MRE11 and EXO1 nucleases degrade reversed forks and elicit MUS81-dependent fork rescue in BRCA2-deficient cells

The breast cancer susceptibility proteins BRCA1 and BRCA2 have emerged as key stabilizing factors for the maintenance of replication fork integrity following replication stress. In their absence, stalled replication forks are extensively degraded by the MRE11 nuclease, leading to chemotherapeutic sensitivity. Here we report that BRCA proteins prevent nucleolytic degradation by protecting replication forks that have undergone fork reversal upon drug treatment. The unprotected regressed arms of reversed forks are the entry point for MRE11 in BRCA-deficient cells. The CtIP protein initiates MRE11-dependent degradation, which is extended by the EXO1 nuclease. Next, we show that the initial limited resection of the regressed arms establishes the substrate for MUS81 in BRCA2-deficient cells. In turn, MUS81 cleavage of regressed forks with a ssDNA tail promotes POLD3-dependent fork rescue. We propose that targeting this pathway may represent a new strategy to modulate BRCA2-deficient cancer cell response to chemotherapeutics that cause fork degradation

Combined PARP and ATR inhibition potentiates genome instability and cell death in ATM-deficient cancer cells.

The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib is FDA approved for the treatment of BRCA-mutated breast, ovarian and pancreatic cancers. Olaparib inhibits PARP1/2 enzymatic activity and traps PARP1 on DNA at single-strand breaks, leading to replication-induced DNA damage that requires BRCA1/2-dependent homologous recombination repair. Moreover, DNA damage response pathways mediated by the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia mutated and Rad3-related (ATR) kinases are hypothesised to be important survival pathways in response to PARP-inhibitor treatment. Here, we show that olaparib combines synergistically with the ATR-inhibitor AZD6738 (ceralasertib), in vitro, leading to selective cell death in ATM-deficient cells. We observe that 24 h olaparib treatment causes cells to accumulate in G2-M of the cell cycle, however, co-administration with AZD6738 releases the olaparib-treated cells from G2 arrest. Selectively in ATM-knockout cells, we show that combined olaparib/AZD6738 treatment induces more chromosomal aberrations and achieves this at lower concentrations and earlier treatment time-points than either monotherapy. Furthermore, single-agent olaparib efficacy in vitro requires PARP inhibition throughout multiple rounds of replication. Here, we demonstrate in several ATM-deficient cell lines that the olaparib and AZD6738 combination induces cell death within 1-2 cell divisions, suggesting that combined treatment could circumvent the need for prolonged drug exposure. Finally, we demonstrate in vivo combination activity of olaparib and AZD6738 in xenograft and PDX mouse models with complete ATM loss. Collectively, these data provide a mechanistic understanding of combined PARP and ATR inhibition in ATM-deficient models, and support the clinical development of AZD6738 in combination with olaparib

Roles For The Dna Damage Checkpoint Protein Hus1 In Tissue Homeostasis And Tumor Suppression

Cancer is aberrant cellular proliferation that arises due to mutations in growth regulatory genes. DNA damage checkpoint proteins are thought to suppress tumorigenesis by preventing mutation accumulation and by inducing senescence in response to oncogenic stimuli. However, checkpoint proteins may be required for cancer cells to survive the increased stress associated with transformation. There are two main mammalian DNA damage checkpoint pathways, the Atm and Atr pathways. While mutations in the Atm pathway results in increased tumorigenesis, roles for the Atr pathway in tumor development are less well understood, in part because deletion of any component of this pathway, including Hus1, results in embryonic lethality. Hus1, a component of the Rad9-Rad1-Hus1 heterotrimeric, PCNA-like sliding clamp, is recruited to sites of DNA damage for optimal phosphorylation of the Atr target Chk1. To investigate the physiological function of the Hus1-dependent Atr pathway, our lab developed two systems that bypass the severe phenotypes associated with germline Hus1 inactivation. A conditional allele can be used for tissue specific deletion of Hus1 in adult mice. I have utilized this approach to identify an essential role for Hus1 in the survival and proliferation of mammary epithelium. Notably, Hus1 inactivation in the mammary gland did not result in tumorigenesis, even when combined with p53 inactivation. p53 deficiency exacerbated the effects of Hus1 loss, resulting in increased cell death without compensatory proliferation, revealing a novel role for p53 in mammary gland tissue regeneration. As a second approach, our lab developed an allelic series in which mice express incrementally reduced Hus1 levels. I used this system to investigate how partial Hus1 impairment affects transformation in cell culture and impacts skin papilloma formation. Reduced Hus1 expression impaired cell transformation by activated oncogenes as measured in cell culture assays. Mice with Hus1 impairment developed significantly fewer and smaller papillomas in a twostep skin carcinogenesis protocol. These results may be due to an inability of Hus1deficient cells to survive the stresses of neoplastic proliferation due to insufficient genome maintenance. These results suggest that reduced Hus1 levels impair tumor development and that the Atr-dependent pathway may be exploited for cancer therapy

Geography of symplectic 4- and 6-manifolds

The geography of minimal symplectic 4-manifolds with arbitrary fundamental group and symplectic 6-manifolds with abelian fundamental group of small rank, and with arbitrary fundamental group are addressed