Joanna I. Loizou

Histone acetylation by Trrap-Tip60 modulates loading of repair proteins and repair of DNA double-strand breaks

DNA is packaged into chromatin, a highly compacted DNA–protein complex; therefore, all cellular processes that use the DNA as a template, including DNA repair, require a high degree of coordination between the DNA-repair machinery and chromatin modification/remodelling, which regulates the accessibility of DNA in chromatin. Recent studies have implicated histone acetyltransferase (HAT) complexes and chromatin acetylation in DNA repair; however, the precise underlying mechanism remains poorly understood. Here, we show that the HAT cofactor Trrap and Tip60 HAT bind to the chromatin surrounding sites of DNA double-strand breaks (DSBs) in vivo. Trrap depletion impairs both DNA-damage-induced histone H4 hyperacetylation and accumulation of repair molecules at sites of DSBs, resulting in defective homologous recombination (HR) repair, albeit with the presence of a functional ATM-dependent DNA-damage signalling cascade. Importantly, the impaired loading of repair proteins and the defect in DNA repair in Trrap-deficient cells can be counteracted by chromatin relaxation, indicating that the DNA-repair defect that was observed in the absence of Trrap is due to impeded chromatin accessibility at sites of DNA breaks. Thus, these data reveal that cells may use the same basic mechanism involving HAT complexes to regulate distinct cellular processes, such as transcription and DNA repair.

The Protein Kinase CK2 Facilitates Repair of Chromosomal DNA Single-Strand Breaks

CK2 was the first protein kinase identified and is required for the proliferation and survival of mammalian cells. Here, we have identified an unanticipated role for CK2. We show that this essential protein kinase phosphorylates the scaffold protein XRCC1 and thereby enables the assembly and activity of DNA single-strand break repair protein complexes in vitro and at sites of chromosomal breakage. Moreover, we show that inhibiting XRCC1 phosphorylation by mutation of the CK2 phosphorylation sites or preventing CK2 activity using a highly specific inhibitor ablates the rapid repair of cellular DNA single-strand breaks by XRCC1. These data identify a direct role for CK2 in the repair of chromosomal DNA strand breaks and in maintaining genetic integrity.

Stereospecific targeting of MTH1 by ( S )-crizotinib as an anticancer strategy

Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.

Interference of papillomavirus E6 protein with single‐strand break repair by interaction with XRCC1

XRCC1 protein is required for the repair of DNA single‐strand breaks and genetic stability, and is essential for viability in mammals. XRCC1 functions as a scaffold protein by interacting and modulating polypeptide components of the single‐strand break repair machinery, including AP endonuclease‐1, DNA ligase IIIα, poly (ADP‐ribose) polymerase, DNA polymerase β and human polynucleotide kinase. We show here that the E6 protein of human papillomavirus type 1, 8 and 16 directly binds XRCC1. When tested in CHO derived XRCC1 ‘knock out’ EM9 cells, co‐expression of human papillomavirus 16 E6 with human XRCC1 reduced the ability of the latter protein to correct the methyl methane sulfate sensitivity of XRCC1 mutant CHO cell line EM9. These data identify a novel link between small DNA tumour viruses and DNA repair pathways, and suggest a novel explanation for the development of genomic instability in tissue cells persistently infected with papillomaviruses.

DNA replication stress: Causes, resolution and disease

DNA replication is a fundamental process of the cell that ensures accurate duplication of the genetic information and subsequent transfer to daughter cells. Various pertubations, originating from endogenous or exogenous sources, can interfere with proper progression and completion of the replication process, thus threatening genome integrity. Coordinated regulation of replication and the DNA damage response is therefore fundamental to counteract these challenges and ensure accurate synthesis of the genetic material under conditions of replication stress. In this review, we summarize the main sources of replication stress and the DNA damage signaling pathways that are activated in order to preserve genome integrity during DNA replication. We also discuss the association of replication stress and DNA damage in human disease and future perspectives in the field.

Competition between NBS1 and ATMIN Controls ATM Signaling Pathway Choice

Ataxia telangiectasia mutated (ATM) protein kinase activation by DNA double-strand breaks (DSBs) requires the Mre11-Rad50-NBS1 (MRN) complex, whereas ATM interactor (ATMIN) protein is required for ATM signaling induced by changes in chromatin structure. We show here that NBS1 and ATMIN proteins compete for ATM binding and that this mechanism controls ATM function. DSB-induced ATM substrate phosphorylation was increased in atmin mutant cells. Conversely, NBS1 deficiency resulted in increased ATMIN-dependent ATM signaling. Thus, the absence of one cofactor increased flux through the alternative pathway. Notably, ATMIN deficiency rescued the cellular lethality of NBS1-deficient cells, and NBS1/ATMIN double deficiency resulted in complete abrogation of ATM signaling and profound radiosensitivity. Hence, ATMIN and NBS1 mediate all ATM signaling by DSBs, and increased ATMIN-dependent ATM signaling explains the different phenotypes of nbs1- and atm-mutant cells. Thus, the antagonism and redundancy of ATMIN and NBS1 constitute a crucial regulatory mechanism for ATM signaling and function.

ATMIN Is Required for Maintenance of Genomic Stability and Suppression of B Cell Lymphoma

Summary Defective V(D)J rearrangement of immunoglobulin heavy or light chain (IgH or IgL) or class switch recombination (CSR) can initiate chromosomal translocations. The DNA-damage kinase ATM is required for the suppression of chromosomal translocations but ATM regulation is incompletely understood. Here, we show that mice lacking the ATM cofactor ATMIN in B cells (ATMINΔB/ΔB) have impaired ATM signaling and develop B cell lymphomas. Notably, ATMINΔB/ΔB cells exhibited defective peripheral V(D)J rearrangement and CSR, resulting in translocations involving the Igh and Igl loci, indicating that ATMIN is required for efficient repair of DNA breaks generated during somatic recombination. Thus, our results identify a role for ATMIN in regulating the maintenance of genomic stability and tumor suppression in B cells.

Histone Acetyltransferase Cofactor Trrap Is Essential for Maintaining the Hematopoietic Stem/Progenitor Cell Pool

The pool of hematopoietic stem/progenitor cells, which provide life-long reconstitution of all hematopoietic lineages, is tightly controlled and regulated by self-renewal and apoptosis. Histone modifiers and chromatin states are believed to govern establishment, maintenance, and propagation of distinct patterns of gene expression in stem cells, however the underlying mechanism remains poorly understood. In this study, we identified a role for the histone acetytransferase cofactor Trrap in the maintenance of hematopietic stem/progenitor cells. Conditional deletion of the Trrap gene in mice resulted in ablation of bone marrow and increased lethality. This was due to the depletion of early hematopoietic progenitors, including hematopoietic stem cells, via a cell-autonomous mechanism. Analysis of purified bone marrow progenitors revealed that these defects are associated with induction of p53-independent apoptosis and deregulation of Myc transcription factors. Together, this study has identified a critical role for Trrap in the mechanism that maintains hematopoietic stem cells and hematopoietic system, and underscores the importance of Trrap and histone modifications in tissue homeostasis.

ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress

Unresolved replication intermediates can block the progression of replication forks and become converted into DNA lesions, hence exacerbating genomic instability. The p53-binding protein 1 (53BP1) forms nuclear bodies at sites of unrepaired DNA lesions to shield these regions against erosion, in a manner dependent on the DNA damage kinase ATM. The molecular mechanism by which ATM is activated upon replicative stress to localize the 53BP1 protection complex is unknown. Here we show that the ATM-INteracting protein ATMIN (also known as ASCIZ) is partially required for 53BP1 localization upon replicative stress. Additionally, we demonstrate that ATM activation is impaired in cells lacking ATMIN and we define that ATMIN is required for initiating ATM signaling following replicative stress. Furthermore, loss of ATMIN leads to chromosomal segregation defects. Together these data reveal that chromatin integrity depends on ATMIN upon exposure to replication-induced stress.

MEK inhibitors block growth of lung tumours with mutations in ataxia–telangiectasia mutated

Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.