RDNA-12. THE FANCONI ANAEMIA (FA) PATHWAY AND GLIOBLASTOMA: A NEW FOUNDATION FOR DNA DAMAGE RESPONSE TARGETED COMBINATIONS

Abstract Treatment resistance in glioblastoma is underpinned by highly interconnected DNA damage response (DDR) processes. The FA-pathway is a fundamental DDR process required for the resolution of replication fork impeding lesions, and we have previously shown that it is inactive in normal brain, but is re-activated in glioblastoma, providing a cancer-specific target for combination DDR therapies. Here, we find that elevated FA-pathway gene expression in gliomas is associated with poor survival (-17.1% 5-year OS, p< 0.0001, n=329–REMBRANT). Furthermore, patient-derived glioblastoma stem cell (GSC) populations, which drive therapeutic resistance, display high FA-pathway expression relative to paired bulk tumour cell populations (mean 2.3-fold higher across genes, p=0.0073). We further show that inhibition of a single DDR process (FA-pathway, PARP, ATR or ATM) increases the susceptibility of glioblastoma cell lines and patient-derived GSCs to current adjuvant therapy. Importantly, clinically approved PARP inhibitor (PARPi) monotherapy stimulates robust FANCD2 mono-ubiquitination, supporting a role of FA-pathway activation in response to current DDR-targeted therapy. In clinically-relevant 3D GSC models, simultaneous inhibition of the FA-pathway (FAPi) and PARP or ATR enhanced temozolomide sensitisation compared to a single DDR inhibitor (DDRi). Furthermore, combined FAPi+PARPi consistently conferred radiosensitisation whilst combined FAPi+ATRi led to a profoundly radiosensitising effect; e.g. sensitizer enhancement ratio (SER0.37) of 3.23 (3.03–3.49, 95% CI). Furthermore, comparison of α/β ratio enhancement suggests dual-DDRi strategies fundamentally alter the response of GSCs, whilst single cell gel electrophoresis & immunofluorescence studies suggest FA-pathway based DDRi combinations profoundly delay the resolution of IR-induced DNA strand breaks at 6 hours post-treatment, with increased persistent DNA double strand breaks at 24 hours. In conclusion, simultaneously targeting the FA-pathway and interconnected DDR processes represents an appealing therapeutic strategy. Additionally, constitutive lack of FA pathway function in some tumours, could serve as a novel predictive biomarker for patient response to PARPi and ATRi currently in clinical trials.

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Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816539115 ◽

2018 ◽

Vol 115 (51) ◽

pp. E11961-E11969 ◽

Cited By ~ 14

Author(s):

Tai-Yuan Yu ◽

Michael T. Kimble ◽

Lorraine S. Symington

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Inhibitory Effects ◽

Checkpoint Signaling ◽

Synthetic Lethal ◽

Strand Breaks ◽

Damage Response ◽

End Resection

The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5′–3′ resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5′ strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1–mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.

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900 Depleting non-canonical, cell-intrinsic PD-L1 signals induces synthetic lethality to small molecule DNA damage response inhibitors in an immune independent and dependent manner

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.900 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A944-A944

Author(s):

Anand Kornepati ◽

Clare Murray ◽

Barbara Avalos ◽

Cody Rogers ◽

Kavya Ramkumar ◽

...

Keyword(s):

Ovarian Cancer ◽

Dna Damage ◽

Dna Repair ◽

Cell Lines ◽

Dna Damage Response ◽

Small Molecule ◽

Treatment Resistance ◽

Programmed Death ◽

Damage Response ◽

Response Biomarker

BackgroundTumor surface-expressed programmed death-ligand 1 (PD-L1) suppresses immunity when it engages programmed death-1 (PD-1) on anti-tumor immune cells in canonical PD-L1/PD-1.1 Non-canonical, tumour-intrinsic PD-L1 signals can mediate treatment resistance2–6 but mechanisms remain incompletely understood. Targeting non-canonical, cell-intrinsic PD-L1 signals, especially modulation of the DNA damage response (DDR), remains largely untapped.MethodsWe made PD-L1 knockout (PD-L1 KO) murine transplantable and human cell lines representing melanoma, bladder, and breast histologies. We used biochemical, genetic, and cell-biology techniques for mechanistic insights into tumor-intrinsic PD-L1 control of specific DDR and DNA repair pathways. We generated a novel inducible melanoma GEMM lacking PD-L1 only in melanocytes to corroborate DDR alterations observed in PD-L1 KO of established tumors.ResultsGenetic tumor PD-L1 depletion destabilized Chk2 and impaired ATM/Chk2, but not ATR/Chk1 DDR. PD-L1KO increased DNA damage (γH2AX) and impaired homologous recombination DNA repair (p-RPA32, BRCA1, RAD51 nuclear foci) and function (DR-GFP reporter). PD-L1 KO cells were significantly more sensitive versus controls to DDR inhibitors (DDRi) against ATR, Chk1, and PARP but not ATM in multiple human and mouse tumor models in vitro and in vivo in NSG mice. PD-1 independent, intracellular, not surface PD-L1 stabilized Chk2 protein with minimal Chek2 mRNA effect. Mechanistically, PD-L1 could directly complex with Chk2, protecting it from PIRH2-mediated polyubiquitination. PD-L1 N-terminal domains Ig-V and Ig-C but not the PD-L1 C-terminal tail co-IP’d with Chk2 and restored Chk1 inhibitor (Chk1i) treatment resistance. Tumor PD-L1 expression correlated with Chk1i sensitivity in 44 primary human small cell lung cancer cell lines, implicating tumor-intrinsic PD-L1 as a DDRi response biomarker. In WT mice, genetic PD-L1 depletion but not surface PD-L1 blockade with αPD-L1, sensitized immunotherapy-resistant, BRCA1-WT 4T1 tumors to PARP inhibitor (PARPi). PARPi effects were reduced on PD-L1 KO tumors in RAG2KO mice indicating immune-dependent DDRi efficacy. Tumor PD-L1 depletion, likely due to impaired DDR, enhanced PARPi induced tumor-intrinsic STING activation (e.g., p-TBK1, CCL5) suggesting potential to augment immunotherapies.ConclusionsWe challenge the prevailing surface PD-L1 paradigm and establish a novel mechanism for cell-intrinsic PD-L1 control of the DDR and gene product expression. We identify therapeutic vulnerabilities from tumor PD-L1 depletion utilizing small molecule DDRi currently being tested in clinical trials. Data could explain αPD-L1/DDRi treatment resistance. Intracellular PD-L1 could be a pharmacologically targetable treatment target and/or response biomarker for selective DDRi alone plus other immunotherapies.ReferencesTopalian SL, Taube JM, Anders RA, Pardoll DM. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer 16:275–287, doi:10.1038/nrc.2016.36 (2016).Clark CA, et al. Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis and autophagy in ovarian cancer and melanoma. Canres 0258.2016 (2016).Gupta HB et al. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer. 1, 16030 (2016).Zhu H, et al. BET bromodomain inhibition promotes anti-tumor immunity by suppressing PD-L1 expression. Cell Rep 16:2829–2837, doi:10.1016/j.celrep.2016.08.032 (2016)Wu B, et al. Adipose PD-L1 modulates PD-1/PD-L1 checkpoint blockade immunotherapy efficacy in breast cancer. Oncoimmunology 7:e1500107, doi:10.1080/2162402X.2018.1500107 (2018)Liang J, et al. Verteporfin inhibits PD-L1 through autophagy and the STAT1-IRF1-TRIM28 signaling axis, exerting antitumor efficacy. Cancer Immunol Res 8:952–965, doi:10.1158/2326-6066.CIR-19-0159 (2020)

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Androgen Receptor Inhibitor Enhances the Antitumor Effect of PARP Inhibitor in Breast Cancer Cells by Modulating DNA Damage Response

Molecular Cancer Therapeutics ◽

10.1158/1535-7163.mct-18-0234 ◽

2018 ◽

Vol 17 (12) ◽

pp. 2507-2518 ◽

Cited By ~ 6

Author(s):

Ahrum Min ◽

Hyemin Jang ◽

Seongyeong Kim ◽

Kyung-Hun Lee ◽

Debora Keunyoung Kim ◽

...

Keyword(s):

Breast Cancer ◽

Dna Damage ◽

Androgen Receptor ◽

Cancer Cells ◽

Dna Damage Response ◽

Breast Cancer Cells ◽

Antitumor Effect ◽

Parp Inhibitor ◽

Damage Response ◽

Receptor Inhibitor

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Phase II trial of the PARP inhibitor, niraparib, in BRCA1-Associated Protein 1 (BAP1) and other DNA damage response (DDR) pathway deficient neoplasms including cholangiocarcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.3_suppl.tps354 ◽

2021 ◽

Vol 39 (3_suppl) ◽

pp. TPS354-TPS354

Author(s):

Thomas J. George ◽

David L. DeRemer ◽

Ji-Hyun Lee ◽

Stephen Staal ◽

Merry Jennifer Markham ◽

...

Keyword(s):

Dna Damage ◽

Solid Tumors ◽

Dna Damage Response ◽

Group Performance ◽

Synthetic Lethality ◽

Parp Inhibitor ◽

Mutant Cell ◽

Objective Response ◽

Eastern Cooperative Oncology Group ◽

Damage Response

TPS354 Background: BRCA1-Associated Protein 1 (BAP1) is a critical regulator of the cell cycle, cellular differentiation, cell death, and DNA damage response. It also acts as a tumor suppressor. Preclinical models demonstrate significant synthetic lethality in BAP1 mutant cell lines and patient xenografts when treated with PARP inhibitors, independent of underlying BRCA status, suggesting this mutation confers a BRCA-like phenotype. BAP1 is mutated, leading to a loss of functional protein, in up to 30% of cholangiocarcinomas as well as several other solid tumors. Methods: This phase 2, open-label, single arm multicenter study aims to exploit the concept of synthetic lethality with the use of the PARP inhibitor niraparib in pts with metastatic relapsed or refractory solid tumors. Eligible pts with measurable metastatic and incurable solid tumors are assigned to one of two cohorts: Cohort A (histology-specific): tumors harboring suspected BAP1 mutations including cholangiocarcinoma, uveal melanoma, mesothelioma or clear cell renal cell carcinoma with tissue available for BAP1 mutational assessment via NGS or Cohort B (histology-agnostic): tumors with known DNA damage response (DDR) mutations (Table) confirmed by CLIA-approved NGS. Other key eligibility criteria include age ≥18 years, adequate cardiac, renal, hepatic function and Eastern Cooperative Oncology Group performance status of 0 to 1. Pts with known BRCA1 or BRCA2 mutations or prior PARPi exposure are excluded. Pts receive niraparib 200-300mg daily (depending on weight and/or platelet count) continuously. Primary endpoint is objective response rate with secondary endpoints of PFS, OS, toxicity and exploratory biomarker determinations. Radiographic response by RECIST criteria is measured every 8 weeks while on treatment. Cohort A has fully enrolled. Cohort B enrollment continues to a maximum of 47 total evaluable subjects with expansion cohorts allowable for histologic or molecular subtypes meeting pre-specified responses. NCT03207347 Clinical trial information: NCT03207347. [Table: see text]

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Mechanisms of PARP inhibitor resistance in cancer and insights into the DNA damage response

Genome Medicine ◽

10.1186/s13073-018-0612-8 ◽

2018 ◽

Vol 10 (1) ◽

Cited By ~ 27

Author(s):

Paola Francica ◽

Sven Rottenberg

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Parp Inhibitor ◽

Damage Response ◽

Inhibitor Resistance

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DNA-PKcs: A Multi-Faceted Player in DNA Damage Response

Frontiers in Genetics ◽

10.3389/fgene.2020.607428 ◽

2020 ◽

Vol 11 ◽

Author(s):

Xiaoqiao Yue ◽

Chenjun Bai ◽

Dafei Xie ◽

Teng Ma ◽

Ping-Kun Zhou

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cell Cycle Checkpoints ◽

Dna Double Strand Breaks ◽

Phosphatidylinositol 3 Kinase ◽

Strand Breaks ◽

Damage Response ◽

Functional Complex ◽

Dependent Protein ◽

Cellular Dna

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a member of the phosphatidylinositol 3-kinase related kinase family, which can phosphorylate more than 700 substrates. As the core enzyme, DNA-PKcs forms the active DNA-PK holoenzyme with the Ku80/Ku70 heterodimer to play crucial roles in cellular DNA damage response (DDR). Once DNA double strand breaks (DSBs) occur in the cells, DNA-PKcs is promptly recruited into damage sites and activated. DNA-PKcs is auto-phosphorylated and phosphorylated by Ataxia-Telangiectasia Mutated at multiple sites, and phosphorylates other targets, participating in a series of DDR and repair processes, which determine the cells’ fates: DSBs NHEJ repair and pathway choice, replication stress response, cell cycle checkpoints, telomeres length maintenance, senescence, autophagy, etc. Due to the special and multi-faceted roles of DNA-PKcs in the cellular responses to DNA damage, it is important to precisely regulate the formation and dynamic of its functional complex and activities for guarding genomic stability. On the other hand, targeting DNA-PKcs has been considered as a promising strategy of exploring novel radiosensitizers and killing agents of cancer cells. Combining DNA-PKcs inhibitors with radiotherapy can effectively enhance the efficacy of radiotherapy, offering more possibilities for cancer therapy.

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Recent advances in the nucleolar responses to DNA double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa713 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9449-9461

Author(s):

Lea Milling Korsholm ◽

Zita Gál ◽

Blanca Nieto ◽

Oliver Quevedo ◽

Stavroula Boukoura ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Ribosomal Dna ◽

Repetitive Sequences ◽

Dna Lesions ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

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CDK and Mec1/Tel1-catalyzed phosphorylation of Sae2 regulate different responses to DNA damage

Nucleic Acids Research ◽

10.1093/nar/gkz814 ◽

2019 ◽

Vol 47 (21) ◽

pp. 11238-11249 ◽

Cited By ~ 4

Author(s):

Tai-Yuan Yu ◽

Valerie E Garcia ◽

Lorraine S Symington

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Phosphorylation Site ◽

Physiological Role ◽

Checkpoint Signaling ◽

Strand Breaks ◽

Checkpoint Kinases ◽

Damage Response ◽

End Resection

Abstract Sae2 functions in the DNA damage response by controlling Mre11-Rad50-Xrs2 (MRX)-catalyzed end resection, an essential step for homology-dependent repair of double-strand breaks (DSBs), and by attenuating DNA damage checkpoint signaling. Phosphorylation of Sae2 by cyclin-dependent kinase (CDK1/Cdc28) activates the Mre11 endonuclease, while the physiological role of Sae2 phosphorylation by Mec1 and Tel1 checkpoint kinases is not fully understood. Here, we compare the phenotype of sae2 mutants lacking the main CDK (sae2-S267A) or Mec1 and Tel1 phosphorylation sites (sae2-5A) with sae2Δ and Mre11 nuclease defective (mre11-nd) mutants. The phosphorylation-site mutations confer DNA damage sensitivity, but not to the same extent as sae2Δ. The sae2-S267A mutation is epistatic to mre11-nd for camptothecin (CPT) sensitivity and synergizes with sgs1Δ, whereas sae2-5A synergizes with mre11-nd and exhibits epistasis with sgs1Δ. We find that attenuation of checkpoint signaling by Sae2 is mostly independent of Mre11 endonuclease activation but requires Mec1 and Tel1-dependent phosphorylation of Sae2. These results support a model whereby CDK-catalyzed phosphorylation of Sae2 activates resection via Mre11 endonuclease, whereas Sae2 phosphorylation by Mec1 and Tel1 promotes resection by the Dna2-Sgs1 and Exo1 pathways indirectly by dampening the DNA damage response.

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