EGFR Mutations Compromise Hypoxia-Associated Radiation Resistance through Impaired Replication Fork–Associated DNA Damage Repair

The humanLMNAgene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations inLMNAresult in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair.

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Radiation Resistance in Glioma Cells Determined by DNA Damage Repair Activity of Ape1/Ref-1

Journal of Radiation Research ◽

10.1269/jrr.09077 ◽

2010 ◽

Vol 51 (4) ◽

pp. 393-404 ◽

Cited By ~ 52

Author(s):

Mamta D. NAIDU ◽

James M. MASON ◽

Raymond V. PICA ◽

Hua FUNG ◽

Louis A. PEÑA

Keyword(s):

Dna Damage ◽

Radiation Resistance ◽

Dna Damage Repair ◽

Glioma Cells ◽

Damage Repair ◽

Repair Activity

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STAG2 Loss Gives Rise to Therapeutically Targetable DNA Damage Repair Defects and Altered Replication Fork Dynamics in Acute Myeloid Leukaemia

Blood ◽

10.1182/blood-2019-129828 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1255-1255

Author(s):

Harmony Estelle Black ◽

Satpal Jhujh ◽

Grant S Stewart ◽

Kienan I Savage ◽

Ken I Mills

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Repair ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Replication Fork ◽

Dna Damage Repair ◽

Replication Forks ◽

Damage Repair ◽

Acute Myeloid

Acute Myeloid Leukaemia (AML) is a genetically heterogeneous disease of the blood and bone marrow primarily affecting older populations. Chemotherapy has been the standard of care for four decades, just 10% of elderly patients show a response and it will be curative in only a third of patients aged 18-60. Therefore, it's necessary to identify new therapeutic targets for improved treatment options. The search for novel mutations within the genomes of karyotypically normal AML patients identified Cohesin's STAG2 as a recurrently mutated gene (6%). Cohesin is a ring-shaped multimeric protein complex which has a multitude of roles including maintaining sister chromatid cohesion, DNA replication and repair, gene expression and chromatin architecture. STAG2 remains the most understudied Cohesin gene, how a mutation affects the ability of Cohesin to fulfil certain roles needs elucidation as well as how STAG2 functions as part of Cohesin to repair DNA breaks, promote faithful replication and control gene expression. Therefore, we sought to explore the consequences of loss-of-function STAG2 mutations on DNA repair and replication using a STAG2 knockout (ΔSTAG2) isogenic cell line model created using CRISPR/Cas9. Immunofluorescent staining of DNA damage repair proteins 53BP1 and RAD51 following 2Gy irradiation (IR) suggests ΔSTAG2 cells have a significant DNA repair defect. The average 53BP1 and RAD51 foci counts were significantly higher (p=0.001) in mutant cells which had three times as many foci at 24 hours after IR. In addition, 40% of ΔSTAG2 cells stain positive for 53BP1 and RAD51 respectively at 24 hours post-IR. This damage persists at 48 and 72 hours, whereas wild type (WT) damage returns to basal levels. Given that our data suggests a HR defect, we assessed the response of WT and ΔSTAG2 cells to PARP inhibitors (PARPi). Interestingly, ΔSTAG2 cells showed significantly greater sensitivity than WT cells to highly potent PARPi Talazoparib with an IC50 of 62nM. We next assayed single DNA fibres to evaluate the implications of a STAG2 mutation on the dynamics of individual replication forks. Interestingly, we observed a significantly reduced number of ongoing replication forks compared to WT cells (p=0.01) with a concomitant increase in the number of stalled forks (p=0.01) in the absence of exogeneous DNA damage. Following treatment with hydroxyurea (HU) this phenotype was exacerbated and ΔSTAG2 cells exhibit significantly more stalled replication forks than WTs (p=0.001). This indicates a role for STAG2 in maintaining replication fork stability and promoting fork restart following replication stress. Importantly, our data identifies a potential DNA damage repair deficiency that sensitises ΔSTAG2 cells to PARPi monotherapy as well as unearthing an essential role for STAG2 in replication fork progression and fork stability. This mutation could therefore be exploited therapeutically and represents a novel therapeutic target/approach for AML patients with mutated STAG2. Disclosures No relevant conflicts of interest to declare.

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