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HUS1 Regulates in Vivo Responses to Genotoxic Chemotherapies

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HUS1 Regulates in Vivo Responses to Genotoxic Chemotherapies Oncogene (2016) 35, 662–669 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc SHORT COMMUNICATION HUS1 regulates in vivo responses to genotoxic chemotherapies G Balmus1, PX Lim1, A Oswald1, KR Hume1,2, A Cassano1, J Pierre1, A Hill1, W Huang3, A August3, T Stokol4, T Southard1 and RS Weiss1 Cells are under constant attack from genotoxins and rely on a multifaceted DNA damage response (DDR) network to maintain genomic integrity. Central to the DDR are the ATM and ATR kinases, which respond primarily to double-strand DNA breaks (DSBs) and replication stress, respectively. Optimal ATR signaling requires the RAD9A-RAD1-HUS1 (9-1-1) complex, a toroidal clamp that is loaded at damage sites and scaffolds signaling and repair factors. Whereas complete ATR pathway inactivation causes embryonic lethality, partial Hus1 impairment has been accomplished in adult mice using hypomorphic (Hus1neo) and null (Hus1Δ1) Hus1 alleles, and here we use this system to define the tissue- and cell type-specific actions of the HUS1-mediated DDR in vivo. Hus1neo/Δ1 mice showed hypersensitivity to agents that cause replication stress, including the crosslinking agent mitomycin C (MMC) and the replication inhibitor hydroxyurea, but not the DSB inducer ionizing radiation. Analysis of tissue morphology, genomic instability, cell proliferation and apoptosis revealed that MMC treatment caused severe damage in highly replicating tissues of mice with partial Hus1 inactivation. The role of the 9-1-1 complex in responding to MMC was partially ATR-independent, as a HUS1 mutant that was proficient for ATR-induced checkpoint kinase 1 phosphorylation nevertheless conferred MMC hypersensitivity. To assess the interplay between the ATM and ATR pathways in responding to replication stress in vivo, we used Hus1/Atm double mutant mice. Whereas Hus1neo/neo and Atm−/− single mutant mice survived low-dose MMC similar to wild-type controls, Hus1neo/neoAtm−/− double mutants showed striking MMC hypersensitivity, consistent with a model in which MMC exposure in the context of Hus1 dysfunction results in DSBs to which the ATM pathway normally responds. This improved understanding of the inter-dependency between two major DDR mechanisms during the response to a conventional chemotherapeutic illustrates how inhibition of checkpoint factors such as HUS1 may be effective for the treatment of ATM-deficient and other cancers. Oncogene (2016) 35, 662–669; doi:10.1038/onc.2015.118; published online 27 April 2015 INTRODUCTION The ATR pathway responds to lesions caused by genotoxins Effective cancer treatment frequently involves DNA-damaging such as ultraviolet-light, HU or MMC, which physically impede agents, such as ionizing radiation (IR), which induces double- replication fork progression, leading to accumulation of strand DNA breaks (DSBs), and various chemotherapeutics that, for RPA-coated single-stranded DNA. This represents the major example, cause interstrand crosslinks (ICLs) (mitomycin C (MMC), recruitment signal for ATR and its partner ATRIP, as well as the Cisplatin), perturb nucleotide metabolism (5-fluoruracil, hydro- RAD9A-RAD1-HUS1 (9-1-1) complex, a toroidal clamp that xyurea (HU)), inhibit topoisomerases (Topotecan), or are clastogenic structurally resembles proliferating cell nuclear antigen. The 9-1- (Bleomycin). Cells respond to such genotoxic threats by activating a 1 complex is loaded at 5′ recessed DNA ends and interacts with DNA damage response (DDR) that consists of a series of signaling TOPBP1 and RHINO, which are required to stimulate ATR kinase pathways that sense genome damage and induce effector activity.8,9 Activated ATR phosphorylates CHK1 and other down- molecules that execute protective responses, including repair, stream effectors to promote cell cycle arrest, replication fork senescence or apoptosis. The mammalian DDR includes two major 1,2 stability and DNA repair. Independently of ATR activation, the pathways, headed by the related kinases ATM and ATR. 9-1-1 complex also directly interacts with base excision repair ATM is activated primarily by DSBs, as well as oxidative stress – factors,10 12 translesion synthesis polymerases13 and members of and chromatin changes, and phosphorylates a host of down- other repair pathways, such as homologous recombination and stream effectors, including checkpoint kinase 2 (CHK2) and p53. In 14 humans, ATM mutations cause Ataxia-Telangiectasia (AT), a mismatch repair. In humans, hypomorphic loss-of-function ATR mutations are genomic instability (GIN) syndrome characterized by extreme IR 15 sensitivity, ataxia due to cerebellar degeneration, telangiectasias, associated with the rare disease Seckel Syndrome. Seckel fi 2,3 patients present with developmental defects, including dwarfism, immune de ciency and sterility. AT patients demonstrate a 16,17 significantly increased risk of hematological malignancies, reflect- microcephaly and cognitive impairment. Mouse models ing the tumor-suppressor functions of ATM. Mouse models of AT targeting the ATR pathway revealed that loss of individual ATR recapitulate many of the human phenotypes.4–6 Consistent with pathway components causes embryonic lethality, highlighting a the concept that ATM is the main responder to DSBs, Atm- continuous need for ATR pathway-mediated protection against – deficient mice succumb to low-dose IR due to gastrointestinal replication stress.18 23 Moreover, mice with partial ATR expression toxicity4 but show normal survival in response to replication stress, show severe phenotypes, including dwarfism, craniofacial defects such as that caused by low-dose MMC.7 and early lethality.24 1Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA; 2Department of Clinical Sciences, Cornell University, Ithaca, NY, USA; 3Department of Microbiology and Immunology, Cornell University, Ithaca, NY, USA and 4Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY, USA. Correspondence: Dr RS Weiss, Department of Biomedical Sciences, Cornell University, T2-006C Veterinary Research Tower, Ithaca, NY 14853, USA. E-mail: [email protected] Received 1 July 2014; revised 8 March 2015; accepted 10 March 2015; published online 27 April 2015 Mitomycin C hypersensitivity in mice with partial Hus1 loss G Balmus et al 663 The role of the ATR pathway in cancer is complex and may differ in several cancer cell lines, particularly those with ATM or p53 depending on the particular stage of carcinogenesis. Some studies defects,38–41 and have shown promising results in clinical have proposed that the ATR pathway is critical for tumor trials.32,42 Thus ATR pathway dependency may represent a key suppression while others suggest an enabling role in supporting vulnerability of cancer cells.42,43 tumor growth.15,25 ATR mutations have been identified in human Because inactivation of genes encoding 9-1-1 complex subunits cancers and ATR pathway mouse mutants also have been causes embryonic lethality, we previously developed a system reported to show increased cancer predisposition in some for partial Hus1 inactivation based on a hypomorphic Hus1 instances,20,26,27 leading to the suggestion that GIN prevention allele (Hus1neo) that expresses wild-type HUS1 at reduced by the ATR pathway may suppress tumor initiation. However, levels.44,45 Hus1neo was combined with null (Hus1Δ1) or wild-type other studies indicate that ATR pathway genes are upregulated in (Hus1+) Hus1 alleles to create a Hus1 allelic series with tumors28,29 and increased expression of DDR proteins, including incrementally decreased Hus1 expression (Hus1+/+4Hus1+/neo4 HUS1 and CHK1, is observed in mouse mammary tumors.30 These Hus1+/Δ14Hus1neo/neo4Hus1neo/Δ1). Hus1+/neo and Hus1+/Δ1 mice findings are consistent with observations that in some contexts are indistinguishable from Hus1+/+ mice and in our studies are the ATR pathway is necessary for cancer cells to tolerate stresses used as normal controls. Hus1neo/Δ1 mice, which show reduced associated with neoplastic proliferation,27,31–33 a phenomenon Hus1 expression in a variety of tissues (Supplementary Figure S1), termed non-oncogene addiction that reflects the importance of are born at expected frequencies and appear grossly normal. They DDR factors in cancers and associated opportunities for ther- display no detectable increase in cancer predisposition but show apeutic intervention.34 Similar to established synthetic lethal elevated micronucleus formation in peripheral blood cells, strategies designed to exploit dependencies in DNA repair- indicating that the reduced Hus1 expression is insufficient for defective cancers,35–37 ATR pathway inhibitors have been used proper genome maintenance.44 Hus1neo/neo cells with intermediate to sensitize cancer cells to common genotoxic chemotherapeutics Hus1 expression show moderate GIN phenotypes. Figure 1. Hypersensitivity to MMC but not IR in mice with partial Hus1 inactivation. Previously described Hus1neo, Hus1Δ1 and Atm knockout mice were utilized in accordance with federal and institutional guidelines, under a protocol approved by the Cornell University Institutional Animal Care and Use Committee. For analysis of DNA damage responses in vivo, mice were subjected to 15 Gy (a) or 9 Gy (b) γ-irradiation using a Mark I Model 68 sealed Cesium source gamma irradiator (JL Shepherd and Associates, San Fernando, CA, USA) or injected intraperitoneally with a single dose of 8 mg (c) or 4 mg (d) per kg body weight MMC (Sigma, St Louis, MO, USA). The mice were subsequently
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