Homologous Recombination Factors Research Articles

The interplay of homology-directed repair pathways in the repair of zebularine-induced DNA-protein crosslinks in Arabidopsis.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions represented by proteins covalently bound to the DNA. Persisting DPCs interfere with fundamental genetic processes such as DNA replication and transcription. Cytidine analog zebularine (ZEB) has been shown to crosslink DNA METHYLTRANSFERASE1 (MET1). Recently, we uncovered a critical role of the SMC5/6-mediated SUMOylation in the repair of DPCs. In an ongoing genetic screen, we identified two additional candidates, HYPERSENSITIVE TO ZEBULARINE 2 and 3, that were mapped to REGULATOR OF TELOMERE ELONGATION 1 (RTEL1) and polymerase TEBICHI (TEB), respectively. By monitoring the growth of hze2 and hze3 plants in response to zebularine, we show the importance of homologous recombination (HR) factor RTEL1 and microhomology-mediated end-joining (MMEJ) polymerase TEB in the repair of MET1-DPCs. Moreover, genetic interaction and sensitivity assays showed the interdependency of SMC5/6 complex, HR, and MMEJ in the homology-directed repair of MET1-DPCs in Arabidopsis. Altogether, we provide evidence that MET1-DPC repair in plants is more complex than originally expected.

Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors

The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4–6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi.

Abstract 711: HDAC3 sustains resistance to hypofractionated radiotherapy in fusion positve rhabdomyosarcoma cells

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. The fusion-positive (FP)-RMS variant expressing chimeric oncoproteins such as PAX3-FOXO1 and PAX7-FOXO1 shows a dismal prognosis with 5-year survival of less than 30% compared to non-metastatic fusion-negative (FN)-RMS variant. In the last years, a lot of interest has focused on new targets identification to improve the radiotherapy (RT) efficacy. HDAC inhibitors (HDACi) radio-sensitize different cancer cell types including RMS. Recently, we reported that MS-275, a Class I and IV HDACi, in combination with RT affected cell survival, reduced colony formation ability, increased DNA damage repair inhibition and reactive oxygen species formation in FP-RMS cells. However, despite promising preclinical studies, HDAC inhibitors (HDACi) achieved only modest success in human clinical trials for solid tumors, frequently showing several toxicities, probably because of the limited specificity of many HDACi. Thus, a major effort is being directed toward identification of HDACi which are selective for HDAC isoforms often uniquely implicated in the radioresistance of specific cancers. In order to identify the class I HDAC responsible of radioresistance in RMS we knocked-down HDAC1, HDAC2, HDAC3, and HDAC8 expression in combination with RT. Interestingly, we observed an increased radiosensitivity only in HDAC3-depleted FP-RMS cells. Thus, we focused on HDAC3 to understand the mechanisms by which it promotes radioresistance in RMS. We observed that HDAC3 is overexpressed in RMS patients and cell lines compared to the normal counterpart. Furthermore, RMS cells are strongly dependent by HDAC3 expression while slight dependency has been observed with other class I HDACs in DepMap portal. We demonstrated that HDAC3 depletion by CRISPR in combination with RT in FP-RMS cells increases apoptosis, reduces colony formation ability, cancer stem cells population and anchorage independent growth and reduces cell growth in vivo and in vitro. Accordingly, HDAC3 depleted cells in combination with RT reduces levels and activation of key player of FP-RMS biology such as MYCN, ERK and AKT. Moreover, HDAC3 KD increases radiotherapy-induced DNA double strand break and impairs DNA repair mechanisms reducing levels and activation of both homologous recombination (HR) and non-homologous end joining (NHEJ) factors such as ATM, RAD51 and DNA-PKcs. Thus, we developed a new potent and highly specific HDAC3 inhibitor (HDAC3i). The new HDAC3i is highly specific in targeting FP-RMS cell growth in vitro while no effects have been observed in normal cells such as myoblasts and lung fibroblast. Moreover, the drug treatment in combination with radiotherapy phenotypically and molecularly recapitulated what observed in HDAC3 depleted cells.The study has been founded by Italian Association for Cancer Research (AIRC) to FM. Citation Format: Matteo Cassandri, Antonella Porrazzo, Silvia Pomella, Simona Camero, Francesca A. Aiello, Lucrezia D'Archivio, Clemens Zwergel, Beatrice Noce, Miriam Tomaciello, Francesca Vulcano, Luisa Milazzo, Francesca Pedini, Michele Signore, Alessandro Fanzani, Cinzia Marchese, Giuseppe Minniti, Sergio Valente, Antonello Mai, Francesca Megiorni, Rossella Rota, Francesco Marampon. HDAC3 sustains resistance to hypofractionated radiotherapy in fusion positve rhabdomyosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 711.

Inhibition of intracellular ATP synthesis impairs the recruitment of homologous recombination factors after ionizing radiation.

Ionizing radiation (IR)-induced double-strand breaks (DSBs) are primarily repaired by non-homologous end joining or homologous recombination (HR) in human cells. DSB repair requires adenosine-5'-triphosphate (ATP) for protein kinase activities in the multiple steps of DSB repair, such as DNA ligation, chromatin remodeling, and DNA damage signaling via protein kinase and ATPase activities. To investigate whether low ATP culture conditions affect the recruitment of repair proteins at DSB sites, IR-induced foci were examined in the presence of ATP synthesis inhibitors. We found that p53 binding protein 1 foci formation was modestly reduced under low ATP conditions after IR, although phosphorylated histone H2AX and mediator of DNA damage checkpoint 1 foci formation were not impaired. Next, we examined the foci formation of breast cancer susceptibility gene I (BRCA1), replication protein A (RPA) and radiation 51 (RAD51), which are HR factors, in G2 phase cells following IR. Interestingly, BRCA1 and RPA foci in the G2 phase were significantly reduced under low ATP conditions compared to that under normal culture conditions. Notably, RAD51 foci were drastically impaired under low ATP conditions. These results suggest that HR does not effectively progress under low ATP conditions; in particular, ATP shortages impair downstream steps in HR, such as RAD51 loading. Taken together, these results suggest that the maintenance of cellular ATP levels is critical for DNA damage response and HR progression after IR.

Abstract 1845 Biochemical characterization of HSF2BP as a meiotic homologous recombination factor

Abstract 1845 Biochemical characterization of HSF2BP as a meiotic homologous recombination factor

Abstract B004: Polymerase theta (Pol θ) is essential to repair a subset of DNA breaks in HR proficient cells

Abstract DNA polymerase theta (Pol θ), functions in theta-mediated end joining (TMEJ), an error-prone DNA double strand break repair pathway. Pol θ is synthetic lethal with homologous recombination (HR) factors, and this finding has led to the development of Pol θ inhibitors and clinical trials to determine their effectiveness in HR-deficient tumors. Despite this, the biological functions of Pol q that promote genome integrity in normal cells are poorly understood. Here, we elucidate an essential role for Pol θ during mammalian interstrand crosslink (ICL) repair. We show that Pol θ is recruited downstream of ICL nucleolytic incision, BRCA2-dependent Rad51 loading, and FBXO5-mediated Rad51 ubiquitylation at sites of unsuccessful HR. Genomic scar analyses reveal that TMEJ is specifically required for repair of clustered ICLs, which are not amenable to HR-mediated repair. Thus, Pol q may be the predominant repair pathway for clustered lesions that generate two-ended double strand breaks during DNA replication. These findings suggest that Pol θ has essential roles in DNA repair in HR proficient cells, suggesting that Pol θ inhibitors may also be beneficial in the treatment of HR proficient tumors. Citation Format: Chelsea M. Smith, Dennis Simpson, Wanjuan Feng, Gaorav Gupta. Polymerase theta (Pol θ) is essential to repair a subset of DNA breaks in HR proficient cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B004.

The COMPASS subunit Spp1 protects nascent DNA at the Tus/Ter replication fork barrier by limiting DNA availability to nucleases

Homologous recombination factors play a crucial role in protecting nascent DNA during DNA replication, but the role of chromatin in this process is largely unknown. Here, we used the bacterial Tus/Ter barrier known to induce a site-specific replication fork stalling in S. cerevisiae. We report that the Set1C subunit Spp1 is recruited behind the stalled replication fork independently of its interaction with Set1. Spp1 chromatin recruitment depends on the interaction of its PHD domain with H3K4me3 parental histones deposited behind the stalled fork. Its recruitment prevents the accumulation of ssDNA at the stalled fork by restricting the access of Exo1. We further show that deleting SPP1 increases the mutation rate upstream of the barrier favoring the accumulation of microdeletions. Finally, we report that Spp1 protects nascent DNA at the Tus/Ter stalled replication fork. We propose that Spp1 limits the remodeling of the fork, which ultimately limits nascent DNA availability to nucleases.

SPIDR is required for homologous recombination during mammalian meiosis.

Meiotic recombinases RAD51 and DMC1 mediate strand exchange in the repair of DNA double-strand breaks (DSBs) by homologous recombination. This is a landmark event of meiosis that ensures genetic diversity in sexually reproducing organisms. However, the regulatory mechanism of DMC1/RAD51-ssDNA nucleoprotein filaments during homologous recombination in mammals has remained largely elusive. Here, we show that SPIDR (scaffold protein involved in DNA repair) regulates the assembly or stability of RAD51/DMC1 on ssDNA. Knockout of Spidr in male mice causes complete meiotic arrest, accompanied by defects in synapsis and crossover formation, which leads to male infertility. In females, loss of Spidr leads to subfertility; some Spidr-/- oocytes are able to complete meiosis. Notably, fertility is rescued partially by ablation of the DNA damage checkpoint kinase CHK2 in Spidr-/- females but not in males. Thus, our study identifies SPIDR as an essential meiotic recombination factor in homologous recombination in mammals.

Pan-Cancer Analysis Identified Homologous Recombination Factor With OB-Fold (HROB) as a Potential Biomarker for Various Tumor Types.

Background: By recruiting the MCM8–MCM9 helicase to DNA damage site, the Homologous Recombination Factor With OB-Fold (HROB) is involved in the repair of inter-strand crosslink and homologous recombination. Previous studies have shown that HROB may play an oncogenic role by promoting cell proliferation and chemoresistance in several tumor types. However, the potential diagnostic and prognostic values of HROB have not been systemically explored in pan-cancer. Methods: We analyzed the expression pattern of HROB among tumor tissues and normal tissues in several public databases, including Human Protein Atlas and the Cancer Genome Atlas (TCGA) and investigated the association between the HROB expression and pathological stage and patient prognosis. We also analyzed the association between HROB expression and cancer stemness and immune infiltration of cancer-associated fibroblasts (CAFs) and CD8+ T cells in pan-cancer. Finally, we explored the potential biological function of HROB through pathway enrichment analysis. Results: In most tumor types, HROB is overexpressed in tumor tissues compared with non-tumor tissues. High HROB expression was correlated with poor prognosis and advanced pathological stages. HROB expression was robustly correlated with cancer stemness. Moreover, significant correlations between CAFs, CD8+ T-cell infiltration, and HROB expression were observed in several tumor types. Pathway enrichment analysis revealed that cell cycle and mitotic-regulated pathways were strongly enriched in HROB co-expressed genes. Conclusion: HROB may be a potential diagnostic and prognostic biomarker in pan-cancer, which may play a role in tumorigenesis and disease progression by affecting the cancer stemness of tumor tissues and immune cell infiltration.

Molecular Characterization of BRCA1 c.5339T>C Missense Mutation in DNA Damage Response of Triple-Negative Breast Cancer

Simple SummaryRecent studies re-classified the c.5339T->C; p.Leu1780Pro (L1780P) BRCA1 missense mutation as “likely pathogenic” in ACMG classification, which shows a high prevalence in the Korean population. This study aims to reveal the molecular mechanisms and therapeutic relevance of BRCA1 L1780P mutation in DNA damaging response of triple-negative breast cancer (TNBC).BRCA1 L1780P BRCT domain mutation has been recognized as a pathogenic mutation in patients with breast cancer. However, the molecular significance of this mutation has not yet been studied in triple-negative breast cancer (TNBC) cells in vitro. We established MDA-MB 231, HCC1937, and HCC1395 TNBC cell lines expressing BRCA1 L1780P mutant. BRCA1 L1780P mutant TNBC cells showed increased migration and invasion capacity, as well as increased sensitivity to olaparib and carboplatin compared to BRCA1 wild-type cells. BRCA1 L1780P mutant TNBC cells showed decreased RAD51 expression and reduced nuclear RAD51 foci formation following carboplatin and olaparib treatment. The molecular interaction between p-ATM and BRCA1 was abrogated following introduction of BRCA1 L1780P mutant plasmid in TNBC cells, suggesting that the BRCA1 L1780P mutation disrupts the p-ATM-BRCA1 protein–protein interaction. We established an olaparib-resistant BRCA1 L1780P mutant TNBC cell line by chronic drug treatment. Olaparib-resistant cell lines showed upregulation of RAD51 expression upon olaparib treatment, and reduction in RAD51 expression in olaparib-resistant cells restored olaparib sensitivity. Collectively, these results suggest that the BRCA1 L1780P mutation impairs RAD51 recruitment by disrupting p-ATM-BRCA1 interaction, which is a crucial molecular factor in homologous recombination and olaparib sensitivity. Further therapeutic targeting of RAD51 in BRCA1 L1780P mutant breast cancer is warranted.

FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxyuridine

Fanconi anemia (FA) is a rare hereditary disorder caused by mutations in any one of the FANC genes. FA cells are mainly characterized by extreme hypersensitivity to interstrand crosslink (ICL) agents. Additionally, the FA proteins play a crucial role in concert with homologous recombination (HR) factors to protect stalled replication forks. Here, we report that the 5-methyl-2’-deoxycytidine (5mdC) demethylation (pathway) intermediate 5-hydroxymethyl-2’-deoxycytidine (5hmdC) and its deamination product 5-hydroxymethyl-2’-deoxyuridine (5hmdU) elicit a DNA damage response, chromosome aberrations, replication fork impairment and cell viability loss in the absence of FANCD2. Interestingly, replication fork instability by 5hmdC or 5hmdU was associated to the presence of Poly(ADP-ribose) polymerase 1 (PARP1) on chromatin, being both phenotypes exacerbated by olaparib treatment. Remarkably, Parp1−/− cells did not show any replication fork defects or sensitivity to 5hmdC or 5hmdU, suggesting that retained PARP1 at base excision repair (BER) intermediates accounts for the observed replication fork defects upon 5hmdC or 5hmdU incorporation in the absence of FANCD2. We therefore conclude that 5hmdC is deaminated in vivo to 5hmdU, whose fixation by PARP1 during BER, hinders replication fork progression and contributes to genomic instability in FA cells.

Top1p targeting by Fob1p at the ribosomal Replication Fork Barrier does not account for camptothecin sensitivity in Saccharomyces cerevisiae cells.

Camptothecin (CPT) is a specific inhibitor of the DNA topoisomerase I (Top1p), currently used in cancer therapy, which induces DNA damage and cell death. Top1p is highly active at the repeated ribosomal DNA locus (rDNA) to relax DNA supercoiling caused by elevated transcription and replication occurring in opposite directions. Fob1p interacts with, and stabilizes, Top1p at the rDNA Replication Fork Barrier (rRFB), where replication and transcription converge. Here, we have investigated if the absence of Fob1p and the consequent loss of Top1p specific targeting to the rRFB impact the sensitivity and the cell cycle progression of wild-type cells to CPT. We have also investigated the consequences of the absence of Fob1p in rad52∆ mutants, which are affected in the repair of CPT-induced DNA damage by homologous recombination. The results show that CPT sensitivity and the global cell cycle progression in cells exposed to CPT is not changed in the absence of Fob1p. Moreover, we have observed in fob1∆ cells treated with CPT that the homologous recombination factor Rad52p still congregates in the shape of foci in the nucleolus, which hosts the rDNA. This suggests that, in the absence of Fob1p, Top1p is still recruited to the rDNA, presumably at sequences other than the rRFB, and its inhibition by CPT leads to recombination events.

DNA Repair Pathways and Their Association With Lethal Prostate Cancer in African American and European American Men.

BackgroundAltered DNA damage response (DDR) has emerged as an important mechanism for the development of aggressive prostate cancer among men of European ancestry but not other ancestry groups. Because common mechanisms for aggressive disease are expected, we explored a large panel of DDR genes and pathways to demonstrate that DDR alterations contribute to development of aggressive prostate cancer in both African American and European American men.MethodsWe performed a case-case study of 764 African American and European American men with lethal or indolent prostate cancer treated at 4 US hospitals. We calculated carrier frequencies of germline pathogenic or likely pathogenic sequence variants within 306 DDR genes, summarized by DDR pathway, and compared lethal cases against indolent cases using 2-sided Fisher’s exact tests. Secondary analysis examined if carrier frequencies differed by ancestry.ResultsLethal cases were more likely to carry a pathogenic sequence variant in a DDR gene compared with indolent cases (18.5% vs 9.6%, P = 4.30 × 10−4), even after excluding BRCA2 (14.6% vs 9.6%, P = .04). The carrier frequency was similar among lethal cases of African (16.7% including and 15.8% excluding BRCA2) and lethal cases of European (19.3% including and 14.2% excluding BRCA2) ancestry. Three DDR pathways were statistically significantly associated with lethal disease: homologous recombination (P = .003), Fanconi anemia (P = .002), and checkpoint factor (P = .02).ConclusionsOur findings suggest that altered DDR is an important mechanism for aggressive prostate cancer not only in men of European but also of African ancestry. Therefore, interrogation of entire DDR pathways is needed to fully characterize and better define genetic risk of lethal disease.

The Shu complex prevents mutagenesis and cytotoxicity of single-strand specific alkylation lesions.

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Thus, our work identifies a novel homologous recombination-based mechanism mediated by the Shu complex for coping with alkylation adducts.

Mechanism of chromosome rearrangement arising from single-strand breaks

Chromosome rearrangements, which are structural chromosomal abnormalities commonly found in human cancer, result from the misrejoining between two or more DNA double-strand breaks arising at different genomic regions. Consequently, chromosome rearrangements can generate fusion genes that promote tumorigenesis. The mechanisms of chromosome rearrangement have been studied using exogenous double-strand break inducers, such as radiation and nucleases. However, the mechanism underlying the occurrence of chromosome rearrangements in the absence of exogenous double-strand break-inducing stimuli is unclear. This study aimed to identify the major source of chromosome rearrangements and the DNA repair pathway that suppresses them. DNA repair factors that potentially suppress gene fusion were screened using The Cancer Genome Atlas dataset. In total, 22 repair factors whose expression levels were negatively correlated with the frequency of gene fusion were identified. More than 60% of these repair factors are involved in homologous recombination, a major double-strand break repair pathway. We hypothesized that DNA single-strand breaks are the source of double-strand breaks that lead to chromosome rearrangements. This study demonstrated that hydrogen peroxide (H2O2)-induced single-strand breaks gave rise to double-strand breaks in a replication-dependent manner. Additionally, H2O2 induced the formation of RPA and RAD51 foci, which indicated that double-strand breaks derived from single-strand breaks were repaired through homologous recombination. Moreover, treatment with H2O2 promoted the formation of radial chromosomes, a type of chromosome rearrangements, only upon the downregulation of homologous recombination factors, such as BRCA1 and CtIP. Thus, single-strand breaks are the major source of chromosome rearrangements when the expression of homologous recombination factors is downregulated.

A Small Molecule RAD51 Inhibitor Preferentially Affects Cells Expressing High Cytidine Deaminase Activity

RAD51 is a homologous recombination factor critical for DNA replication, recombination, and DNA double strand break repair. RAD51 forms nucleoprotein filaments at damaged DNA sites or corrupted DNA replication forks to mediate DNA strand exchange, culminating in recombinational repair of breaks and replication fork restart. RAD51 is essential for the repair of DNA lesions induced by agents that stall or collapse DNA replication forks. In this context, RAD51 is an important factor in resistance to DNA replication stress induced by the cellular cytidine deaminase AICDA (also known as AID). AICDA is a DNA-directed cytidine deaminase that normally acts to initiate somatic hypermutation and immunoglobulin class switching in activated B-lymphocytes. However, AICDA is often constitutively overexpressed in tumor cells in a range of cancer types. In transformed cells the cytidine deamination activity of AICDA, and the resulting DNA base pair mismatches, cause tumor cell hypermutation. As a byproduct of its mutational activity, AICDA also destabilizes DNA replication, causing a high concentration of DNA double stranded breaks in AIDCA-expressing cells. This leads to an obligate dependency on RAD51 for DSB repair and cell survival. We previously demonstrated that RAD51 depletion induces acute sensitivity to AICDA activity in transformed cells. We now describe the mechanistic characterization of a small molecule RAD51 inhibitor - CYT01A- that is preferentially cytotoxic to AICDA expressing cells. CYT01A is a potent RAD51 inhibitor (EC50 ~150nM) that promotes DNA replication catastrophe selectively in cells expressing AICDA. Exposure to CYT01A in cell culture leads to the depletion of nuclear RAD51, resulting in diminished nuclear foci formation in response to DNA damage. We demonstrate a dose-dependent cytotoxic response to CYT01A that was reflected by RAD51 foci suppression. We further demonstrate compromised RAD51 nucleoprotein filament formation, evidenced by persistence of Replication Protein A (RPA) foci following CYT01A treatment. RAD51 depletion and reduced foci formation correlates with a reduction in homologous recombination activity. Finally, we show that CYT01A treated cells undergo replication catastrophe, associated with caspase3/7 activation. Collectively, these data indicate that CYT01A prevents RAD51 transport into the nucleus and thereby inhibits DNA repair activity. This demonstrates the feasibility of targeting RAD51 to induce cell death selectively in cells with high DNA replication stress, such as AICDA overexpressing tumor cells. These findings form the basis for a new synthetic lethal therapeutic approach, targeting the AICDA/RAD51 axis in cancers that exhibit high levels of AICDA activity DisclosuresMills:Cyteir Therapeutics: Employment, Equity Ownership. Cyr:Cyteir Therapeutics: Employment, Equity Ownership. Maclay:Cyteir Therapeutics: Employment, Equity Ownership. Day:Cyteir Therapeutics: Employment, Equity Ownership. Khalil:Cyteir Therapeutics: Consultancy, Equity Ownership.

Oct4 confers stemness and radioresistance to head and neck squamous cell carcinoma by regulating the homologous recombination factors PSMC3IP and RAD54L

Head and neck squamous cell carcinoma (HNSCC) is often being diagnosed at an advanced stage, conferring a poor prognosis. The probability of local tumor control after radiotherapy depends on the eradication of cancer stem cells (CSCs) with activated DNA repair. This study provides evidence that the CSC-related transcription factor Oct4 contributes to HNSCC radioresistance by regulating DNA damage response and the CSC phenotype. Knockdown of Oct4 A isoform reduced self-renewal capacity in HNSCC and led to partial tumor cell radiosensitization caused by transcriptional downregulation of the cell cycle checkpoint kinases CHK1 and WEE1 and homologous recombination (HR) repair genes PSMC3IP and RAD54L. Besides, PARP inhibition with Olaparib selectively radiosensitized Oct4 A knockout, but not wild-type HNSCC cells. This finding links Oct4 A to the HR-mediated DNA repair mechanisms. In turn, knockdown of PSMC3IP and RAD54L reduced the HNSCC self-renewal capacity and clonogenic cell survival after irradiation, suggesting the interplay between DNA repair and the CSC phenotype. Similar to the effect of Oct4 knockdown, overexpression of Oct4 also resulted in significant HNSCC radiosensitization and increased DNA damage, suggesting that Oct4-dependent regulation of DNA repair depends on its fine-tuned expression. In line with this observation, HNSCC patients with high and low nuclear Oct4 expression at the invasive tumor front exhibited better loco-regional tumor control after postoperative radio(chemo)therapy compared to the intermediate expression subgroup. Thus, we found that the Oct4-driven transcriptional program plays a critical role in regulating HNSCC radioresistance, and a combination of radiotherapy with PARP inhibitors may induce synthetic lethality in Oct4-deregulated tumors.

Abstract PO-032: Mechanistic studies of hypoxia as a driver of genomic instability in prostate cancer

Abstract Prostate cancer (PCa) is the 2nd most prevalent cancer in men and has poor patient outcome upon transition from localized disease to the castration-resistant and metastatic state. Numerous studies have indicated intratumoral hypoxia as a key driver in disease progression across multiple cancers and in prostate cancer. Hypoxia is associated with local recurrence, metastatic capacity, androgen independence and neuroendocrine differentiation (Ashton and Bristow, Brit.J.Rad, 2020). Crucially, among localized PCa patients, hypoxia and genomic instability have an independent and additive effect on biochemical relapse and bioinformatic studies suggest that hypoxia co-segregates mutation signatures representing defects in homologous recombination (HR) and mismatch repair (MMR) DNA repair pathways (Bhandari et al., Nat Comms, 2020). In order to investigate whether hypoxia directly drives genome instability in prostate cancer, mechanistic studies using hTERT-immortalized, primary prostate epithelial cells isogenic for c-MYC, BRCA2, or TP53/RB (SV40-transduced as PNT2 cells) were studied as to the relationship between hypoxia, inherent versus acquired genetic instability, and DNA repair capacity in-vitro and in-situ. Chronic hypoxia (20 generation doublings at 0.2% O2) was sufficient to drive increased copy number alterations (CNAs; based on low-pass whole genome sequencing - WGS) in selected clones by up to 30% in TP53/RB null cells, but not hTERT immortalized models. This finding is now being quantitated within a clonal evolution model and genotype-phenotype associations are being validated using functional transformation and invasion assays. Associated mechanistic studies have revealed that the hypoxic suppression of homologous recombination (HR) factor transcript and protein abundance (e.g up to ~60% reduction of RAD51/BRCA2 in PC3, DU145 and PNT2 cells) and associated with reduced RAD51 nuclear foci induction or defective DR-GFP reconstitution, is highly-dependent on cell density. This suggests that reduced HR and MMR repair capacity may exist in situ within hypoxic tumor oxygen gradients with close cell-cell interactions; in situ studies of RAD51 and other DNA repair proteins across hypoxic gradients in primary human tumors will be presented. These translational studies will accompany the HYPROGEN trial in Manchester in which patients who present with treatment-naïve M1 bone metastatic disease are given oral pimonidazole prior to prostate and bone metastasis biopsies to directly study genomic instability (using WGS) and hypoxia (pimonidazole staining and RNA hypoxic signatures) to understand these relationships in paired primary-metastatic samples. This data will provide a mechanistic basis for the adverse prognosis seen in patients whose tumors have both hypoxia and genome instability, as well as fresh evidence for stratification and treatment intensification of localized PCa patients with hypoxic tumors. (Supported by a core grant from Cancer Research UK to RGB) Citation Format: Jack Ashton, Richard Rebello, Stephen Lyons, Robert G. Bristow. Mechanistic studies of hypoxia as a driver of genomic instability in prostate cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-032.

RAD51 Inhibition Induces R-Loop Formation in Early G1 Phase of the Cell Cycle.

R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.

A Recently Discovered Essential Factor for DNA Homologous Recombination:RNA Polymerase III

End resection is a central step in DNA homologous recombination (HR) and the HR-mediated repair of DNA double-strand breaks (DSBs), which degrades 5’-end strands at DSBs by several kilo-bases and thus creates 3’-end single-strand DNA overhangs. A critical long-standing question is how the 3’-end strands are protected during end resection. Now, this question is answered. Liu et al. found that the protection of 3’-end strands is achieved by the formation of an RNA-DNA hybrid. RNA polymerase III is responsible for catalyzing the RNA strand in the hybrid. Thus, RNA polymerase III is an essential factor for HR, and the RNA-DNA hybrid is an essential HR intermediate.