ATR/ATM-Mediated Phosphorylation of BRCA1 T1394 Promotes Homologous Recombinational Repair and G2-M Checkpoint Maintenance.

BRCA1 maintains genome integrity and suppresses tumorigenesis by promoting homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSB) and DNA damage-induced cell-cycle checkpoints. Phosphorylation of BRCA1 by ATM, ATR, CHK2, CDK, and PLK1 kinases has been reported to regulate its functions. Here we show that ATR and ATM-mediated phosphorylation of BRCA1 on T1394, a highly conserved but functionally uncharacterized site, is a key modification for its function in the DNA damage response (DDR). Following DNA damage, T1394 phosphorylation ensured faithful repair of DSBs by promoting HR and preventing single-strand annealing, a deletion-generating repair process. BRCA1 T1394 phosphorylation further safeguarded chromosomal integrity by maintaining the G2-M checkpoint. Moreover, multiple patient-derived BRCA1 variants of unknown significance were shown to affect T1394 phosphorylation. These results establish an important regulatory mechanism of BRCA1 function in the DDR and may have implications in the development or prognosis of BRCA1-associated cancers. SIGNIFICANCE: This study identifies a BRCA1 phosphorylation event critical for its DNA repair function and reveals the functional defects of several BRCA1 variants of unknown significance.

The Regulation of Homologous Recombination by Helicases.

Homologous recombination is essential for DNA repair, replication and the exchange of genetic material between parental chromosomes during meiosis. The stages of recombination involve complex reorganization of DNA structures, and the successful completion of these steps is dependent on the activities of multiple helicase enzymes. Helicases of many different families coordinate the processing of broken DNA ends, and the subsequent formation and disassembly of the recombination intermediates that are necessary for template-based DNA repair. Loss of recombination-associated helicase activities can therefore lead to genomic instability, cell death and increased risk of tumor formation. The efficiency of recombination is also influenced by the 'anti-recombinase' effect of certain helicases, which can direct DNA breaks toward repair by other pathways. Other helicases regulate the crossover versus non-crossover outcomes of repair. The use of recombination is increased when replication forks and the transcription machinery collide, or encounter lesions in the DNA template. Successful completion of recombination in these situations is also regulated by helicases, allowing normal cell growth, and the maintenance of genomic integrity.

Synthesis and Characterization of Novel BMI1 Inhibitors Targeting Cellular Self-Renewal in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) represents one of the most lethal cancers worldwide due to therapy resistance and disease recurrence. Tumor relapse following treatment could be driven by the persistence of liver cancer stem-like cells (CSCs). The protein BMI1 is a member of the polycomb epigenetic factors governing cellular self-renewal, proliferation, and stemness maintenance. BMI1 expression also correlates with poor patient survival in various cancer types. OBJECTIVE: We aimed to elucidate the extent to which BMI1 can be used as a potential therapeutic target for CSC eradication in HCC. METHODS: We have recently participated in characterizing the first known pharmacological small molecule inhibitor of BMI1. Here, we synthesized a panel of novel BMI1 inhibitors and examined their ability to alter cellular growth and eliminate cancer progenitor/stem-like cells in HCC with different p53 backgrounds. RESULTS: Among various molecules examined, RU-A1 particularly downregulated BMI1 expression, impaired cell viability, reduced cell migration, and sensitized HCC cells to 5-fluorouracil (5-FU) in vitro. Notably, long-term analysis of HCC survival showed that, unlike chemotherapy, RU-A1 effectively reduced CSC content, even as monotherapy. BMI1 inhibition with RU-A1 diminished the number of stem-like cells in vitro more efficiently than the model compound C-209, as demonstrated by clonogenic assays and impairment of CSC marker expression. Furthermore, xenograft assays in zebrafish showed that RU-A1 abrogated tumor growth in vivo. CONCLUSIONS: This study demonstrates the ability to identify agents with the propensity for targeting CSCs in HCC that could be explored as novel treatments in the clinical setting.

BMI-1 Targeting Interferes with Patient-Derived Tumor-Initiating Cell Survival and Tumor Growth in Prostate Cancer.

PURPOSE: Current prostate cancer management calls for identifying novel and more effective therapies. Self-renewing tumor-initiating cells (TICs) hold intrinsic therapy resistance and account for tumor relapse and progression. As BMI-1 regulates stem cell self-renewal, impairing BMI-1 function for TIC-tailored therapies appears to be a promising approach. EXPERIMENTAL DESIGN: We have previously developed a combined immunophenotypic and time-of-adherence assay to identify CD49bhiCD29hiCD44hi cells as human prostate TICs. We utilized this assay with patient-derived prostate cancer cells and xenograft models to characterize the effects of pharmacologic inhibitors of BMI-1. RESULTS: We demonstrate that in cell lines and patient-derived TICs, BMI-1 expression is upregulated and associated with stem cell-like traits. From a screened library, we identified a number of post-transcriptional small molecules that target BMI-1 in prostate TICs. Pharmacologic inhibition of BMI-1 in patient-derived cells significantly decreased colony formation in vitro and attenuated tumor initiation in vivo, thereby functionally diminishing the frequency of TICs, particularly in cells resistant to proliferation- and androgen receptor-directed therapies, without toxic effects on normal tissues. CONCLUSIONS: Our data offer a paradigm for targeting TICs and support the development of BMI-1-targeting therapy for a more effective prostate cancer treatment. Clin Cancer Res; 22(24); 6176-91. ©2016 AACR.