Germ-line mutations in BRCA1 predispose to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired recombinatorial DNA repair. Here we identify 53BP1 as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Importantly, loss of 53BP1 partially restores the homologous recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.

DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G 2 /M. These data support a crucial role for BMI1 in the cellular response to DNA damage.The induction of a DNA break leads to activation of multiple signaling pathways that lead to local modification of chromatin structure and recruitment of DNA repair complexes (18,22,55). Histone H2AX is rapidly phosphorylated near sites of DNA breaks by ATM, ATR, and DNA-PK (39, 54) and can spread to encompass a region of chromatin covering several megabases (40, 41).H2AX phosphorylation facilitates the recruitment of other proteins, including MDC1 (52) and the E3 ubiquitin ligases RNF8 and RNF168, which in turn participate locally in the K63-linked polyubiquitination of histones H2A and H2AX (23,32,50,51). Polyubiquitinated K63-linked histones provide a recognition element that recruits RAP80 through its ubiquitin interaction motifs (28,49,56). RAP80 can then promote the recruitment of other DNA repair factors such as BRCA1 and Abraxas, which are essential for efficient repair. RNF8 and RNF168 function are also required for proper localization of 53BP1, although the exact mechanism is unclear (12,23,32,51). 53BP1 recruitment to regions of DNA damage is dependent upon its Tudor domains, which have been found to specifically interact with methylated histone residues (6, 24, 42). A model has been proposed in which RNF8-and RNF168-mediated ubiquitination of histones confers local changes in chromatin structure, leading to exposure of methylated lysine residues in core histones, allowing the subsequent recruitment of 53BP1 (50). Enzymes involved in deubiquitination, such as BRCC36, USP3, and USP28, are also critical for efficient DNA repair, demonstrating that a dynamic regulation of ubiquitin conjugation and hydrolysis is necessary for optimal DNA repair (37,46,47,61).Polycomb group proteins BMI1 and RING1B/RNF2 form an active heterodimer E3 ligase that catalyzes the monoubiquitination of histone H2A at Lysine 119. (7,8,44,53,57). This activity is important for BMI1-mediated transcriptional silencing during organism development and cellular differentiation (27,48,58). Ubiquitination of H2A at lysine 119 is also induced locally at sites of DNA damage, both at s...

SYNOPSIS Ubiquitin-like protein, containing PHD and RING finger domains-1 (UHRF1) is required for cell cycle progression and epigenetic regulation. In this study, we show that depleting cancer cells of UHRF1 causes activation of the DNA damage response pathway, cell cycle arrest in G2/M and apoptosis dependent on caspase-8. The DNA damage response in cells depleted of UHRF1 is illustrated by: phosphorylation of histone H2AX on serine 139, phosphorylation of CHK2 on threonine 68, phosphorylation of CDC25 on serine 216 and phosphorylation of CDK1 on tyrosine 15. Moreover, we find that UHRF1 accumulates at sites of DNA damage suggesting that the cell cycle block in UHRF1 depleted cells is due to an important role in damage repair. The consequence of UHRF1 depletion is apoptosis: cells undergo activation of caspases 8 and 3 and depletion of caspase-8 prevents cell death induced by UHRF1 knock-down. Interestingly, the cell cycle block and apoptosis occurs in p53 containing and deficient cells. From these studies we conclude that UHRF1 links epigenetic regulation with DNA replication.

Fusions involving the oncogenic gene RET have been observed in thyroid and lung cancers. Here we report RET gene alterations, including amplification, missense mutations, known fusions, novel fusions, and rearrangements in breast cancer. Their frequency, oncogenic potential, and actionability in breast cancer are described. Two out of eight RET fusions (NCOA4-RET and a novel RASGEF1A-RET fusion) and RET amplification were functionally characterized and shown to activate RET kinase and drive signaling through MAPK and PI3K pathways. These fusions and RET amplification can induce transformation of non-tumorigenic cells, support xenograft tumor formation, and render sensitivity to RET inhibition. An index case of metastatic breast cancer progressing on HER2-targeted therapy was found to have the NCOA4-RET fusion. Subsequent treatment with the RET inhibitor cabozantinib led to a rapid clinical and radiographic response. RET alterations, identified by genomic profiling, are promising therapeutic targets and are present in a subset of breast cancers.

Introduction. For patients with non-small cell lung cancer (NSCLC) tobenefitfromALKinhibitors,sensitiveandspecificdetectionofALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. Materials and Methods. Hybrid-capture-based CGP using nextgenerationsequencingwasperformedinthe course ofclinicalcare of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements.