Dr. wael mansour

Here, we present a functional assay to detect the repair switch to the alternative PARP1-dependent end joining (PARP1-EJ) pathway and the associated susceptibility to PARPi-mediated radiosensitization in freshly collected tumor samples from prostate cancer (PCa) patients, thereby facilitating the selection of patients who should benefit from combined PARPi plus radiotherapy (RT) treatment. Our optimized ex-vivo approach sustains tumor slices for up to 15 days under culture conditions that maintain proliferation and oxygenation rates, as measured by EdU incorporation and pimonidazole staining, respectively. We present a robust system to analyze DSB repair using, for the first time in an ex vivo tumor slice setting, two DSB-markers simultaneously i.e. γH2AX and 53BP1. A computer-based processing method (i) controls variations in DNA content and slicing on the number of repair foci and (ii) measures the PARPi-mediated enhancement ratio on DSB foci numbers to ensure inter-patient-comparability. We validated this approach using a PC3 xenograft model with its previously described repair switch to PARP1-EJ. More importantly, we show that approximately 30% of the analyzed tumor tissue samples collected from PCa patients display a switch to PARP1-EJ, as indicated by the enhanced number of residual γH2AX/53BP1 foci exclusively after PARPi+RT. Furthermore, normal prostatic tissues show no repair switch to PARP1-EJ, indicating that this repair switch and its associated radiosensitizing effect is tumor-specific. Collectively, we present here a predictive assay for the switch to PARP1-EJ that enables individualization of anti-cancer treatment using a combination of RT and radiosensitizing anticancer agents such as PARPi in PCa.

BACKGROUND: Disruptor of telomeric silencing 1-like (DOT1L) is a non-SET domain containing methyltransferase known to catalyze mono-, di-, and tri-methylation of histone 3 on lysine 79 (H3K79me). DOT1L-mediated H3K79me has been implicated in chromatin-associated functions including gene transcription, heterochromatin formation, and DNA repair. Recent studies have uncovered a role for DOT1L in the initiation and progression of leukemia and other solid tumors. The development and availability of small molecule inhibitors of DOT1L may provide new and unique therapeutic options for certain types or subgroups of cancer.

METHODS: In this study, we examined the role of DOT1L in DNA double-strand break (DSB) response and repair by depleting DOT1L using siRNA or inhibiting its methyltransferase activity using small molecule inhibitors in colorectal cancer cells. Cells were treated with different agents to induce DNA damage in DOT1L-depleted or -inhibited cells and analyzed for DNA repair efficiency and survival. Further, rectal cancer patient samples were analyzed for H3K79me3 levels in order to determine whether it may serve as a potential marker for personalized therapy.

RESULTS: Our results indicate that DOT1L is required for a proper DNA damage response following DNA double-strand breaks by regulating the phosphorylation of the variant histone H2AX (γH2AX) and repair via homologous recombination (HR). Importantly, we show that small molecule inhibitors of DOT1L combined with chemotherapeutic agents that are used to treat colorectal cancers show additive effects. Furthermore, examination of H3K79me3 levels in rectal cancer patients demonstrates that lower levels correlate with a poorer prognosis.

CONCLUSIONS: In this study, we conclude that DOT1L plays an important role in an early DNA damage response and repair of DNA double-strand breaks via the HR pathway. Moreover, DOT1L inhibition leads to increased sensitivity to chemotherapeutic agents and PARP inhibition, which further highlights its potential clinical utility. Our results further suggest that H3K79me3 can be useful as a predictive and or prognostic marker for rectal cancer patients.

Despite high cure rates, about 20% of patients with advanced germ cell tumors (GCTs) fail cisplatin-based chemotherapy. High levels of DNA methylation have been identified in GCTs and linked to cisplatin resistance. Here, we examined the effects of DNA hypomethylating 5-azacitidine (5-aza) on two embryonal carcinoma cell lines (NCCIT, 2102Ep) and their cisplatin-resistant isogenic derivatives. Effects on cell viability and cisplatin sensitivity were assessed by the trypan blue exclusion method. Western blotting was used to examine induction of apoptosis 5-aza and results were validated by flow cytometry. Single agent treatment with 5-aza strongly impacted viability and induced apoptosis at low nanomolar concentrations, both in cisplatin-sensitive and -resistant cell lines. 5-aza exerted an immediate apoptotic response, followed by a prolonged inhibitory effect on cell viability and cell-cycle progression. Sequential treatment with 5-aza and cisplatin reduced cellular survival of the cisplatin-resistant sublines already at nanomolar concentrations, suggesting a partial restoration of cisplatin sensitivity by the compound. 5-aza demonstrated anti-tumor activity as a single agent at low nanomolar concentrations in GCT cells, irrespective of cisplatin-sensitivity. 5-aza may also have the potential at least to partially restore cisplatin-sensitivity in non-seminoma cells, supporting the hypothesis that combining DNA demethylating agents with cisplatin-based chemotherapy may be a valid therapeutic approach in patients with refractory GCTs.

Here we report that PTEN contributes to DNA double-strand break (DSB) repair via homologous recombination (HR), as evidenced by (i) inhibition of HR in a reporter plasmid assay, (ii) enhanced sensitivity to mitomycin-C or olaparib and (iii) reduced RAD51 loading at IR-induced DSBs upon PTEN knockdown. No association was observed between PTEN-status and RAD51 expression either in-vitro or in-vivo in a tissue microarray of 1500 PTEN-deficient prostate cancer (PC) samples. PTEN depletion and sustained activation of AKT sequestered CHK1 in the cytoplasm, thus impairing the G2/M-checkpoint after irradiation. Consistently, AKT inhibition recovered the G2/M-checkpoint and restored HR efficiency in PTEN-depleted cells. We show that, although PTEN loss correlates with a worse prognosis, it may predict for improved response of PC patients to radiotherapy. Further, we provide evidence for the use of PTEN as a biomarker for predicting the response to PARP inhibitors as radiosensitizing agents in prostate cancer. Collectively, these data implicate PTEN in maintaining genomic stability by delaying G2/M-phase progression of damaged cells, thus allowing time for DSB repair by HR. Furthermore, we identify PTEN-status in PC as a putative predictor of (i) radiotherapy response and (ii) response to treatment with PARP inhibitor alone or combined with radiotherapy.

Here we report that BCL2 blocks DNA double strand break (DSB) repair via nonhomologous end-joining (NHEJ), through sequestration of KU80 protein outside the nucleus. We find that this effect is associated with a repair switch to the error-prone PARP1-dependent end-joining (PARP1-EJ). We present in-vitro proof-of-concept for therapeutic targeting of this switch using PARP inhibitor to specifically enhance the radiosensitivity of BCL2-overexpressing cells. Given its erroneous behavior, PARP1-EJ might allow for the accumulation of genetic alterations and tumor progression. Consistently, we report an inverse correlation between BCL2 expression and biochemical recurrence-free survival of 10.259 prostate cancer (PCa) patients who underwent primary radical-prostatectomy for localized disease. Further, we evaluated retrospectively the impact of BCL2 expression on clinical outcome of 1.426 PCa patients, who had been given salvage radiotherapy at relapse after radical prostatectomy. In line with its role in blocking NHEJ, BCL2 over-expressers showed significantly better response to salvage radiotherapy compared to low-expressers. Collectively, our findings identify BCL2 status in PCa as a putative predictor of (i) radiotherapy response and (ii) response to treatment with PARP inhibitor olaparib as a radiosensitizing agent.

End processing at DNA double strand breaks (DSB) is a decisive step in repair pathway selection. Here, we investigated the role of 53BP1/RIF1 in limiting BRCA1/CtIP-mediated end resection to control DSB repair pathway choice. ATM orchestrates this process through 53BP1 phosphorylation to promote RIF1 recruitment. As cells enter S/G2-phase, end resection is activated, which displaces pATM from DSB sites and diminishes 53BP1 phosphorylation and RIF1 recruitment. Consistently, the kinetics of ATM and 53BP1 phosphorylation in S/G2-phase concur. We show that defective 53BP1/RIF1-mediated DSB end-protection in G1-phase stimulates CtIP/MRE11-dependent end-resection, which requires Polo-like kinase 3. This end resection activity in G1 was shown to produce only short tracks of ssDNA overhangs, as evidenced by the findings that in 53BP1 depleted cells, (i) RPA focus intensity was significantly lower in G1 compared to that in S/G2 phase, and (ii) EXO1 knockdown did not alter either number or intensity of RPA foci in G1 but significantly decreased the RPA focus intensity in S/G2 phase. Importantly, we report that the observed DSB end resection in G1 phase inhibits DNA-PK-dependent nonhomologous end joining but is not sufficient to stimulate HR. Instead, it switches the repair to the alternative PARP1-dependent end joining pathway.

End processing at DNA double strand breaks (DSB) is a decisive step in repair pathway selection. Here, we investigated the role of 53BP1/RIF1 in limiting BRCA1/CtIP-mediated end resection to control DSB repair pathway choice. ATM orchestrates this process through 53BP1 phosphorylation to promote RIF1 recruitment. As cells enter S/G2-phase, end resection is activated, which displaces pATM from DSB sites and diminishes 53BP1 phosphorylation and RIF1 recruitment. Consistently, the kinetics of ATM and 53BP1 phosphorylation in S/G2-phase concur. We show that defective 53BP1/RIF1-mediated DSB end-protection in G1-phase stimulates CtIP/MRE11-dependent end-resection, which requires Polo-like kinase 3. This end resection activity in G1 was shown to produce only short tracks of ssDNA overhangs, as evidenced by the findings that in 53BP1 depleted cells, (i) RPA focus intensity was significantly lower in G1 compared to that in S/G2 phase, and (ii) EXO1 knockdown did not alter either number or intensity of RPA foci in G1 but significantly decreased the RPA focus intensity in S/G2 phase. Importantly, we report that the observed DSB end resection in G1 phase inhibits DNA-PK-dependent nonhomologous end joining but is not sufficient to stimulate HR. Instead, it switches the repair to the alternative PARP1-dependent end joining pathway.

PURPOSE: The aim of this study was to elucidate the impact of DNA damage response (DDR) proteins 53BP1 and BRCA1 on the double-strand break (DSB)-repair choice. This is important not only in order to understand the underlying mechanisms of DSB-repair pathway regulation but also to determine the therapeutic implications for BRCA1-associated tumors.

MATERIALS AND METHODS: Human tumor cell lines A549 and HeLa were used. Non-homologous end-joining (NHEJ) and homologous recombination (HR) were assessed using NHEJ and HR reporter constructs. Colocalization of HR-proteins RPA and RAD51 with 53BP1 was evaluated by confocal microscopy and 3D-analysis.

RESULTS: We demonstrate a specific crosstalk between 53BP1 and BRCA1. While 53BP1 does not colocalize with RPA or RAD51 and prohibits the recruitment of BRCA1 to DSBs to stimulate NHEJ, BRCA1 promotes the 53BP1 displacement specifically in S/G2-phase to allow end-resection, initiating HR. HR-efficiency was restored in BRCA1-depleted cells upon additional 53BP1-knockdown. Further, we found that 53BP1-mediated end protection precedes BRCA1-dependent end-resection.

CONCLUSION: These results demonstrate that the interplay between 53BP1/NHEJ and BRCA1/HR is of great relevance for tumor treatment, as the 53BP1 status would be highly important for the treatment response of BRCA1-associated tumors.

There is a need to develop new, more efficient therapies for head and neck cancer (HNSCC) patients. It is currently unclear whether defects in DNA repair genes play a role in HNSCCs' resistance to therapy. PARP1 inhibitors (PARPi) were found to be "synthetic lethal" in cancers deficient in BRCA1/2 with impaired homologous recombination. Since tumors rarely have these particular mutations, there is considerable interest in finding alternative determinants of PARPi sensitivity. Effectiveness of combined irradiation and PARPi olaparib was evaluated in ten HNSCC cell lines, subdivided into HR-proficient and HR-deficient cell lines using a GFP-based reporter assay. Both groups were equally sensitive to PARPi alone. Combined treatment revealed stronger synergistic interactions in the HR-deficient group. Because HR is mainly active in S-Phase, replication processes were analyzed. A stronger impact of treatment on replication processes (p = 0.04) and an increased number of radial chromosomes (p = 0.003) were observed in the HR-deficient group. We could show that radiosensitization by inhibition of PARP1 strongly correlates with HR competence in a replication-dependent manner. Our observations indicate that PARP1 inhibitors are promising candidates for enhancing the therapeutic ratio achieved by radiotherapy via disabling DNA replication processes in HR-deficient HNSCCs.

There is a need to develop new, more efficient therapies for head and neck cancer (HNSCC) patients. It is currently unclear whether defects in DNA repair genes play a role in HNSCCs' resistance to therapy. PARP1 inhibitors (PARPi) were found to be "synthetic lethal" in cancers deficient in BRCA1/2 with impaired homologous recombination. Since tumors rarely have these particular mutations, there is considerable interest in finding alternative determinants of PARPi sensitivity. Effectiveness of combined irradiation and PARPi olaparib was evaluated in ten HNSCC cell lines, subdivided into HR-proficient and HR-deficient cell lines using a GFP-based reporter assay. Both groups were equally sensitive to PARPi alone. Combined treatment revealed stronger synergistic interactions in the HR-deficient group. Because HR is mainly active in S-Phase, replication processes were analyzed. A stronger impact of treatment on replication processes (p = 0.04) and an increased number of radial chromosomes (p = 0.003) were observed in the HR-deficient group. We could show that radiosensitization by inhibition of PARP1 strongly correlates with HR competence in a replication-dependent manner. Our observations indicate that PARP1 inhibitors are promising candidates for enhancing the therapeutic ratio achieved by radiotherapy via disabling DNA replication processes in HR-deficient HNSCCs.

Despite the significant contribution of radiotherapy to non-small lung cancer (NSCLC), radioresistance still occurs. One of the major radioresistance mechanisms is the hyperactivation of the PI3K/Akt pathway in which Akt facilitates the repair of DNA double-strand breaks (DSBs) through the stimulation of DNA-PKcs. We investigated if targeting PI3K would be a potential approach for enhancing the radiosensitivity of K-RAS mutated (K-RASmut) NSCLC cell lines A549 and H460. Short-term (1-2 h) pre-treatment of cells with the PI3K inhibitor PI-103 (1 μM) inhibited Akt/DNA-PKcs activity, blocked DSBs repair and induced radiosensitivity, while long-term (24 h) pre-treatment did not. Lack of an effect after 24 h of PI-103 pre-treatment was due to reactivation of K-Ras/MEK/ERK-dependent Akt. However, long-term treatment with the combination of PI-103 and MEK inhibitor PD98059 completely blocked reactivation of Akt and impaired DSBs repair through non-homologous end joining (NHEJ) leading to radiosensitization. The effect of PI3K inhibition on Akt signaling was also tested in A549 mouse xenografts. P-Akt and P-DNA-PKcs were inhibited 30 min post-irradiation in xenografts, which were pretreated by PI-103 30 min before irradiation. However, Akt was reactivated 30 min post-irradiation in tumors, which were pre-treated for 3 h with PI-103 before irradiation. After a 24 h pretreatment with PI-103, a significant reactivation of Akt was achieved 24 h after irradiation. Thus, due to MEK/ERK-dependent reactivation of Akt, targeting PI3K alone is not a suitable approach for radiosensitizing K-RASmut NSCLC cells, indicating that dual targeting of PI3K and MEK is an efficient approach to improve radiotherapy outcome.

Despite the significant contribution of radiotherapy to non-small lung cancer (NSCLC), radioresistance still occurs. One of the major radioresistance mechanisms is the hyperactivation of the PI3K/Akt pathway in which Akt facilitates the repair of DNA double-strand breaks (DSBs) through the stimulation of DNA-PKcs. We investigated if targeting PI3K would be a potential approach for enhancing the radiosensitivity of K-RAS mutated (K-RASmut) NSCLC cell lines A549 and H460. Short-term (1-2 h) pre-treatment of cells with the PI3K inhibitor PI-103 (1 μM) inhibited Akt/DNA-PKcs activity, blocked DSBs repair and induced radiosensitivity, while long-term (24 h) pre-treatment did not. Lack of an effect after 24 h of PI-103 pre-treatment was due to reactivation of K-Ras/MEK/ERK-dependent Akt. However, long-term treatment with the combination of PI-103 and MEK inhibitor PD98059 completely blocked reactivation of Akt and impaired DSBs repair through non-homologous end joining (NHEJ) leading to radiosensitization. The effect of PI3K inhibition on Akt signaling was also tested in A549 mouse xenografts. P-Akt and P-DNA-PKcs were inhibited 30 min post-irradiation in xenografts, which were pretreated by PI-103 30 min before irradiation. However, Akt was reactivated 30 min post-irradiation in tumors, which were pre-treated for 3 h with PI-103 before irradiation. After a 24 h pretreatment with PI-103, a significant reactivation of Akt was achieved 24 h after irradiation. Thus, due to MEK/ERK-dependent reactivation of Akt, targeting PI3K alone is not a suitable approach for radiosensitizing K-RASmut NSCLC cells, indicating that dual targeting of PI3K and MEK is an efficient approach to improve radiotherapy outcome.

The CHD1 gene, encoding the chromo-domain helicase DNA-binding protein-1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double-strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of CtIP to chromatin and subsequent end resection during DNA DSB repair. Our data support a role for CHD1 in opening the chromatin around the DSB to facilitate the recruitment of homologous recombination (HR) proteins. Consequently, depletion of CHD1 specifically affects HR-mediated DNA repair but not non-homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may serve as a marker for prostate cancer patient stratification and the utilization of targeted therapies such as PARP inhibitors, which specifically target tumors with HR defects.

PURPOSE: The aim of this study was to elucidate the impact of DNA damage response (DDR) proteins 53BP1 and BRCA1 on the double-strand break (DSB)-repair choice. This is important not only in order to understand the underlying mechanisms of DSB-repair pathway regulation but also to determine the therapeutic implications for BRCA1-associated tumors.

MATERIALS AND METHODS: Human tumor cell lines A549 and HeLa were used. Non-homologous end-joining (NHEJ) and homologous recombination (HR) were assessed using NHEJ and HR reporter constructs. Colocalization of HR-proteins RPA and RAD51 with 53BP1 was evaluated by confocal microscopy and 3D-analysis.

RESULTS: We demonstrate a specific crosstalk between 53BP1 and BRCA1. While 53BP1 does not colocalize with RPA or RAD51 and prohibits the recruitment of BRCA1 to DSBs to stimulate NHEJ, BRCA1 promotes the 53BP1 displacement specifically in S/G2-phase to allow end-resection, initiating HR. HR-efficiency was restored in BRCA1-depleted cells upon additional 53BP1-knockdown. Further, we found that 53BP1-mediated end protection precedes BRCA1-dependent end-resection.

CONCLUSION: These results demonstrate that the interplay between 53BP1/NHEJ and BRCA1/HR is of great relevance for tumor treatment, as the 53BP1 status would be highly important for the treatment response of BRCA1-associated tumors.

Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2), which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2), on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks.

Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2), which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2), on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks.

Cellular chromosomal DNA is the principal target through which ionising radiation exerts it diverse biological effects. This chapter summarises the relevant DNA damage signalling and repair pathways used by normal and tumour cells in response to irradiation. Strategies for tumour radiosensitisation are reviewed which exploit tumour-specific DNA repair deficiencies or signalling pathway addictions, with a special focus on growth factor signalling, PARP, cancer stem cells, cell cycle checkpoints and DNA replication. This chapter concludes with a discussion of DNA repair-related candidate biomarkers of tumour response which are of crucial importance for implementing precision medicine in radiation oncology.

In response to replication stress ATR signaling through CHK1 controls the intra-S checkpoint and is required for the maintenance of genomic integrity. Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double strand breaks and interstrand crosslinks. In addition, HR, with its key player RAD51, provides critical support for the recovery of stalled forks during replication. High levels of RAD51 are regularly found in various cancers, yet little is known about the effect of the increased RAD51 expression on intra-S checkpoint signaling. Here, we describe a role for RAD51 in driving genomic instability caused by impaired replication and intra-S mediated CHK1 signaling by studying an inducible RAD51 overexpression model as well as 10 breast cancer cell lines. We demonstrate that an excess of RAD51 decreases I-Sce-I mediated HR despite formation of more RAD51 foci. Cells with high RAD51 levels display reduced elongation rates and excessive dormant origin firing during undisturbed growth and after damage, likely caused by impaired CHK1 activation. In consequence, the inability of cells with a surplus of RAD51 to properly repair complex DNA damage and to resolve replication stress leads to higher genomic instability and thus drives tumorigenesis.

Malignant tumors of the rectum are treated by neoadjuvant radiochemotherapy. This involves a combination of 5-fluorouracil (5-FU) and double stranded DNA-break (DSB)-inducing radiotherapy. Here we explored how 5-FU cooperates with DSB-induction to achieve sustainable DNA damage in colorectal cancer (CRC) cells. After DSB induction by neocarzinostatin, phosphorylated histone 2AX (γ-H2AX) rapidly accumulated but then largely vanished within a few hours. In contrast, when CRC cells were pre-treated with 5-FU, gammaH2AX remained for at least 24 hours. GFP-reporter assays revealed that 5-FU decreases the efficiency of homologous recombination (HR) repair. However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci. Thus, we propose that 5-FU interferes with the continuation of HR repair, e. g. the synthesis of new DNA strands. Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa. Inhibition of HR by targeting Rad51 enhanced DNA damage upon DSB-inducing treatment, outlining an alternative way of enhancing therapeutic efficacy. Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy.

Malignant tumors of the rectum are treated by neoadjuvant radiochemotherapy. This involves a combination of 5-fluorouracil (5-FU) and double stranded DNA-break (DSB)-inducing radiotherapy. Here we explored how 5-FU cooperates with DSB-induction to achieve sustainable DNA damage in colorectal cancer (CRC) cells. After DSB induction by neocarzinostatin, phosphorylated histone 2AX (γ-H2AX) rapidly accumulated but then largely vanished within a few hours. In contrast, when CRC cells were pre-treated with 5-FU, gammaH2AX remained for at least 24 hours. GFP-reporter assays revealed that 5-FU decreases the efficiency of homologous recombination (HR) repair. However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci. Thus, we propose that 5-FU interferes with the continuation of HR repair, e. g. the synthesis of new DNA strands. Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa. Inhibition of HR by targeting Rad51 enhanced DNA damage upon DSB-inducing treatment, outlining an alternative way of enhancing therapeutic efficacy. Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy.

Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3'-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.

Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3'-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.