Purpose To investigate the safety and efficacy of durvalumab, a human monoclonal antibody that binds programmed cell death ligand-1 (PD-L1), and the role of PD-L1 expression on clinical response in patients with advanced urothelial bladder cancer (UBC). Methods A phase 1/2 multicenter, open-label study is being conducted in patients with inoperable or metastatic solid tumors. We report here the results from the UBC expansion cohort. Durvalumab (MEDI4736, 10 mg/kg every 2 weeks) was administered intravenously for up to 12 months. The primary end point was safety, and objective response rate (ORR, confirmed) was a key secondary end point. An exploratory analysis of pretreatment tumor biopsies led to defining PD-L1–positive as ≥ 25% of tumor cells or tumor-infiltrating immune cells expressing membrane PD-L1. Results A total of 61 patients (40 PD-L1–positive, 21 PD-L1–negative), 93.4% of whom received one or more prior therapies for advanced disease, were treated (median duration of follow-up, 4.3 months). The most common treatment-related adverse events (AEs) of any grade were fatigue (13.1%), diarrhea (9.8%), and decreased appetite (8.2%). Grade 3 treatment-related AEs occurred in three patients (4.9%); there were no treatment-related grade 4 or 5 AEs. One treatment-related AE (acute kidney injury) resulted in treatment discontinuation. The ORR was 31.0% (95% CI, 17.6 to 47.1) in 42 response-evaluable patients, 46.4% (95% CI, 27.5 to 66.1) in the PD-L1–positive subgroup, and 0% (95% CI, 0.0 to 23.2) in the PD-L1–negative subgroup. Responses are ongoing in 12 of 13 responding patients, with median duration of response not yet reached (range, 4.1+ to 49.3+ weeks). Conclusion Durvalumab demonstrated a manageable safety profile and evidence of meaningful clinical activity in PD-L1–positive patients with UBC, many of whom were heavily pretreated.

Purpose: Histone deacetylase (HDAC) inhibitors have emerged recently as promising anticancer agents. They arrest cells in the cell cycle and induce differentiation and cell death. The antitumor activity of HDAC inhibitors has been linked to their ability to induce gene expression through acetylation of histone and nonhistone proteins. However, it has recently been suggested that HDAC inhibitors may also enhance the activity of other cancer therapeutics, including radiotherapy. The purpose of this study was to evaluate the ability of HDAC inhibitors to radiosensitize human melanoma cells in vitro. Experimental Design: A panel of HDAC inhibitors that included sodium butyrate (NaB), phenylbutyrate, tributyrin, and trichostatin A were tested for their ability to radiosensitize two human melanoma cell lines (A375 and MeWo) using clonogenic cell survival assays. Apoptosis and DNA repair were measured by standard assays. Results: NaB induced hyperacetylation of histone H4 in the two melanoma cell lines and the normal human fibroblasts. NaB radiosensitized both the A375 and MeWo melanoma cell lines, substantially reducing the surviving fraction at 2 Gy (SF2), whereas it had no effect on the normal human fibroblasts. The other HDAC inhibitors, phenylbutyrate, tributyrin, and trichostatin A had significant radiosensitizing effects on both melanoma cell lines tested. NaB modestly enhanced radiation-induced apoptosis that did not correlate with survival but did correlate with functional impairment of DNA repair as determined based on the host cell reactivation assay. Moreover, NaB significantly reduced the expression of the repair-related genes Ku70 and Ku86 and DNA-dependent protein kinase catalytic subunit in melanoma cells at the protein and mRNA levels. Normal human fibroblasts showed no change in DNA repair capacity or levels of DNA repair proteins following NaB treatment. We also examined g-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, g-H2AX foci persisted long after ionizing exposure in the NaB-treated cells. Conclusions: HDAC inhibitors radiosensitize human tumor cells by affecting their ability to repair the DNA damage induced by ionizing radiation and that g-H2AX phosphorylation can be used as a predictive marker of radioresponse.

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Purpose Nivolumab, an anti-PD-1 immune checkpoint inhibitor, improved overall survival versus everolimus in a phase 3 trial of previously treated patients with metastatic renal cell carcinoma (mRCC). We investigated immunomodulatory activity of nivolumab in a hypothesis-generating prospective mRCC trial. Experimental Design Nivolumab was administered intravenously every 3 weeks at 0.3, 2, or 10 mg/kg to previously treated patients and 10 mg/kg to treatment-naïve patients with mRCC. Baseline and on-treatment biopsies and blood were obtained. Clinical activity, tumor-associated lymphocytes, PD-L1 expression (Dako immunohistochemistry; ≥5% vs. <5% tumor membrane staining), tumor gene expression (Affymetrix U219), serum chemokines, and safety were assessed. Results In 91 treated patients, median overall survival (95% CI) was 16.4 months (10.1–not reached [NR]) for nivolumab 0.3 mg/kg, NR for 2 mg/kg, 25.2 months (12.0–NR) for 10 mg/kg, and NR for treatment-naïve patients. Median percent change from baseline in tumor-associated lymphocytes was 69% (CD3+), 180% (CD4+), and 117% (CD8+). Of 56 baseline biopsies, 32% had ≥5% PD-L1 expression, and there was no consistent change from baseline to on-treatment biopsies. Transcriptional changes in tumors on treatment included up-regulation of interferon-γ–stimulated genes (e.g., CXCL9). Median increases in chemokine levels from baseline to C2D8 were 101% (CXCL9) and 37% (CXCL10) in peripheral blood. No new safety signals were identified. Conclusion Immunomodulatory effects of PD-1 inhibition were demonstrated through multiple lines of evidence across nivolumab doses. Biomarker changes from baseline reflect nivolumab pharmacodynamics in the tumor microenvironment. These data may inform potential combinations.

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The bcl-2 proto-oncogene is frequently expressed in human cancer. Although bcl-2 was first cloned as the t(14;18) translocation breakpoint from human follicular B-cell lymphoma, it has become apparent that many cell types express bcl-2 because of transcriptional regulation. As such, several transcription factors have been demonstrated to activate expression of bcl-2, including NF-B. We investigated the role of NF-B1 (p50) homodimers in the expression of Bcl-2 in two murine B-cell lymphoma cell lines: LY-as, an apoptosis-proficient line with low Bcl-2 protein expression and no nuclear NF-B activity, and LY-ar, a nonapoptotic line with constitutive p50 homodimer activity and 30 times more Bcl-2 protein expression than LY-as. We found that nuclear p50 homodimer activity correlated with Bcl-2 expression in these cell types and identified several sites within the bcl-2 5-flanking region that p50 was capable of binding. In vitro transcription revealed that recombinant p50 enhanced the production of run-off transcripts from the bcl-2 P1 promoter. Additional in vitro transcription experiments suggested the sites by which p50 afforded this effect. We conclude that the p50 homodimer is capable of transcriptional activation of the bcl-2 gene and suggest that its nuclear activity contributes to the expression of bcl-2 in LY-ar cells.

Myc is a transcription factor that features prominently in cancer. The oncogenicity of Myc stems from its ability to regulate expression of genes required for cell growth and proliferation. Although the mechanisms through which Myc activates transcription have been extensively studied, less is known about how Myc represses transcription. Recently, we reported that a conserved element within Myc-MbIII-is important for transcriptional repression. Here, we investigate the mechanism through which MbIII contributes to repression. We show that Myc represses transcription of target genes Id2 and Gadd153 by a process that involves histone deacetylation. We show that MbIII is important for repression of these genes and present evidence that this element contributes to repression by recruiting the histone deacetylase HDAC3 to the Id2 and Gadd153 promoters. These results describe a mechanistic role for MbIII in transcription, and reveal that recruitment of HDAC3 is a process by which Myc represses gene activity. [Cancer Res 2008;68(10):3624-9]