Targeting TOPK sensitises tumour cells to radiation-induced damage by enhancing replication stress

T-LAK-originated protein kinase (TOPK) overexpression is a feature of multiple cancers, yet is absent from most phenotypically normal tissues. As such, TOPK expression profiling and the development of TOPK-targeting pharmaceutical agents have raised hopes for its future potential in the development of targeted therapeutics. Results presented in this paper confirm the value of TOPK as a potential target for the treatment of solid tumours, and demonstrate the efficacy of a TOPK inhibitor (OTS964) when used in combination with radiation treatment. Using H460 and Calu-6 lung cancer xenograft models, we show that pharmaceutical inhibition of TOPK potentiates the efficacy of fractionated irradiation. Furthermore, we provide in vitro evidence that TOPK plays a hitherto unknown role during S phase, showing that TOPK depletion increases fork stalling and collapse under conditions of replication stress and exogenous DNA damage. Transient knockdown of TOPK was shown to impair recovery from fork stalling and to increase the formation of replication-associated single-stranded DNA foci in H460 lung cancer cells. We also show that TOPK interacts directly with CHK1 and Cdc25c, two key players in the checkpoint signalling pathway activated after replication fork collapse. This study thus provides novel insights into the mechanism by which TOPK activity supports the survival of cancer cells, facilitating checkpoint signalling in response to replication stress and DNA damage.

Single Molecule Imaging of T-DNA Intermediates Following Agrobacterium tumefaciens Infection in Nicotiana benthamiana

Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement.

MICRORNA REGULATORS OF THE SENESCENCE TRANSCRIPTOME

Cellular senescence is a state of indefinite growth arrest triggered in response to sublethal stresses such as telomere shortening, DNA damage, oxidative injury, oncogene activation, and hypoxia. Compared with proliferating cells, senescent cells are enlarged, display heterochromatic DNA foci, and express distinct subsets of proteins, including the enzyme β-galactosidase (β-gal). Previously, we identified transcriptome signature of senescent cells. We asked if these transcripts might be regulated by microRNAs (miRNAs). To address this question, we identified six miRNAs (miR-129-5p, -19a-3p, -128-3p, -124-3p, -340-5p, and -27b-3p) as potential regulators of subsets of transcripts differentially expressed during senescence. RT-qPCR analysis indicated that miR-129-5p, -19a-3p, -128-3p, -124-3p, and -340-5p were downregulated in senescent cells. We modulated these miRNAs in proliferating WI-38 fibroblasts and found that miRNA antagomirs did not show significant changes in β-gal activity. Interestingly, however, overexpression of miR-124-3p or miR-340-5p increased β-gal activity. We conclude that despite the decrease of miR-124-3p and miR-340-5p in senescent cells, their overexpression enhanced senescence as indicated by β-gal activity. Future analyses will focus on the mechanisms through which these miRNAs induce senescence and their physiologic and pathologic impacts in vivo.

Functional Interaction between FANCD2 and ATM in the DNA Damage Response.

Fanconi Anemia is a multigenic cancer susceptibility syndrome. The FA pathway controls the monoubiquitination of FANCD2, and its targeting to damage-associated DNA foci. Cells that are deficient in the FA pathway are highly sensitive to bi-functional crosslinking agents such as mitomycin-C (MMC). Although FANCD2 monoubiquitination is activated by DNA damage, how this is coupled to the DNA damage response is unknown. Previous studies suggest a functional interaction between FANCD2 and the protein kinase ATM (ataxia telangiectasia mutated). Recently we have shown that the C-terminus of FANCD2 is required for its function (Montes de Oca et al., Blood2005:105, 1003). A C-terminal truncation of FANCD2 (FANCD2-C) fails to correct the MMC sensitivity FANCD2−/− cells despite its monoubiquitination and assembly into DNA damage foci. In the present study we demonstrate that FANCD2 forms a protein complex with the ATM kinase and that this interaction requires the FANCD2 C-terminus. The association of ATM with FANCD2 occurs in the absence of DNA damage, and does not require FANCD2 to be monoubiquitinated. In immunoprecipitation experiments involving the FANCD2-C mutant, ATM fails to co-precipitate, suggesting the C-terminus of FANCD2 is required for recruiting ATM to sites of DNA damage. In addition, we have identified a new ATM phosphorylation site on FANCD2, Thr691. Using a phospho-specific antibody to FANCD2 Thr691 we have determined that siRNA knockdown of ATM results in loss of phosphorylation of Thr691, and ionizing radiation (IR) activates the phosphorylation of Thr691. Mutation of Thr691 to Alanine destroys this ATM phosphorylation site. Interestingly, this FANCD2 mutant also fails to correct the MMC sensitivity FANCD2−/− cells even though it can be monoubiquitinated and targeted to DNA foci following DNA damage, a phenotype observed for the FANCD2-C mutant. Next we examined the phosphorylation of Thr691 in FANCD2-C. In FANCD2 constructs expressing a mutated or truncated C-terminus, phosphorylation of Thr691 is delayed following exposure to MMC and the cells are sensitive to DNA damage. Following exposure to ionizing radiation the ATM kinase phosphorylates itself on Ser1981. Using a phospho-specific antibody to ATM Ser1981 we evaluated the phosphorylation of ATM in response to DNA damage caused by IR and MMC. Western analysis shows that phosphorylation of ATM on Ser1981 following IR is the same in FANCD2 and FANCD2-C cells as well as uncorrected FAND2−/− cells. ATM foci formation following IR also appears identical in all three FANCD2 cell lines based upon immunoflourescence microscopy. However in cells damaged with MMC, there is a delay in Ser1981 phosphorylation and fewer ATM foci in the uncorrected and FANCD2-C cells compared to cells corrected with intact FANCD2. We propose a model for FANCD2 function where the targeting of FANCD2 to DNA damage inducible foci is required for the proper coordination of some ATM-dependent DNA repair and checkpoint responses. For instance, FANCD2 may be required for targeting ATM to a subset of double strand breaks - namely, those breaks associated with an adjacent interstrand crosslink. Thus targeting of ATM to MMC-induced DNA damage requires FANCD2 but recruitment to IR-induced double strand breaks does not, depending instead on NBS1 and the M/R/N complex.