bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A007: Ser/Thr kinase BUB1 stabilizes DNAPKcs in response to radiation induced DNA double strand break repair
Authors: Sriramulu Sushmitha , Thoidingjam Shivani , Brown Stephen , Siddiqui Farzan , Movsas Benjamin , Green Michael , Speers Corey , Nyati Shyam
Published: January 2024

Ser/Thr kinase BUB1 is known to regulate chromosomal segregation during mitosis. However, its exact role in DNA damage repair in response to radiotherapy remained elusive. Here, we demonstrate that inhibition of BUB1 kinase activity (BAY1816032) or genomic depletion (CRISPR) lead to radiation sensitization in triple negative breast cancer and lung cancer cell lines and in TNBC tumor xenograft models. Mechanistic studies demonstrated reduced DNA-DSB repair (γ-H2AX foci assay), and effect on the NHEJ pathway by the bioluminescent DNA repair reporter (BLRR). Gene expression analysis show that BUB1 expression correlates tightly with key members of the NHEJ machinery including H2AX, ATM, 53BP1, DNAPKcs, Ku70, Ku80, Lig4 among others. Cycloheximide-chase experiments demonstrate that BUB1 ablation (pharmacological or genomic) stabilizes DNAPKcs after irradiation. The half-lives (t1/2) of total-DNAPK, phospho-DNAPK could not be estimated in BUB1 ablated cells post radiotherapy. Conversely, we observed BUB1 stabilization following radiotherapy alone while pre-treatment with BUB1i or DNAPKi caused robust degradation of BUB1 (significantly shorter t1/2). Nuclear and chromatin fractionation assays revealed an increase in the phospho- and total- DNAPK, ATM, and KAP1 in nuclear fractions and in chromatin enriched fractions in BUB1 inhibited or depleted cells. There was no significant change in the levels of Ku70 and Ku80 in the nuclear fractions or in the chromatin enriched fractions. This data indicates that BUB1 may be involved in the activation and recruitment of key NHEJ proteins (ATM, DNAPKcs) to DSBs. Further experiments are underway to determine how BUB1 stabilizes DNAPKcs and regulate chromatin loading of key NHEJ factors following radiation. Additionally, we demonstrate that BUB1 mediate apoptotic cell death of cancer cells post-radiation. Our findings suggest that BUB1 may serve as a novel molecular target for radiosensitization in various solid cancers including TNBC.

Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Stephen Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. Ser/Thr kinase BUB1 stabilizes DNAPKcs in response to radiation induced DNA double strand break repair [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 A007.
Abstract A009: DNA damage-induced senescence as a driver of glioblastoma recurrence
Authors: Burma Sandeep , Tomimatsu Nozomi , Cristofaro Luis , Kanji Suman , Jordan Benjamin , Mukherjee Bipasha
Published: January 2024

Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR), yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. An understanding of mechanisms driving GBM recurrence is critical for improving therapy. During GBM therapy, both the tumor and the normal brain tissue surrounding the tumor are treated with up to 60 Gy of IR. IR is a potent inducer of senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting proteases, cytokines and growth factors that create a senescence-associated secretory phenotype (SASP). We have shown previously that IR-induced senescence of astrocytes in the brain leads to the secretion of SASP factors that promote the growth and invasiveness of tumor cells in mouse models of GBM (Fletcher-Sananikone, et al, Cancer Research, 2021). Following up on this study, we irradiated a panel of GBM cell lines and found that a significant fraction of these cells senesced rapidly in a p21-dependent manner. Senescent glioma cells upregulated SASP genes in a NF-kB-dependent manner, prominently, the interleukin IL6, which is an activator of the JAK-STAT pathway. Conditioned media from senescent GBM cells activated the JAK-STAT pathway in non-senescent GBM cells and promoted tumor cell proliferation and radiation resistance. Interestingly, conditioned media from senescent GBM cells could also activate the NF-kB pathway and induce SASP in non-senescent cells. These results indicate that senescent GBM cells can activate pro-tumorigenic pathways in their non-senescent counterparts and SASP can spread from senescent to non-senescent cells. Bioinformatic analyses of the transcriptomic profiles of irradiated GBM cells and the TCGA database revealed that the inhibitor of apoptosis protein cIAP2 is a critical survival factor for senescent glioma cells. We find that targeting cIAP2 using Smac mimetics triggers apoptosis of senescent GBM cells with minimal toxicity towards normal brain cells like astrocytes. Upregulation of cIAP2 in irradiated tumor cells was also seen in multiple PDX models of GBM. Using these PDX lines, we demonstrate that adjuvant senolytic treatment can delay or even prevent recurrence in pre-clinical mouse models of GBM. In sum, our findings illustrate how senescence of both stromal and tumor cells promote GBM recurrence via different mechanisms and underscore the potential utility of adjuvant senolytic therapy for blunting GBM recurrence after radiotherapy.

Citation Format: Sandeep Burma, Nozomi Tomimatsu, Luis Cristofaro, Suman Kanji, Benjamin Jordan, Bipasha Mukherjee. DNA damage-induced senescence as a driver of glioblastoma recurrence [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 A009.
Abstract A013: Selective HDAC6 inhibition by SP-2-225 enhances the innate anti-tumor immune response after radiation exposure
Authors: Grindrod Scott , Noonepalle Satish , Li Xingtang , Quiceno David , Jung Mira , Dritschilo Anatoly , Villagra Alejandro
Published: January 2024

Circulating tumor DNA fragments and other antigens resulting from radiation therapy cause activation of the STING pathway and initiate an inflammatory anti-tumor immune response. However, when pro-inflammatory immune cells interact with the tumor microenvironment, they are rapidly converted to their anti-inflammatory, pro-tumor state associated with wound healing. HDAC6 presents an important target due to its role in immune regulatory processes involved in antitumor immune responses. We have shown that SP-2-225, a selective inhibitor of HDAC6, regulates macrophages to recognize, phagocyte, and present antigens to the adaptive immune system. Treatment with SP-2-225 enhances the production of cancer-associated antigens and macrophage antigen cross-presentation to T cells. Through a non-cytotoxic mechanism, SP-2-225 significantly reduced the tumor volume in syngeneic SM1 melanoma and 4T1 breast models. Tumor analysis from treated animals showed a shift in the M1/M2 ratio of the infiltrating macrophages as compared to controls, demonstrating that SP-2-225 caused a pro-inflammatory, antitumor shift in the macrophage equilibrium present in the tumor microenvironment. The putative mechanism was identified through adoptive cell therapy where macrophages from naive animals were harvested and treated ex-vivo with SP-2-225. Reimplantation of these SP-2-225 ex-vivo treated macrophages resulted in reduced tumor volumes when compared to both vehicle and untreated macrophage animal cohorts. We propose that this selective inhibitor of HDAC6 offers potential in combination with radiation therapy for cancer.

Citation Format: Scott Grindrod, Satish Noonepalle, Xingtang Li, David Quiceno, Mira Jung, Anatoly Dritschilo, Alejandro Villagra. Selective HDAC6 inhibition by SP-2-225 enhances the innate anti-tumor immune response after radiation exposure [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 A013.
Abstract A017: Targeting Tlk2 impairs breast cancer growth and engages immune responses
Authors: Villamor-Payá Marina , Caballé Adrià , Smak Jordann , Benmebarek Mohamed-Reda , Attolini Camille Stephan-Otto , Stracker Travis H.
Published: January 2024

The Tousled like kinases 1 and 2 (TLK1 and TLK2) are evolutionarily conserved Ser/Thr kinases that are required for genome stability and normal development in numerous organisms. Both kinases contribute to histone deposition by targeting the histone chaperones ASF1A and ASF1B and are regulated by the DNA damage response (DDR). TLK1/2 expression is ubiquitous and often elevated in human cancers with suppressed immune gene expression. In some cancer types, elevated TLK levels correlate with decreased disease-free survival. In cultured cells, TLK depletion causes replication stress, DNA damage and altered chromatin maintenance. TLK1/2 depletion increases the efficacy of DNA damage checkpoint inhibitors and PARP inhibitors and induces the Alternative-lengthening of telomeres pathway and innate immune signaling. Collectively, this data suggests that TLK activity may be a druggable anti-cancer target. To test this, we generated a conditional Tlk2 allele in mice and introduced the Mouse Mammary Tumor Virus-Polyoma Middle Tumor-antigen (MMTV-PyMT) transgene to generate breast cancers. Deletion of Tlk2 in developing breast cancer resulted in impaired primary tumor growth and a complete inhibition of lung metastasis. Characterization of the tumor microenvironment revealed elevated immune infiltration that includes activated T cells with higher levels of PD-1, which could potentially sensitize these tumors to immune checkpoint blockade. To test this possibility, we established a syngeneic breast cancer model to allow us to combine Tlk2 depletion or deletion with DDR inhibitor and anti-PD-1 therapy in vivo. Preliminary data supports the strategy of targeting Tlk2 in cancer and we will share current data from tumor models and preliminary characterization of small molecule inhibitors.

Citation Format: Marina Villamor-Payá, Adrià Caballé, Jordann Smak, Mohamed-Reda Benmebarek, Camille Stephan-Otto Attolini, Travis H. Stracker. Targeting Tlk2 impairs breast cancer growth and engages immune responses [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 A017.
Abstract A024: Epigenetic control of topoisomerase 1 activity presents a cancer vulnerability
Authors: Lee Tae-Hee , Qiao Xuan C. , Wu Tongyu , Kuzin Vladislav , Zhou Xianzhen , Ramanarayanan Vijayalalitha , Baranello Laura , Oberdoerffer Philipp
Published: January 2024

Genome integrity is inherently at risk due to the torsional constraints imposed by DNA transactions. Topoisomerase 1 (TOP1) is essential for genomic stability by removing DNA supercoils generated during replication and transcription. Aberrant stabilization of TOP1:DNA cleavage complexes (TOP1ccs), however, result in DNA lesions that drive transcription-associated mutation burden and are a cytotoxic consequence of many clinically used chemotherapeutic agents. What protects genomic regions prone to elevated torsional stress from the cytotoxic potential of TOP1 enzymes remains unknown. Here, we identify macroH2A1.1, the only nucleosome component with an inherent ability to bind the TOP1cc repair-essential poly(ADP-ribose) (PAR) protein modification, as an essential means to establish a TOP1-permissive chromatin environment. MacroH2A1.1 is one of two alternatively spliced isoforms of the macro-histone variant macroH2A1, and comparative proteomics between the two isoforms uncovered a macroH2A1.1-specific and PARP activity-dependent interaction with the TOP1cc repair effector TDP1 as well as downstream base excision repair (BER) factors XRCC1 and LIG3. Consequently, macroH2A1.1 loss or inactivation of its PAR-binding domain resulted in impaired XRCC1 recruitment to damaged DNA and defective TOP1cc turnover, while the macroH2A1.2 splice isoform is unable to bind PAR or protect from TOP1ccs. Genome-wide mapping of macroH2A1.1, TOP1 and TOP1-DNA adducts identified macroH2A1.1 as a regulatory hub for TOP1 activity that ensures transcriptional integrity by preventing excessive TOP1cc accumulation at transcription start sites. Impaired macroH2A1.1 splicing, a frequent cancer feature, correlated with sensitivity to TOP1 poisons in a pharmaco-genomic screen in breast cancer cells, and macroH2A1.1 inactivation mirrored this effect. Finally, we find that aberrant macroH2A1 alternative splicing in cancer correlates with BER-related mutation signatures. We propose that macroH2A1 alternative splicing serves as an epigenetic modulator of TOP1-associated genome maintenance and a potential cancer vulnerability.

Citation Format: Tae-Hee Lee, Xuan C. Qiao, Tongyu Wu, Vladislav Kuzin, Xianzhen Zhou, Vijayalalitha Ramanarayanan, Laura Baranello, Philipp Oberdoerffer. Epigenetic control of topoisomerase 1 activity presents a cancer vulnerability [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 A024.
Abstract A028: Alcohol induces DNA damage and genomic instability in MCF-7 breast cancer cells and primary mammary tissues
Authors: Yang Xiaohe
Published: January 2024

Alcohol consumption has been associated with increased breast cancer risk. Previous studies indicated that alcohol-induced DNA damage plays a central role in carcinogenesis. However, the underlying molecular mechanisms remain unclear. This study aimed to examine the effect of alcohol exposure on key DNA damage/repair pathways in both in vitro and in vivo settings. When MCF-7 breast cancer cells were exposed to 0.1 ~ 0.8% alcohol, the treatment induced concentration-dependent DNA damage, as indicated by the increased 8-OHdG (8-hydroxy 2 deoxyguanosine) production and p-H2AX foci formation. Signal transduction analysis showed that alcohol induced DNA damage signals, including p53, p21, E2F-1 and CHK1, and the markers in DNA repair pathways, including XLF, Rad50 and DNA-PK. p53 knockdown in MCF-7 cells resulted in enhanced DNA damage and DNA repair responses, which underscores the role of p53 in alcohol-induced cellular regulation. We also demonstrated that alcohol induces sister chromatid exchanges (SCEs) in MCF-7 cells, and ALDH2 knockout enhanced alcohol induced SCEs, which suggests that alcohol-induced acetaldehyde (AA) is a key mediator in this process. Moreover, we examined the DNA damage markers in mammary tissues of FVB/N mice exposed to alcohol and showed that alcohol exposure in vivo induced similar patterns of DNA damage and repair responses. Taken together, our data highlights the contribution of alcohol-induced DNA damage in cellular injury and the consequent tumorigenesis, which provide fundamental support for further studies on alcohol-induced DNA damage and carcinogenesis.

Citation Format: Xiaohe Yang. Alcohol induces DNA damage and genomic instability in MCF-7 breast cancer cells and primary mammary tissues [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 A028.
Abstract A032: Identifying the binding partners of a long noncoding RNA involved in the DNA damage response
Authors: Correa Michele Ramos , Castillo Jonathan , Marconett Crystal N.
Published: January 2024

Lung adenocarcinoma (LUAD) is the most common histological subtype of the deadliest cancer in the United States. This cancer can arise from a high rate of mutations exaggerated by dysregulation in DNA damage and repair signaling that cause genomic instability. LUAD has largely been studied in the context of protein-coding genes, however, recent studies have implicated long noncoding RNAs (lncRNAs) as critical regulators of gene expression in the DNA damage response, controlling the repair mechanism response's magnitude, duration, and resolution. My lab has identified the lncRNA LINC00261 as a tumor suppressor in LUAD that regulates the activation of the DNA damage repair system; however, its exact mechanisms of action remain unknown. We found that the reintroduction of LINC00261 in LUAD is associated with G2/M cell cycle arrest and phosphorylation-mediated activation of key DNA damage response pathway components. We hypothesize that LINC00261 protects DNA fidelity through direct interaction with repair components and aim to obtain insights into the essential functions that LINC00261 plays in the DNA damage response. We observe that LINC00261 is differentially highly expressed upon increased DNA damage with UV-light and ionizing radiation alongside enhanced expression of DDR components like p53. We have conducted initial RNA-FISH and ChIRP-MS studies to characterize the molecular mechanisms of this lncRNA. We demonstrate that subcellular localization of LINC00261 is found to be distributed in both the cytoplasm and nucleus, inferring an ability to function in cis and in trans to interact with partners locally and genome wide. Initial ChIRP mass spectrometry pilot data shows significant enrichment for DNA replication and p53 pathway activity. These findings contribute valuable information to understanding the role of LINC00261 in the DNA damage response and have implications for how lncRNA-mediated dysregulation of DNA repair leads to genomic instability and oncogenic activation.

Citation Format: Michele Ramos Correa, Jonathan Castillo, Crystal N. Marconett. Identifying the binding partners of a long noncoding RNA involved in the DNA damage response [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 A032.
Abstract B004: Polymerase theta (Pol θ) is essential to repair a subset of DNA breaks in HR proficient cells
Authors: Smith Chelsea M. , Simpson Dennis , Feng Wanjuan , Gupta Gaorav
Published: January 2024

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.
Abstract B016: Developing diagnostic tools via mutational spectra analysis and multiomic plasma profiling from liquid biopsies to identify potential signatures in cancer in Trinidad and Tobago
Authors: Ramlachan Nicole , West Samuel M.
Published: January 2024

Cancer, especially lung cancer is a leading cause of nosocomial deaths worldwide. Non-small cell lung cancer (NSCLC) has a five-year survival rate of only 24%. Early diagnoses markedly improves survival with personalized cancer therapies using non-invasive techniques for asymptomatic detection of mutational spectrum-related by genotyping NSCLC patients, even prior to symptoms. Analysis of conformational change in proteins due to mutations in oncogenes allow for mutation-specific treatment with specialized cancer drugs. In addition, tumor mutational burden (TMB) is a promising predictive biomarker for cancer immunotherapy as high TMB patients respond better to immune checkpoint inhibitors cancer drugs. Treatment planning for non-small cell lung cancer generally requires testing for EGFR, ALK, ROS1, BRAF, MET, RET and KRAS gene alterations or whole genome analysis to assess TMB. The Caribbean region remains disadvantaged by the lack of available genetic testing mainly due to accessibility. We propose the genotyping of 30 stage III-IV NSCLC patients via next-generation sequencing (NGS) to improve personalized therapies. Through the analysis of tumor-specific alterations amongst a complete mutation spectrum analysis, including single nucleotide variants (SNVs), insertions, deletions, copy number variations (CNVs), and methylation alterations, we would generate data to be used as an accurate form of cancer genotyping for diagnostic purposes. We intend to include these genes as biomarkers in a mutational spectrum analysis, to be collated into a predictive DNA assay to be used for the first time as a diagnostic/prognostic tool in a medically underserved community. We intend to also identify TMB-high patients without cost-prohibitive whole genome sequencing by focusing on DNA damage repair (DDR) genes. There is an increasing utilization of agnostic testing which involves the analysis of all potentially actionable genes across all tumor and cancer types. The invention of genomic-tailored specialized cancer treatments has resulted in a spectacular improvement in disease outcomes for a significant portion of cancer patients, which we hope to offer to improve clinical outcomes and prognosis in the racially diverse population of Trinidad and Tobago.

Citation Format: Nicole Ramlachan, Samuel M. West. Developing diagnostic tools via mutational spectra analysis and multiomic plasma profiling from liquid biopsies to identify potential signatures in cancer in Trinidad and Tobago [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 B016.
Abstract B019: BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response
Authors: Jiang Haoyang , Zhang Tianpeng , Kaur Hardeep , Shi Tao , Krishnan Aravind , Kwon Youngho , Sung Patrick , Greenberg Roger
Published: January 2024

Alternative Lengthening of Telomeres (ALT) is a homology-directed repair (HDR) mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. ALT cells exhibit more endogenous replication stress and DNA damage at the telomere compared to ALT-negative cells. The Bloom Syndrome helicase (BLM) is critical for ALT and promotes replication stress and DNA damage specifically at telomeres in ALT cells. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication stress-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2 commensurate with the appearance of telomere C-strand specific single-stranded DNA. Moreover, DNA2 nuclease deficiency increased 5’-flap formation in a BLM-dependent manner, and BLM promoted ALT in response to telomere C-strand, but not G-strand, nicks. These findings define the seminal events in the ALT DNA damage response, linking aberrant lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers (Jiang et al. submitted). These findings will be discussed and how BLM activity is a key determinant of the specific vulnerability of ALT cells to FANCM inhibition, which is currently being developed to target ALT-dependent cancers.

Citation Format: Haoyang Jiang, Tianpeng Zhang, Hardeep Kaur, Tao Shi, Aravind Krishnan, Youngho Kwon, Patrick Sung, Roger Greenberg. BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response [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 B019.
Abstract B021: Assessing replication stress as an actionable therapeutic opportunity in chordoma
Authors: Punturi Nindo , Leung Wendy , Levy Joan , Zou Lee , Cote Gregory M. , Freed Dan M.
Published: January 2024

Chordoma is a rare bone cancer with a high rate of disease recurrence and no approved systemic therapies. Tumor profiling and functional genomics studies suggest a significant number of chordomas are characterized by molecular alterations associated with increased replication stress, and a dependence on replication stress response pathways. We confirm here in preclinical models that chordomas exhibit a replication stress phenotype, which creates an actionable therapeutic vulnerability exploitable by induction of replication stress and/or inhibition of the ATR-dependent replication stress response. A panel of chordoma cell lines was treated with gemcitabine or the ATR inhibitor (ATRi) elimusertib (BAY 1895344). In a subset of cell lines, gemcitabine or elimusertib demonstrated low-nanomolar EC50 values. Upon treatment with the ATRi ceralasertib (AZD6738), DNA fiber assays revealed a mild decrease in replication fork speed and a significant increase in fork asymmetry in cells sensitive to ATR inhibition, suggesting possible fork stalling or collapse. Alkaline and neutral comet assays were performed to determine the presence of genome-wide ssDNA and/or dsDNA breaks, respectively, with orthogonal evaluation of H2A.X activation during S-phase to assess DNA strand integrity after ATR and DNA synthesis inhibition. In a panel of chordoma PDX models, striking differential sensitivity to ATR inhibition was observed. In sensitive models, elimusertib (50 mg/kg, po, bidx3) promoted 85-90% tumor growth inhibition, in contrast to resistant models, where elimusertib treatment had no effect on tumor growth. PDX models sensitive to ATR inhibition were also highly sensitive to gemcitabine, further suggesting gemcitabine induces an ATR-dependent replication stress response in chordoma models. Indeed, combining gemcitabine with ATRi revealed exceptional synergy in chordoma cell lines. Collectively, our data indicate that a subset of chordomas are characterized by intrinsic replication stress that can be therapeutically exploited through the synergistic activity of gemcitabine and ATRi.

Citation Format: Nindo Punturi, Wendy Leung, Joan Levy, Lee Zou, Gregory M. Cote, Dan M. Freed. Assessing replication stress as an actionable therapeutic opportunity in chordoma [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 B021.
Abstract B022: C11ORF68 is essential for γH2AX formation after replication stress
Authors: Toh Clarissa Chin Min , Hoang Mai Phuong , Jeyasekharan Anand
Published: January 2024

Introduction: Unresolved replication stress (RS) often leads to genomic instability – a feature of many diseases including cancer. RS can activate downstream DNA damage response (DDR) phenotypes such as phosphorylation of histone H2AX (γH2AX). Therefore, understanding the molecular foundation of DDR is crucial for identifying potential biomarkers or therapeutic targets. Our lab has identified an uncharacterized protein that is symmetrically dimethylated by PRMT5 under hydroxyurea (HU) induced RS. Therefore, we hypothesize that C11ORF68 is involved in the RS and DNA damage response. Materials and methods: RS and DNA damaging agents including HU and etoposide were used to induce RS or DNA damage in various cell lines. C11ORF68 was depleted using siRNA and dicer siRNA (dsiRNA) systems. Immunofluorescence and immunoblotting were used to evaluate DDR phenotypes. Results: One of the earliest phenomenon in DDR is phosphorylation of histone H2AX (γH2AX). Depletion of C11ORF68 under HU-induced RS reduced γH2AX foci formation as compared to cells treated with control siRNA or dsiRNA. Since HU affects cells entering S-phase, we evaluated if this is a cell cycle-related effect by using other DNA damaging agents that target cells in all phases of the cell cycle. Similarly, depletion of C11ORF68 under DNA damaging conditions showed reduced γH2AX foci formation. This may be due to a lack of upstream PIKK kinases activation as knockdown of C11ORF68 also attenuated phosphorylation of PIKKs important for H2AX phosphorylation after treatment with DNA damaging agents and RS-inducing agents. Conclusions: Our data suggests that C11ORF68 it appears to modulate early DDR even before H2AX phosphorylation and upstream PIKK activation. We are currently investigating if C11ORF68 is involved in initial events after DNA damage or RS such as PARylation events or MRN sensing of DNA breaks.

Citation Format: Clarissa Chin Min Toh, Mai Phuong Hoang, Anand Jeyasekharan. C11ORF68 is essential for γH2AX formation after replication stress [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 B022.
Abstract B027: Preclinical efficacy of Tumor Treating Fields (TTFields) applied with PARP inhibitors or carboplatin in ovarian cancer
Authors: Martinez-Conde Antonia , Ene Hila , Ben-Meir Kerem , Frechtel-Gerzi Roni , Dor-On Eyal , Haber Adi , Giladi Moshe , Weinberg Uri , Palti Yoram
Published: January 2024

Purpose: Ovarian cancer is the leading cause of death among cancers of the female reproductive system. Surgery and platinum-based adjuvant chemotherapy followed by PARP inhibitor (PARPi) maintenance therapy is standard treatment for most patients diagnosed with ovarian cancer. The mechanism of action of PARPi and platinum-based therapy involves the induction of DNA damage, which requires the Fanconi Anemia (FA)- BRCA pathway for repair, making these agents most beneficial in cancers with BRCA mutations. However, 75% of patients with ovarian cancer have BRCA wild type tumors and therefore their benefit from PARPi is limited. TTFields are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cancer cell death. Recent research demonstrated TTFields induce a state of BRCAness in multiple cancer types, as well as anticancer effects in ovarian cancer preclinical models. This study investigated the impact of TTFields concomitant with carboplatin or PARPi in ovarian cancer cell lines. Methods: A2780 (BRCA wild type), OVCAR3 (BRCA mutated), and A2780cis (platinum-resistant) ovarian cancer cells were treated with TTFields (1 V/cm RMS, 200 kHz, 72 h), alone or concomitant with application of carboplatin or a PARPi (olaparib or niraparib). Efficacy assessments included cell count, colony formation, and apoptosis induction. The overall effect was calculated by multiplying the effects on cell count and colony formation. Results: TTFields treatment reduced cell count, increased overall effect, and elevated apoptosis. Carboplatin, olaparib, and niraparib exhibited dose-dependent effects in all cell lines. When TTFields were applied together with these drugs, a synergistic interaction was observed in the BRCA wild-type A2780 cells and platinum-resistant A2780cis cells, while an additive effect was noted in the BRCA mutant OVCAR-3 cells. Conclusions: This data implies potential advantages for TTFields concomitant with platinum-based chemotherapy and PARPi in ovarian cancer, even without underlying BRCA mutations. This aligns with the BRCAness state induced by TTFields and suggests a benefit in platinum resistant cancer. Overall, TTFields may augment the efficacy of ovarian cancer treatment in both adjuvant and maintenance settings.

Citation Format: Antonia Martinez-Conde, Hila Ene, Kerem Ben-Meir, Roni Frechtel-Gerzi, Eyal Dor-On, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Preclinical efficacy of Tumor Treating Fields (TTFields) applied with PARP inhibitors or carboplatin in ovarian cancer [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 B027.
Abstract B028: Preclinical effects of Tumor Treating Fields (TTFields) applied with cisplatin for treatment of cervical cancer
Authors: Frechtel-Gerzi Roni , Gerasimova Daria , Zeevi Einav , Monin Roni , Fishman Hila , Schlachet-Drukerman Inbar , Mumblat Helena , Martinez-Conde Antonia , Dor-On Eyal , Tzchori Itai , Haber Adi , Giladi Moshe , Weinberg Uri , Palti Yoram , Palmer Greg , Secord Angeles A.
Published: January 2024

Introduction: Though largely preventable, cervical cancer remains a significant health issue. Treatment of early-stage disease usually involves surgery; while systemic therapy is often required for advanced, recurrent, or metastatic cases. Most patients with advanced disease receive cisplatin-containing chemotherapy combinations in the first-line setting. TTFields are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. Concomitant treatment with TTFields and cisplatin has shown benefit in other tumor types. This in vitro study assessed the effects of TTFields in cervical cancer cells and explored their efficacy together with cisplatin. Methods: Cervical cancer cell lines were exposed to TTFields (0.5 V/cm for Ca Ski cells, 1 V/cm RMS for HeLa and SiHa cells; 72 h) at varying frequencies (100-400 kHz) followed by cell count assays. TTFields (200 kHz) were then applied with a range of cisplatin doses, and cell count, colony formation, and apoptosis were evaluated. Overall effect was calculated as the product of the relative reductions in cell count and colony formation. TTFields-treated cells were also examined for formation of γH2AX (a DNA damage marker) using fluorescent microscopy and for the expression of Fanconi Anemia (FA)-BRCA pathway proteins involved in repairing cisplatin-induced DNA damage via Western blot. Results: TTFields treatment reduced cell count in all cervical cancer cell lines tested. A frequency of 200 kHz was determined effective and used for all experiments. Cisplatin exhibited dose-response effects, which were augmented when TTFields were co-applied. TTFields also induced DNA damage in the cells and downregulated the expression of FA-BRCA pathway proteins. Conclusions: These preclinical findings suggest that TTFields should be explored as a potential treatment for cervical cancer. TTFields-induced downregulation of the FA-BRCA pathway creates a conditional vulnerability that may account for the enhanced efficacy seen when TTFields are applied together with cisplatin, providing mechanistic insight.

Citation Format: Roni Frechtel-Gerzi, Daria Gerasimova, Einav Zeevi, Roni Monin, Hila Fishman, Inbar Schlachet-Drukerman, Helena Mumblat, Antonia Martinez-Conde, Eyal Dor-On, Itai Tzchori, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti, Greg Palmer, Angeles A. Secord. Preclinical effects of Tumor Treating Fields (TTFields) applied with cisplatin for treatment of cervical cancer [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 B028.
Abstract IA007: Targeting the hypoxia-induced DDR to improve radiotherapy response
Authors: Hammond Ester
Published: January 2024

Regions of hypoxia (insufficient oxygen) occur to some extent in most if not all solid tumors. Within hypoxic regions the levels of oxygen are heterogeneous and can reach near anoxic levels, often referred to as radiobiological hypoxia. Cells experiencing radiobiological hypoxia are significantly more resistant to radiation-induced DNA damage and negatively impact radiotherapy response. A number of clinical studies have demonstrated that hypoxia is a significant indicator of poor patient response. Radiobiological hypoxia, unlike milder hypoxic conditions, leads to a robust DNA damage response although interestingly, this occurs in the absence of detectable DNA damage. Instead, the hypoxia-induced DDR has been attributed to the accumulation of replication stress. Previously, we demonstrated that hypoxia-induced replication stress originated from reduced nucleotide availability due to the oxygen-dependency of ribonucleotide reductase. More, recently we have implicated the accumulation of R-loops in contributing to not only the hypoxia-induced DDR but also transcriptional stress. Numerous factors play key roles in maintaining R-loops and preventing the accumulation of DNA damage including the DNA/RNA helicase SETX. Using SETX has an exemplar, we have demonstrated that targeting R-loop factors leads to increased DNA damage in hypoxic conditions. Currently, we are testing the hypothesis that hypoxia specific R-loop factors are potential therapeutic targets which offer the possibility of increasing the radiosensitivity of hypoxic cells.

Citation Format: Ester Hammond. Targeting the hypoxia-induced DDR to improve radiotherapy response [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 IA007.
Abstract IA009: Scars of faulty DNA repair in cancer whole genomes
Authors: Setton Jeremy , Powell Simon , Imielinski Marcin
Published: January 2024

Cancer genomes provide a record of the genetic alterations acquired from DNA damage and DNA repair defects during normal cell development and carcinogenesis. Genome-wide somatic alteration patterns in BRCA1-deficient (BRCA1d) and BRCA2-deficient (BRCA2d) cancers are attributed to a deficiency in homologous recombination (HR), a major pathway for the repair of double-strand breaks (DSBs) in human cells. Some of these mutational patterns may reflect specific error-prone repair mechanisms of DSB repair that cells use in the absence of HR. Such mutational patterns can provide biomarkers of HR-deficiency and help identify clinically relevant therapeutic vulnerabilities. HRD is has been associated with specific DNA rearrangements and cytogenetic aberrations. Paradoxically, the types of DNA rearrangements specifically associated with HR-deficient (HRD) cancers only minimally impact chromosomal structure. Addressing this, we combined a genome graph analysis of short-read whole genome sequencing (WGS) profiles across thousands of tumors with deep linked-read (LR) WGS of 46 BRCA1- or BRCA2-mutant breast cancers to discover a distinct class of HR deficiency-enriched rearrangements called reciprocal pairs. LR WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long molecule data. While one (cis) outcome corresponded to the copy and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10kb) duplications at each junction. We propose an HR-independent replication restart repair mechanism to explain the full spectrum of reciprocal pair outcomes. LR WGS additionally identified single-strand annealing (SSA) as a BRCA2-deficiency specific repair pathway in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- vs. BRCA2-deficient genomes. In conclusion, our data reveal classes of BRCA1- and BRCA2-deficiency specific rearrangements as a source of cytogenetic aberrations in HRD cells.

Citation Format: Jeremy Setton, Simon Powell, Marcin Imielinski. Scars of faulty DNA repair in cancer whole genomes [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 IA009.
Abstract IA015: Using systems approaches to interrogate DNA double strand break signaling and repair for optimal tumor cell killing
Authors: Kruswick Alex , Kooij Bert van de , Emmons Tiffany R. , Sriram Ganapathy , Martin Emma D. , Arvind Anjali , van Attikum Haico , Yaffe Michael B.
Published: January 2024

Repair of DNA double strand breaks (DSBs) occurs through multiple distinct repair pathways including canonical and microhomology-mediated/Pol Q-dependent end joining (c-NHEJ and alt-NHEJ), homologous recombination (HR), and single strand annealing (SSA). Exactly how a particular DSB dictates which pathway is selected for repair, and the extent to which different DSB repair pathways cross-talk with each other is incompletely understood, but appears to be influenced by the specific type of DNA ends, the extent of DNA resection, the cell state and stage of the cell cycle, and the chromatin context around the break. To simultaneously interrogate multiple DNA DSB repair pathways in a systems-based manner at the single cell level, we built upon the pioneering DSB repair reporter systems created by the Jasin, Stark, Scharenberg and Certo labs to create several new CRISPR-based DNA DSB reporter systems that readout the repair of a single genomic DNA DSB by error-free c-NHEJ, mutagenic c- and alt-NHEJ, HR or SSA using multi-color fluorescence-based imaging and flow cytometry approaches. Using these DSB reporter systems we show that competition between short-range and long-range end resection influences the choice between repair by HR versus SSA, demonstrate accepted and novel functions for a variety of DNA damage sensors and repair proteins in NHEJ and HR, and illustrate the feasibility of using this system to interrogate the effects of patient-specific mutations in DNA repair proteins on DNA DSB repair pathway choice. Finally, we show that DNA DSBs created by topoisomerase inhibitor treatment in live but DNA-damaged tumor cells are capable of activating anti-tumor immune responses by signaling through both canonical and non-canonical DNA break sensing and response pathways.

Citation Format: Alex Kruswick, Bert van de Kooij, Tiffany R. Emmons, Ganapathy Sriram, Emma D. Martin, Anjali Arvind, Haico van Attikum, Michael B. Yaffe. Using systems approaches to interrogate DNA double strand break signaling and repair for optimal tumor cell killing [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 IA015.
Abstract IA023: The role of RTF2 in replication and replication stress response
Authors: Smogorzewska Agata , Broton Cayla , Blobel Nicolas J. , Conti Brooke
Published: January 2024

The replisome faithfully coordinates the essential process of genome duplication despite genomic sequences or lesions that slow or stall replication fork progression, collectively termed replication stress. Such lesions threaten the accurate transmission of the genome; as such, cells have evolved many mechanisms of replication fork protection and repair to ensure that the genome is fully replicated within the confines of S phase. Our group has previously characterized the essential protein Replication Termination Factor 2 (RTF2) as one such facilitator of replication control during both unperturbed and stalled conditions. During unperturbed replication, RTF2 localizes the Ribonuclease H2 heterotrimer (RNase H2) to replication forks, where RNase H2 facilitates the removal of ribonucleotides aberrantly incorporated into the genome to ensure replication fork speeds and promote the completion of genome duplication. When the replisome encounters fork-stalling lesions, however, RTF2-RNase H2 are removed from the replisome through the action of proteasome shuttle proteins DNA Damage Inducible 1 and 2 (DDI1/2) to promote replication restart dependent upon PRIM1, the primase subunit of DNA polymerase α. Further, we find that cells depleted of RTF2 accumulate mitotic markers, including phosphorylated FOXM1 and cyclin B1, during S phase and exhibit abnormal nuclear structures. Together, these findings suggest that RTF2-deficient cells bypass the S/G2 checkpoint, in which ATR functions to suppress CDK1 activity and mitotic progression while replication is ongoing and implicate RTF2 as a mediator of replication checkpoint control.

Citation Format: Agata Smogorzewska, Cayla Broton, Nicolas J. Blobel, Brooke Conti. The role of RTF2 in replication and replication stress response [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 IA023.