Transcriptional activators, several different coactivators, and general transcription factors are necessary to access specific loci in the dense chromatin structure to allow precise initiation of RNA polymerase II (Pol II) transcription. Histone acetyltransferase (HAT) complexes were implicated in loosening the chromatin around promoters and thus in gene activation. Here we demonstrate that the 2 MDa GCN5 HAT-containing metazoan TFTC/STAGA complexes contain a histone H2A and H2B deubiquitinase activity. We have identified three additional subunits of TFTC/STAGA (ATXN7L3, USP22, and ENY2) that form the deubiquitination module. Importantly, we found that this module is an enhancer of position effect variegation in Drosophila. Furthermore, we demonstrate that ATXN7L3, USP22, and ENY2 are required as cofactors for the full transcriptional activity by nuclear receptors. Thus, the deubiquitinase activity of the TFTC/STAGA HAT complex is necessary to counteract heterochromatin silencing and acts as a positive cofactor for activation by nuclear receptors in vivo.

Breast cancers are composed of molecularly distinct subtypes with different clinical outcomes and responses to therapy. To discover potential therapeutic targets for the poor prognosis-associated triple-negative breast cancer (TNBC), gene expression profiling was carried out on a cohort of 130 breast cancer samples. Polo-like kinase 1 (PLK1) was found to be significantly overexpressed in TNBC compared with the other breast cancer subtypes. High PLK1 expression was confirmed by reverse phase protein and tissue microarrays. In triple-negative cell lines, RNAi-mediated PLK1 depletion or inhibition of PLK1 activity with a small molecule (BI-2536) induced an increase in phosphorylated H2AX, G 2 -M arrest, and apoptosis. A soft-agar colony assay showed that PLK1 silencing impaired clonogenic potential of TNBC cell lines. When cells were grown in extracellular matrix gels (Matrigel), and exposed to BI-2536, apoptosis was observed specifically in TNBC cancerous cells, and not in a normal cell line. When administrated as a single agent, the PLK1 inhibitor significantly impaired tumor growth in vivo in two xenografts models established from biopsies of patients with TNBC. Most importantly, the administration of BI-2536, in combination with doxorubicin þ cyclophosphamide chemotherapy, led to a faster complete response compared with the chemotherapy treatment alone and prevented relapse, which is the major risk associated with TNBC. Altogether, our observations suggest PLK1 inhibition as an attractive therapeutic approach, in association with conventional chemotherapy, for the management of patients with TNBC. Cancer Res; 73(2); 813-23. Ó2012 AACR.

The multisubunit SAGA coactivator complex facilitates access of general transcription factors to DNA through histone acetylation mediated by GCN5. USP22 (ubiquitin-specific protease 22) was recently described as a subunit of the human SAGA complex that removes ubiquitin from monoubiquitinated histone H2B and H2A in vitro. Here we demonstrate an allosteric regulation of USP22 through multiple interactions with different domains of other subunits of the SAGA deubiquitination module (ATXN7, ATXN7L3, and ENY2). Downregulation of ATXN7L3 by short hairpin RNA (shRNA) specifically inactivated the SAGA deubiquitination activity, leading to a strong increase of global H2B ubiquitination and a moderate increase of H2A ubiquitination. Thus, SAGA is the major H2Bub deubiquitinase in human cells, and this activity cannot be fully compensated by other deubiquitinases. Here we show that the deubiquitination activity of SAGA is required for full activation of SAGA-dependent inducible genes. Interestingly, the reduction of the SAGA deubiquitination activity and the parallel increase in H2B ubiquitation at inducible target genes before activation do not induce aberrant gene expression. Our data together indicate that different dynamic equilibriums of H2B ubiquitination/deubiquitination are established at different gene regulatory elements and that H2B ubiquitination changes are necessary but not sufficient to trigger parallel activation of gene expression.

PurposeUveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies. Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments. Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2. Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics. In this aim, we have investigated the efficacy of the Bcl-2/Bcl-XL inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines.Experimental DesignFour well characterized UM PDXs were used for in vivo experiments. S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent. Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC).ResultsS44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect. However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models. In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts. Finally, IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expression were not modified after S44563 administration.ConclusionThe novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine. These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM.

The last 20 years have witnessed the identification of an increasing number of druggable oncogenic drivers and the development and clinical use of specific inhibitors against these targets. Unfortunately, patients treated with targeted therapies consistently develop resistance and progression under treatment. Hence, important scientific, pharmaceutical and medical research efforts are directed towards understanding the mechanisms of acquired resistance to explore new therapeutic pathways. The MATCH-R clinical trial enrolls patients with oncogene-driven cancer who have had previous clinical response to targeted therapy and subsequently experienced disease progression. In the framework of this project, Gustave Roussy and XenTech are joining forces to develop a panel of patient-derived xenografts (PDXs) derived from biopsies collected from these patients at the stage of acquired resistance. These PDX models will be used to improve knowledge on the mechanisms underlying resistance to treatment and to evaluate response to new treatments. In this perspective, the development of 75 PDX-AR (Active Resistance) models is planned over 3 years. All the models are maintained under the same therapeutic pressure the parental tumor was submitted to at the time of biopsy, and will be subjected to extensive phenotypic and genotypic characterization. The following models have been established so far: • ENDx-MR-004-AR (endometrial): resistant to the combination of MEK and MDM2 inhibitors; • LCx-MR-007-AR: (NSCLC): resistant to third generation EGFR inhibitor (osimertinib); • UREx-MR-015A-AR (ureter) and VEx-MR-086A-AR (bladder): resistant to a FGFR inhibitor (erdafitinib); • PARx-MR-010-AR (parotid): resistant to a NOTCH Inhibitor; • TCx-MR-122-AR (colon): resistant to an ATR inhibitor. To favor successful xenograft establishment, the first two passages were performed without drug treatment, which was applied from the third passage on. When doing so, some models showed resistance from the first passage under treatment, whereas others showed stabilization under treatment at the first passages and rapidly acquired resistance over passages. These different behaviors might underlie different mechanisms of resistance, irreversible (monoclonal) for the former, reversible (polyclonal) for the latter. Parallel to the development of UREx-MR-015A-AR, we developed the UREx-MR-015B-SD (stable disease) model from a biopsy collected from a different metastasis in the same patient, but stabilized by the therapy. Comparative analysis of these two models will provide important insights into the mechanisms of resistance to FGFR inhibitors. The MATCH-R PDX project will provide a unique preclinical platform for identifying resistance mechanisms to current targeted therapies and developing next generation therapeutic strategies. Citation Format: Olivier Déas, Ludovic Bigot, Guillaume Lang, Yohann Loriot, Fabrice Andre, Jean Charles Soria, Benjamin Besse, Stefano Cairo, Marie Tavernier, Katell Mevel, Enora Le Ven, Jean-Gabriel Judde, Luc Friboulet. Development of preclinical models to accelerate the identification of next generation treatments for patients with acquired resistance to targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2147.

Muscle mass correlated strongly with coordination and isometric strength in untreated rats. Anabolic effect, fiber phenotype modification, and perturbation in neuromuscular communication with clenbuterol improved muscle mass, but it altered physical capacities.

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Breast cancers are composed of molecularly distinct subtypes with different clinical outcomes and responses to therapy. Our aim is to discover potential therapeutic targets for triple negative breast cancer (TNBC), a poor prognosis subgroup with no targeted therapy available yet. Gene expression profiling on a cohort of 160 breast cancers including 40 TNBC revealed that the polo-like kinase 1 (PLK1), a protein kinase, is specifically over-expressed in TNBC compared to the other breast cancer subtypes. High PLK1 expression was confirmed at a proteomic level by reverse phase protein array and tissue microarray. In TNBC cell lines grown as monolayers, RNAi-mediated PLK1 depletion or small compound (BI-2536)-mediated PLK1 inhibition induced an increase in phosphorylated H2AX, G2/M arrest and apoptosis, resulting in a decrease in cell viability. In addition, a soft-agar colony assay showed that PLK1 silencing impaired tumorigenicity of TNBC cell lines. With cells grown in extracellular matrix gels (Matrigel), BI-2536 induced apoptosis specifically in TNBC cancerous versus “normal” cell lines. The in vivo anti-tumor effect of BI-2536 was investigated in two TNBC xenografts derived from patient's biopsies. When administrated as a single agent, the PLK1 inhibitor significantly impaired the tumor growth. Most importantly, administration of BI-2536 in combination with adriamycin + cyclophosphamide chemotherapy led to 100% complete response (9/9 mice). Altogether, our observations point out that PLK1 may represent an attractive therapeutic target, in association with conventional chemotherapy, for the management of patients with TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2938. doi:1538-7445.AM2012-2938