An Intrinsically Disordered Region of the Acetyltransferase p300 with Similarity to Prion-Like Domains Plays a Role in Aggregation

Several human diseases including neurodegenerative disorders and cancer are associated with abnormal accumulation and aggregation of misfolded proteins. Proteins with high tendency to aggregate include the p53 gene product, TAU and alpha synuclein. The potential toxicity of aberrantly folded proteins is limited via their transport into intracellular sub-compartments, the aggresomes, where misfolded proteins are stored or cleared via autophagy. We have identified a region of the acetyltransferase p300 that is highly disordered and displays similarities with prion-like domains. We show that this region is encoded as an alternative spliced variant independently of the acetyltransferase domain, and provides an interaction interface for various misfolded proteins, promoting their aggregation. p300 enhances aggregation of TAU and of p53 and is a component of cellular aggregates in both tissue culture cells and in alpha-synuclein positive Lewy bodies of patients affected by Parkinson disease. Down-regulation of p300 impairs aggresome formation and enhances cytotoxicity induced by misfolded protein stress. These data unravel a novel activity of p300, offer new insights into the function of disordered domains and implicate p300 in pathological aggregation that occurs in neurodegeneration and cancer

Targeting the epigenetic readers in Ewing Sarcoma inhibits the oncogenic transcription factor EWS/Fli1.

Ewing Sarcoma is a rare bone and soft tissue malignancy affecting children and young adults. Chromosomal translocations in this cancer produce fusion oncogenes as characteristic molecular signatures of the disease. The most common case is the translocation t (11; 22) (q24;q12) which yields the EWS-Fli1 chimeric transcription factor. Finding a way to directly target EWS-Fli1 remains a central therapeutic approach to eradicate this aggressive cancer. Here we demonstrate that treating Ewing Sarcoma cells with JQ1(+), a BET bromodomain inhibitor, represses directly EWS-Fli1 transcription as well as its transcriptional program. Moreover, the Chromatin Immuno Precipitation experiments demonstrate for the first time that these results are a consequence of the depletion of BRD4, one of the BET bromodomains protein from the EWS-Fli1 promoter. In vitro, JQ1(+) treatment reduces the cell viability, impairs the cell clonogenic and the migratory abilities, and induces a G1-phase blockage as well as a time- and a dose-dependent apoptosis. Furthermore, in our in vivo model, we observed a tumor burden delay, an inhibition of the global vascularization and an increase of the mice overall survival. Taken together, our data indicate that inhibiting the BET bromodomains interferes with EWS-FLi1 transcription and could be a promising strategy in the Ewing tumors context

Transcription factor clusters regulate genes in eukaryotic cells

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression

Effects of a Fluorescent Myosin Light Chain Phosphatase Inhibitor on Prostate Cancer Cells

Myosin light chain phosphatase (MLCP) is an enzyme important to regulation of cell cycle and motility that is shown to be upregulated in aggressive prostate cancer cells and tissue. We developed a fluorescent small molecule inhibitor of MLCP using structure based design in recombinant protein phosphatase 1C. Several best fit compounds were synthesized and evaluated by their inhibition of MLCP/32P-MLC dephosphorylation, which resulted in the identification of novel MLCP inhibitors. Androgen dependent (AD) and castration resistant prostate cancer cell (CRPC) lines were treated with the lead inhibitor resulting in decreased growth rate, reduced DNA synthesis, and G2/M cell cycle arrest. Moreover, CRPC cell lines showed an increased sensitivity to drug treatment having GI50 values four times lower than the AD prostate cancer cell line. This was reinforced by reduced BrdU DNA incorporation into CRPC cells compared to AD cells. β-actin disruption was also seen at much lower drug concentrations in CR cells which caused a dose dependent reduction in cellular chemotaxis of PC-3 cells. Since there are currently few clinical therapeutics targeting CR prostate cancer, MLCP represents a new target for preclinical and clinical development of new potential therapeutics which inhibit this disease phenotype

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation

The first European interdisciplinary Ewing sarcoma research summit

The European Network for Cancer Research in Children and Adolescents (ENCCA) provides an interaction platform for stakeholders in research and care of children with cancer. Among ENCCA objectives is the establishment of biology-based prioritization mechanisms for the selection of innovative targets, drugs, and prognostic markers for validation in clinical trials. Specifically for sarcomas, there is a burning need for novel treatment options, since current chemotherapeutic treatment protocols have met their limits. This is most obvious for metastatic Ewing sarcoma (ES), where long term survival rates are still below 20%. Despite significant progress in our understanding of ES biology, clinical translation of promising laboratory results has not yet taken place due to fragmentation of research and lack of an institutionalized discussion forum. To fill this gap, ENCCA assembled 30 European expert scientists and five North American opinion leaders in December 2011 to exchange thoughts and discuss the state of the art in ES research and latest results from the bench, and to propose biological studies and novel promising therapeutics for the upcoming European EWING2008 and EWING2012 clinical trials

Insurance payer is associated with length of stay after traumatic brain injury.

OBJECTIVES: Timely provision of postacute care (PAC) rehabilitation is critical for achieving functional recovery after traumatic brain injury (TBI). Medicaid coverage is a predictor of prolonged hospital length of stay (LOS) after TBI, a proxy for decreased PAC access. Among Medicaid patients with TBI, it is unknown whether coverage under a managed care organization (MCO) or fee-for-service (FFS) model predicts differences in LOS. STUDY DESIGN: Discharge data for patients with TBI from 318 California hospitals between 2017 and 2019 were obtained. We used multivariable regression models, treating mortality/hospice disposition as competing risks, to evaluate associations between insurance type and LOS, adjusting for sociodemographic factors and illness severity. Sensitivity analysis was conducted in patients with severe TBI identified by receipt of intracranial pressure monitoring or trauma craniotomy/craniectomy. Adjusted HRs (aHRs) were reported. METHODS: The California Department of Health Care Access and Information Patient Discharge Dataset was queried for patients with TBI using International Classification of Diseases, Tenth Revision, Clinical Modification codes. Exclusion criteria were younger than 18 years or older than 65 years, payer other than private insurance (PI) or Medicaid, death or hospice discharge within 5 days of hospitalization, presence of a do-not-resuscitate order, and nonemergency admission. RESULTS: A total of 39,834 patients were analyzed (FFS, 24.2%; MCO, 33.2%; PI, 42.6%). Competing risk regressions showed that Medicaid models were associated with longer LOS compared with PI (FFS: aHR, 0.80; 95% CI, 0.80-0.83; MCO: aHR, 0.92; 95% CI, 0.87-0.96). Compared with MCOs, FFS was associated with longer LOS in the overall cohort (aHR, 0.88; 95% CI, 0.85-0.91) and in the severe TBI subgroup (aHR, 0.90; 95% CI, 0.82-0.99). CONCLUSIONS: Medicaid FFS is associated with increased LOS in hospitalized patients with TBI compared with Medicaid MCOs, suggesting decreased PAC access

Combinatorial drug screening identifies Ewing sarcoma-specific sensitivities

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1-dependent manner. Mol Cancer Ther; 16(1); 88-101. ©2016 AACR

YK-4-279 Inhibits ERG and ETV1 Mediated Prostate Cancer Cell Invasion

Background: Genomic rearrangements involving the ETS family of transcription factors occur in 40–70 % of prostate cancer cases. ERG and ETV1 are the most common ETS members observed in these genetic alterations. The high prevalence of these rearrangements and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. Methods and Findings: We recently reported the development of YK-4-279, a small molecule inhibitor of EWS-FLI1 oncoprotein in Ewing’s Sarcoma. Since ERG and ETV1 belong to the same class of ETS factors as FLI1, we tested the ability of YK-4-279 to inhibit biological functions of ERG and ETV1 proteins in prostate cancer. YK-4-279 inhibited ERG and ETV1 mediated transcriptional activity in a luciferase assay. YK-4-279 also decreased ERG and ETV1 downstream target mRNA and protein expression in ETV1-fusion positive LNCaP and ERG fusion positive VCaP cells. YK-4-279 reduced the motility of LNCaP cells in a scratch assay and the invasive phenotype of both LNCaP and VCaP cells in a HUVEC invasion assay. Fusion-negative PC3 cells were unresponsive to YK-4-279. SiRNA mediated ERG knockdown in VCaP cells resulted in a loss of drug responsiveness. Concurrently, transient ERG expression in PC-3 cells resulted in increased invasive potential, which was reduced by YK-4-279. Conclusion: These data demonstrate that YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostat

Insulin-Like Growth Factor 1 Receptor as a Therapeutic Target in Ewing Sarcoma: Lack of Consistent Upregulation or Recurrent Mutation and a Review of the Clinical Trial Literature

The insulin-like growth factor 1 receptor (IGF-1R) has been considered an important therapeutic target in Ewing sarcoma (ES), generating a need to identify the subset of patients most likely to respond to IGF-1R inhibitors. We assessed IGF-1R expression in ES cell lines and patient tumors to understand the variable clinical responses to anti-IGF-1R therapy. Using ligand-binding displacement, we measured between 13,000 and 40,000 receptors per cell in ES cell lines. We used ELISA to quantify IGF-1R in patient tumors, which expressed 4.8%  ± 3.7 to 20.0%  ± 0.2 of the levels in a positive control cell line overexpressing IGF-1R. Flow cytometry showed markedly reduced IGF-1R expression in ES cell lines compared to a standard positive control cell line. The IGF1R gene was sequenced in 47 ES tumor samples and 8 ES cell lines; only one tumor sample showed a nonsynonymous mutation, R1353H, in a region with low functional impact. Finally, we assessed IGF-1R pathway activity in the ES stem cell (ESSC) population, to characterize its potential for resistance to anti-IGF-1R therapy, using Luminex technology. We found no significant differences in IGF-1R pathway activity between ESSCs and the total cell population. Overall, our findings suggest that IGF-1R as a therapeutic target in this sarcoma may require reevaluation