No abstract

Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)–infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.

Manipulation of the activity of the p53 tumor suppressor pathway has demonstrated potential benefit in preclinical mouse tumor models and has entered human clinical trials. We describe here an improved, extensive small-molecule chemical compound library screen for p53 pathway activation in a human cancer cell line devised to identify hits with potent antitumor activity. We uncover six novel small-molecule lead compounds, which activate p53 and repress the growth of human cancer cells. Two tested compounds suppress in vivo tumor growth in an orthotopic mouse model of human B-cell lymphoma. All compounds interact with DNA, and two activate p53 pathway in a DNA damage signaling-dependent manner. A further screen of a drug library of approved drugs for medicinal uses and analysis of gene-expression signatures of the novel compounds revealed similarities to known DNA intercalating and topoisomerase interfering agents and unexpected connectivities to known drugs without previously demonstrated anticancer activities. These included several neuroleptics, glycosides, antihistamines and adrenoreceptor antagonists. This unbiased screen pinpoints interference with the DNA topology as the predominant mean of pharmacological activation of the p53 pathway and identifies potential novel antitumor agents.

Activation of the p53 pathway has been considered a therapeutic strategy to target cancers. We have previously identified several p53-activating small molecules in a cell-based screen. Two of the compounds activated p53 by causing DNA damage, but this modality was absent in the other four. We recently showed that one of these, BMH-21, inhibits RNA polymerase I (Pol I) transcription, causes the degradation of Pol I catalytic subunit RPA194, and has potent anticancer activity. We show here that three remaining compounds in this screen, BMH-9, BMH-22, and BMH-23, cause reorganization of nucleolar marker proteins consistent with segregation of the nucleolus, a hallmark of Pol I transcription stress. Further, the compounds destabilize RPA194 in a proteasome-dependent manner and inhibit nascent rRNA synthesis and expression of the 45S rRNA precursor. BMH-9-and BMH-22-mediated nucleolar stress was detected in ex vivo-cultured human prostate tissues indicating good tissue bioactivity. Testing of closely related analogues showed that their activities were chemically constrained. Viability screen for BMH-9, BMH-22, and BMH-23 in the NCI60 cancer cell lines showed potent anticancer activity across many tumor types. Finally, we show that the Pol I transcription stress by BMH-9, BMH-22, and BMH-23 is independent of p53 function. These results highlight the dominant impact of Pol I transcription stress on p53 pathway activation and bring forward chemically novel lead molecules for Pol I inhibition, and, potentially, cancer targeting. Mol Cancer Ther; 13(11); 2537-46. Ó2014 AACR.

Cellular DNA damage triggers the DNA damage response pathway and leads to enforcement of cell cycle checkpoints, which are essential for the maintenance of genomic integrity and are activated in early stages of tumorigenesis. A special feature of prostate cancer is its high incidence and multifocality. To address the functionality of DNA damage checkpoints in the prostate, we analyzed the responses of human primary prostate epithelial cells (HPECs) and freshly isolated human prostate tissues to ␥-irradiation. We find that ␥-irradiation activates the ataxia telangiectasia mutated-associated DNA damage response pathway in the HPECs but that the clearance of phosphorylated histone H2AX (␥H2AX) foci is delayed. Surprisingly, ␥-irradiated HPECs were unable to enforce cell cycle checkpoint arrest and had sustained cyclin-dependent kinase 2 (Cdk2)-associated kinase activity because of a lack of inhibitory Cdk phosphorylation by Wee1A tyrosine kinase. We further show that HPECs express low levels of Wee1A and that ectopic Wee1A efficiently rescues the checkpoints. We recapitulate the absence of checkpoint responses in epithelium of ex vivo irradiated human prostate tissue despite robust induction of ␥H2AX. The findings show that prostate epithelium has a surprising inability to control checkpoint arrest, the lack of which may predispose to accrual of DNA lesions.p53 ͉ irradiation ͉ cyclin-dependent kinase