adolescente , Adulto , Anilidas/efectos adversos , Anilidas/farmacocinética , Animales , Modelos Animales de Enfermedad , Femenino , Genes de Neurofibromatosis 1 , Humanos , Masculino , Ratones , Ratones Mutantes , Neurofibroma Plexiforme/genética , Neurofibroma Plexiforme/patología , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , Dimensión del Dolor , Estudios Prospectivos , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/efectos adversos , Piridinas/farmacocinética , Calidad de Vida , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Investigación Biomédica Traslacional , Adulto JovenRESUMEN
Neurofibromatosis Type 2 (NF2) is an autosomal dominant genetic syndrome caused by mutations in the NF2 tumor suppressor gene resulting in multiple schwannomas and meningiomas. There are no FDA approved therapies for these tumors and their relentless progression results in high rates of morbidity and mortality. Through a combination of high throughput screens, preclinical in vivo modeling, and evaluation of the kinome en masse, we identified actionable drug targets and efficacious experimental therapeutics for the treatment of NF2 related schwannomas and meningiomas. These efforts identified brigatinib (ALUNBRIG®), an FDA-approved inhibitor of multiple tyrosine kinases including ALK, to be a potent inhibitor of tumor growth in established NF2 deficient xenograft meningiomas and a genetically engineered murine model of spontaneous NF2 schwannomas. Surprisingly, neither meningioma nor schwannoma cells express ALK. Instead, we demonstrate that brigatinib inhibited multiple tyrosine kinases, including EphA2, Fer and focal adhesion kinase 1 (FAK1). These data demonstrate the power of the de novo unbiased approach for drug discovery and represents a major step forward in the advancement of therapeutics for the treatment of NF2 related malignancies.
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Neoplasias Meníngeas/genética , Meningioma/genética , Neurilemoma/genética , Neurofibromina 2/deficiencia , Neurofibromina 2/genética , Compuestos Organofosforados/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Proliferación Celular , Humanos , Mutación , Neurilemoma/patologíaRESUMEN
Loss of the tumor suppressor NF1 leads to activation of RAS effector pathways, which are therapeutically targeted by inhibition of mTOR (mTORi) or MEK (MEKi). However, therapeutic inhibition of RAS effectors leads to the development of drug resistance and ultimately disease progression. To investigate molecular signatures in the context of NF1 loss and subsequent acquired drug resistance, we analyzed the exomes, transcriptomes, and kinomes of Nf1-mutant mouse tumor cell lines and derivatives of these lines that acquired resistance to either MEKi or mTORi. Biochemical comparisons of this unique panel of tumor cells, all of which arose in Nf1+/- mice, indicate that loss of heterozygosity of Nf1 as an initial genetic event does not confer a common biochemical signature or response to kinase inhibition. Although acquired drug resistance by Nf1-mutant tumor cells was accompanied by altered kinomes and irreversibly altered transcriptomes, functionally in multiple Nf1-mutant tumor cell lines, MEKi resistance was a stable phenotype, in contrast to mTORi resistance, which was reversible. Collectively, these findings demonstrate that Nf1-mutant tumors represent a heterogeneous group biochemically and undergo broader remodeling of kinome activity and gene expression in response to targeted kinase inhibition.
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Mutación , Neurofibromatosis 1/genética , Neurofibromatosis 1/metabolismo , Neurofibromina 1/genética , Proteínas Quinasas/metabolismo , Transcriptoma , Animales , Línea Celular Tumoral , Biología Computacional , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Modelos Biológicos , Neurofibromatosis 1/tratamiento farmacológico , Neurofibromatosis 1/patología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de SeñalRESUMEN
BACKGROUND: Aberrant Myc expression plays a critical role in various tumors, including non-Hodgkin lymphoma (NHL). Myc-positive lymphoma is clinically aggressive, more resistant to chemotherapy, and associated with high mortality. OBJECTIVE: The current study aimed to show inhibition of aurora A kinase (AURKA) may overcome resistance to chemotherapy and improve outcomes in Myc-overexpressing lymphoma. METHODS: Myc-overexpressing lymphoma cell lines were evaluated by trypan blue, annexin V/propidium iodide staining, and western blotting for cytotoxicity, cell cycle, apoptosis, and Myc-associated protein expression, respectively, in the presence of cyclophosphamide with or without MLN8237, an AURKA inhibitor. Immunofluorescence for apoptosis-inducing factor (AIF) and acridine orange staining were used to analyze levels of autophagy. EµMyc genetically modified mouse model and xenograft models bearing Myc-overexpressing lymphoma cells were used to determine the efficacy of cyclophosphamide, MLN8237, or the combination in chemosensitive and chemoresistant tumors. RESULTS: In our in vitro experiments using chemoresistant lymphoma cells, MLN8237 and cyclophosphamide showed synergistic effects. Mice bearing lymphoma xenograft had rapid disease progression with median survival of ~ 35 days when treated with cyclophosphamide alone. In contrast, the combination of cyclophosphamide and MLN8237 induced complete tumor regression in all mice, which led to improvement in survival compared with the single agent control (p = 0.022). Kinome analysis of tumors treated with MLN8237 showed global suppression of various kinases. CONCLUSION: Our data demonstrate that AURKA inhibition induces synthetic lethality and overcomes chemoresistance in Myc-overexpressing lymphoma. The combination of MLN8237 and conventional chemotherapy showed promising safety and anti-tumor activities in preclinical models of Myc-positive NHL.
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Antineoplásicos/uso terapéutico , Aurora Quinasa A/antagonistas & inhibidores , Azepinas/farmacología , Ciclofosfamida/farmacología , Linfoma no Hodgkin/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/genética , Pirimidinas/farmacología , Animales , Apoptosis , Ciclo Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Quimioterapia Combinada , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Linfoma no Hodgkin/genética , Ratones , Ratones Desnudos , Ratones Transgénicos , Mutación/genéticaRESUMEN
Screening of an inhibitor library targeting kinases and epigenetic regulators identified several molecules having antiproliferative synergy with extraterminal domain (BET) bromodomain (BD) inhibitors (JQ1, OTX015) in triple-negative breast cancer (TNBC). GSK2801, an inhibitor of BAZ2A/B BDs, of the imitation switch chromatin remodeling complexes, and BRD9, of the SWI/SNF complex, demonstrated synergy independent of BRD4 control of P-TEFb-mediated pause-release of RNA polymerase II. GSK2801 or RNAi knockdown of BAZ2A/B with JQ1 selectively displaced BRD2 at promoters/enhancers of ETS-regulated genes. Additional displacement of BRD2 from rDNA in the nucleolus coincided with decreased 45S rRNA, revealing a function of BRD2 in regulating RNA polymerase I transcription. In 2D cultures, enhanced displacement of BRD2 from chromatin by combination drug treatment induced senescence. In spheroid cultures, combination treatment induced cleaved caspase-3 and cleaved PARP characteristic of apoptosis in tumor cells. Thus, GSK2801 blocks BRD2-driven transcription in combination with BET inhibitor and induces apoptosis of TNBC. IMPLICATIONS: Synergistic inhibition of BDs encoded in BAZ2A/B, BRD9, and BET proteins induces apoptosis of TNBC by a combinatorial suppression of ribosomal DNA transcription and ETS-regulated genes.