Search

Prostate cancer is a heterogeneous disease whose progression is linked to genome instability. However, the impact of this instability on the noncoding genome and its three-dimensional organization to aid progression is unclear. Using primary benign and tumor tissue, we find a high concordance in higher-order three-dimensional genome organization. This concordance argues for constraints to the topology of prostate tumor genomes. Nonetheless, we identified changes in focal chromatin interactions, typical of loops bridging noncoding cis-regulatory elements, and showed how structural variants can induce these changes to guide cis-regulatory element hijacking. Such events resulted in opposing differential expression of genes found at antipodes of rearrangements. Collectively, these results argue that changes to focal chromatin interactions, as opposed to higher-order genome organization, allow for aberrant gene regulation and are repeatedly mediated by structural variants in primary prostate cancer. Significance: This work showcases how the noncoding genome can be hijacked by focal insults to its three-dimensional organization that contribute to prostate cancer oncogenesis.

Fanconi anemia is an inherited genome instability syndrome characterized by interstrand cross-link hypersensitivity, congenital defects, bone marrow failure, and cancer predisposition. Although DNA repair mediated by Fanconi anemia genes has been extensively studied, how inactivation of these genes leads to specific cellular phenotypic consequences associated with Fanconi anemia is not well understood. Here we report that Fanconi anemia stem cells in the C. elegans germline and in murine embryos display marked nonhomologous end joining (NHEJ)–dependent radiation resistance, leading to survival of progeny cells carrying genetic lesions. In contrast, DNA cross-linking does not induce generational genomic instability in Fanconi anemia stem cells, as widely accepted, but rather drives NHEJ-dependent apoptosis in both species. These findings suggest that Fanconi anemia is a stem cell disease reflecting inappropriate NHEJ, which is mutagenic and carcinogenic as a result of DNA misrepair, while marrow failure represents hematopoietic stem cell apoptosis. Significance: This study finds that Fanconi anemia stem cells preferentially activate error-prone NHEJ-dependent DNA repair to survive irradiation, thereby conferring generational genomic instability that is instrumental in carcinogenesis.

Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive TP53−/− and BRCA1−/− epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance. Significance: This study shows that BRCA1-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.

Chromosomal instability (CIN) comprises continual gain and loss of chromosomes or parts of chromosomes and occurs in the majority of cancers, often conferring poor prognosis. Because of a scarcity of functional studies and poor understanding of how genetic or gene expression landscapes connect to specific CIN mechanisms, causes of CIN in most cancer types remain unknown. High-grade serous ovarian carcinoma (HGSC), the most common subtype of ovarian cancer, is the major cause of death due to gynecologic malignancy in the Western world, with chemotherapy resistance developing in almost all patients. HGSC exhibits high rates of chromosomal aberrations and knowledge of causative mechanisms would represent an important step toward combating this disease. Here we perform the first in-depth functional characterization of mechanisms driving CIN in HGSC in seven cell lines that accurately recapitulate HGSC genetics. Multiple mechanisms coexisted to drive CIN in HGSC, including elevated microtubule dynamics and DNA replication stress that can be partially rescued to reduce CIN by low doses of paclitaxel and nucleoside supplementation, respectively. Distinct CIN mechanisms indicated relationships with HGSC-relevant therapy including PARP inhibition and microtubule-targeting agents. Comprehensive genomic and transcriptomic profiling revealed deregulation of various genes involved in genome stability but were not directly predictive of specific CIN mechanisms, underscoring the importance of functional characterization to identify causes of CIN. Overall, we show that HGSC CIN is complex and suggest that specific CIN mechanisms could be used as functional biomarkers to indicate appropriate therapy. Significance: These findings characterize multiple deregulated mechanisms of genome stability that lead to CIN in ovarian cancer and demonstrate the benefit of integrating analysis of said mechanisms into predictions of therapy response.

Aberrant activation of histone methyltransferase EZH2 and ribosome synthesis strongly associate with cancer development and progression. We previously found that EZH2 regulates RNA polymerase III–transcribed 5S ribosomal RNA gene transcription. However, whether EZH2 regulates ribosome synthesis is still unknown. Here, we report that EZH2 promotes ribosome synthesis by targeting and silencing a long noncoding RNA PHACTR2-AS1. PHACTR2-AS1 directly bound ribosome DNA genes and recruited histone methyltransferase SUV39H1, which in turn triggered H3K9 methylation of these genes. Depletion of PHACTR2-AS1 resulted in hyperactivation of ribosome synthesis and instability of ribosomal DNA, which promoted cancer cell proliferation and metastasis. Administration of PHACTR2-AS1-30nt-RNA, which binds to SUV39H1, effectively inhibited breast cancer growth and lung metastasis in mice. PHACTR2-AS1 was downregulated in breast cancer patients, where lower PHACTR2-AS1 expression promoted breast cancer development and correlated with poor patient outcome. Taken together, we demonstrate that PHACTR2-AS1 maintains a H3K9 methylation-marked silent state of ribosomal DNA genes, comprising a regulatory axis that controls breast cancer growth and metastasis. Significance: These findings reveal that EZH2 mediates ribosomal DNA stability via silencing of PHACTR2-AS1, representing a potential therapeutic target to control breast cancer growth and metastasis.

Efficient DNA damage repair (DDR) is crucial for a cell as aberrant repair can create deleterious mutations and genomic instability while unrepaired damage leads to cell apoptosis. Cells with altered DDR are more inclined to develop a variety of diseases, including cancers Cancer therapies including radiotherapy and chemotherapy rely on double strand break (DBS) formation to drive the killing of rapidly cycling tumor cells. As efficient DNA repair pathways can enable tumor cells to survive treatment-induced DNA damage, the identification of new actors participating in efficient DDR will help design alternative therapeutic approaches. Here we report that the well-known DNA sensor of innate immunity Stimulator of Interferon Genes (STING) is directly involved in the modulation of DSB repair efficiency in breast cancer cells. STING partly resides at the inner membrane of the nucleus where it interacts with core proteins of DNA-damage complexes and promotes its assembly with chromatin. STING silencing decreases DNA repair and cell viability, while STING overexpression protects cancer cells from genotoxic treatment. STING involvement in DNA repair is independent of the canonical STING-triggered pro-inflammatory response. In line with this observation, while overall STING expression level is positively correlated with favorable outcome in breast cancer patients, high STING expression in breast and ovarian cancer patients treated with adjuvant chemotherapy is associated with poor prognosis. This newly-identified function of STING elucidates a cell autonomous tumor-promoting mechanism complementary and independent from its well-documented role in tumor immunosurveillance. Citation Format: Laura Cheradame, Chiara Guerrera, Jean-Gabriel Judde, Vincent Goffin, Stefano Cairo. Nuclear STING localization induces chemoresistance in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-06.

Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. The clinical success of PARP inhibitors has highlighted the potential of targeting these dependencies therapeutically to induce synthetic lethality. We have developed novel RAD51 inhibitors to selectively target cancers that have elevated levels of DNA damage and/or reduced levels of DNA repair. RAD51 is a critical component of the homologous recombination (HR) pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown anti-cancer activity of RAD51 inhibitors in preclinical xenograft mouse models of lymphomas that have elevated rates of DNA mutations due to aberrant expression of Activation Induced Cytidine Deaminase (AID). Breast cancer, due to high rates of genomic instability, may potentially be treated in this fashion. Here we present the characterization of our RAD51 inhibitors in breast cancer. In a murine cell line model of metastatic breast cancer, 4T1, treatment with CYT-0851 resulted in a reduction in homologous recombination activity in a sister chromatid exchange (SCE) assay at a similar rate observed in a human derived cell line (HEK293T) suggesting conservation of mechanism in human and murine models. In the same cell line, treatment with CYT-0851 and a DNA damaging agent, carboplatin, resulted in cellular toxicity above what was observed with CYT-0851, indicating that RAD51 inhibition coupled with genomic instability is cytotoxic in this breast cancer model. PARP inhibitors have been shown to induce genomic instability as measured by an increase in HR activity in SCE assays. We therefore hypothesized that our RAD51 inhibitor could act as a sensitizer to PARP inhibitors. Combinatorial treatment of 4T1 cells with CYT-0851 and olaparib reduced homologous recombination activity in the SCE assay. Additionally, combination therapy led to increased accumulation of gH2AX foci after ionizing radiation, and increased tail moment on neutral Comet assay. Chou-Talalay synergy assay demonstrated synergistic activity between olaparib and CYT-0851 in 4T1. To determine potential drug synergy in human models, a matrix study was performed with CYT-0851 and olaparib in three cell lines of triple negative breast cancer (HCC1937, HCC1143, and BT20). Concentration-dependent synergy was observed in all cell lines tested. These data suggest that CYT-0851 may be active as a mono therapy and a combinatorial therapy with PARP inhibitors in breast cancer. Overall, we conclude that the synergy observed in vitro indicates significant potential for RAD51 inhibition in future cancer treatment strategies and warrants future exploration in vivo. Citation Format: Joey L Guy, Tyler Maclay, Melinda Day, Monika L Burness, Kevin Mills. RAD51 inhibition using CYT-0851, shows anti-cancer activity in cellular models of breast cancer and acts synergistically with PARP inhibitors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-05.

Triple negative breast cancer (TNBC) is highly proliferative and genomically unstable, making these tumors particularly sensitive to anti-proliferative chemotherapies. For nearly 20 years, inhibitors of cyclin/cyclin dependent kinases (CDK) that target proliferation have been investigated for the treatment of a myriad of neoplasms with limited success due to dose limiting toxicities as a result of generally targeting CDKs. Agents that can selectively target proliferation and mitosis within cancer cells without inducing systemic toxicity should greatly improve patient outcomes from TNBC. Recently, selective CDK7 inhibitors have entered clinical trials for solid tumors promising to diminish toxicity by primarily inhibiting CDK7. Due to resistance mechanisms that will inevitably manifest during treatment, understanding the pathways controlled by CDK7 inhibition is imperative. CDK7 forms a complex with MAT1 and cyclin H to phosphorylate serine 2 and 7 on the C terminal domain of RNA Polymerase II and promote pause release during transcription of TNBC driver genes. With selective covalent CDK7 inhibitors such as THZ1, RNA Polymerase II remains un-phosphorylated reducing the expression of genes necessary for mitosis, proliferation and viability. While this has been proposed to be due to inhibition of super-enhancer function, we show here that inhibition of CDK7 increases genomic instability of TNBC cell lines by promoting multi-nucleation and micro-nucleation. This is associated with an arrest at G2/M as well as the suppression of mitosis-related gene expression. Together, these data reveal that the mechanism of action of CDK7 inhibitors is similar to conventional anti-mitotics. Because resistance mechanisms frequently occur in cancer settings, we also generated two models of resistance to CDK7 inhibitors using the TNBC models, MDA-MB-231 and MDA-MB-468. We show the transporter ABCG2 can be up-regulated with the development of resistance and that inhibition of this transporter restores sensitivity to CDK7 inhibition. These data suggest that blockade of multi-drug resistance proteins such as ABCG2, may be useful for ensuring responsiveness to CDK7 inhibitors. Further analysis of gene expression changes occurring in response to such inhibitors should reveal additional key target genes that define therapeutic response in this disease. Understanding the full effects of CDK7 inhibition and resistance mechanisms may lead to a selective approach for targeting proliferation of TNBC and improving patient outcomes from this disease. Citation Format: Bryan M Webb, Melyssa Shively, Katrina Piemonte, Jennifer M Sahni, Ruth A Keri. Cyclin dependent kinase 7 ( CDK7) inhibition promotes genomic instability and mitotic catastrophe in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-04-01.

Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer that lacks effective targeted therapies with the main therapeutic option being cytotoxic chemotherapies, which have toxic side effects on patient’s health. Less toxic alternatives that could target key pathways that are altered in TNBCs could lead to better outcomes for the patients. Important distinguishing traits of TNBCs is the high amounts of genomic instability and high number of alterations in key proteins in DNA repair and cell proliferation that helps the cancer grow, survive and progress. Previous findings from our lab indicate that Imipramine Blue, a derivative of anti-depressant imipramine, is a potent inhibitor of proliferation and DNA repair pathways in TNBC. Therefore, we tested the therapeutic efficacy of Imipramine on TNBCs using various in vitro assays for viability, migration, invasion as well as in vivo xenograft assays for tumor growth. Our results demonstrate that Imipramine inhibits cancer cell viability and progression in vitro. Similarly, in vivo xenograft assays showed reduced tumor growth in pre-clinical models upon imipramine treatment. Importantly, at the molecular level, Imipramine treatment suppressed key players in both cell proliferation, and DNA repair pathways, including FOXM1; a proto-oncogene that is a master regulator of both cell proliferation and DNA Repair pathways. These findings suggest that Imipramine may be a potential therapeutic option for TNBC patients. A clinical trial is ongoing to test the efficacy of Imipramine in Triple Negative Breast Cancer patients. Citation Format: Santosh Timilsina, Subapriya Rajamanickam, Ismail Jatoi, Yidong Chen, Ratna Vadlamudi, Virginia Kaklamani, Manjeet K Rao. Imipramine, an FDA approved anti-depressant, suppresses triple negative breast cancer growth and progression through inhibition of FOXM1 and DNA repair pathways [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-05.

Chromosomal instability (CIN) is one of the major forms of genomic instability in various human cancers and is recognized as a common hallmark of tumorigenesis and heterogeneity. However, some malignant tumors show a paucity of chromosomal alterations, suggesting that tumor progression and evolution can occur in the absence of CIN. It is unclear whether CIN is stable between precursor lesions, primary tumor, and metastases or if it evolves during these steps. In this review, we describe the influence of CIN on the various steps in tumor initiation and development. Given the recognized significant effects of CIN in cancer, CIN-targeted therapeutics could have a major impact on improving clinical outcomes.

The role of transcriptional regulator ten-eleven translocation methylcytosine dioxygenease 1 (TET1) has not been well characterized in lung cancer. Here we show that TET1 is overexpressed in adenocarcinoma and squamous cell carcinomas. TET1 knockdown reduced cell growth in vitro and in vivo and induced transcriptome reprogramming independent of its demethylating activity to affect key cancer signaling pathways. Wild-type p53 bound the TET1 promoter to suppress transcription, while p53 transversion mutations were most strongly associated with high TET1 expression. Knockdown of TET1 in p53-mutant cell lines induced senescence through a program involving generalized genomic instability manifested by DNA single- and double-strand breaks and induction of p21 that was synergistic with cisplatin and doxorubicin. These data identify TET1 as an oncogene in lung cancer whose gain of function via loss of p53 may be exploited through targeted therapy–induced senescence. Significance: These studies identify TET1 as an oncogene in lung cancer whose gain of function following loss of p53 may be exploited by targeted therapy–induced senescence. See related commentary by Kondo, p. 1751

The cellular DNA replication stress response functions to stabilize DNA replication forks and inhibits genome instability and tumorigenesis induced by oncogenes. However, the specific proteins required for resolving oncogenic stress remain poorly understood. Here we report that Smarcal1 and Zranb3, closely related replication fork–remodeling proteins, have nonredundant functions in resolving Myc-induced DNA replication stress. In Myc-overexpressing primary cells, significant differences in replication fork stalling, collapse, and DNA damage were detected between cells deficient in Smarcal1 or Zranb3, leading to changes in proliferation and apoptosis. These differences were also reflected in Myc-induced lymphoma development; haploinsufficiency of Smarcal1 resulted in accelerated lymphomagenesis, whereas haploinsufficiency of Zranb3 inhibited lymphoma development. Complete loss of either protein resulted in disparate survival outcomes. Our results reveal that endogenous replication stress from Myc in primary cells requires both alleles of Smarcal1 and Zranb3 and demonstrate the requirement of both proteins to stabilize replication forks upon Myc dysregulation in a nonredundant manner. Significance: Smarcal1 and Zranb3 are essential, but nonredundant, for responding to DNA replication stress and stabilizing replication forks following Myc overexpression. See related commentary by Sotiriou and Halazonetis, p. 1297

Defects in the DNA damage repair system result in increased genomic instability and have recently been implicated as being drivers of tumorigenesis in both familial and sporadic breast cancers. To maintain genomic integrity, cells have a DNA damage response (DDR) mechanism that functions to repair damaged DNA efficiently and commits cells to death if damage is irreparable. Failure of this mechanism results in genomic instability and cancer predisposition. Widespread chromosomal instability is a characteristic feature of Triple-Negative Breast Cancer (TNBC), making it difficult to decipher between genes that drive cancer development from those that play a bystander role. Little is known about what gives rise to the extensive genomic instability of TNBC, and presents a major deficit in our scientific and clinical knowledge. Oncogene induced hyper-proliferation results in replication-associated double strand breaks (DSBs) that engage an Mre11-Rad50-Nbs1 complex-dependent DDR. Classically, the oncogene induced DDR is believed to suppress tumorigenesis due to downstream activation of p53. Using genetically engineered primary mammary epithelial cell models, we demonstrate p53-independent effects of the Mre11-dependent DDR in suppressing proliferation and DNA damage induced by diverse oncogenic drivers. Single cell whole genome sequencing in Her2/Neu expressing primary mammary epithelial cells reveals a landscape of stochastic copy number aberrations induced by oncogenic stress that becomes enriched for a genomic scar pattern of larger-size deletions in cells with Mre11 hypomorphism. We identify Mre11 pathway hypomorphism in a subset of basal-like breast cancers (BLBC), which confers vulnerability to specific DNA damaging agents and DDR inhibitors in murine models of p53-deficient BLBC. Thus, assessing the functional status of the Mre11-dependent DDR pathway in p53-mutant breast cancers may provide an opportunity for therapeutic exploitation. Citation Format: Fagan-Solis KD, Simpson DA, Kapustina M, Martelotto L, Reis-Filho JS, Petrini JH, Gupta GP. A p53-independent DNA damage response that regulates breast cancer phenotypes [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-06-08.

Triple negative breast cancer (TNBC) is highly proliferative and genomically unstable, making these tumors particularly sensitive to anti-proliferative chemotherapies. While efficacious, these drugs induce dose limiting toxicities. Agents that can selectively target proliferation within cancer cells without inducing systemic toxicity should greatly improve patient outcomes from TNBC. In this regard, we have found that TNBC cells are particularly vulnerable to suppression of the transcriptional regulator, LIN9, that controls cell cycle progression. LIN9 mRNA is overexpressed in 66% of TNBCs and is correlated with worse patient outcomes. Moreover, suppression of LIN9 expression induces multi-nucleation, micronucleation, mitotic catastrophe, and cell death and/or senescence. While transcription factors are generally considered “undruggable, LIN9 expression can be pharmacologically suppressed by blocking the activity of cyclin dependent kinase 7 (CDK7) with the selective, covalent CDK7 inhibitor, THZ1. CDK7 inhibitors inactivate RNA polymerase 2 and destabilize the super-enhancer mediated expression of oncogenes. Treatment of three TNBC cell lines (MDA-MB-231, MDA-MB-468, and HCC38) with THZ1 induces G2/M arrest and phenocopies genetic silencing of LIN9. Use of live cell imaging revealed that THZ1 increases the duration of mitosis and also leads to mitosis-associated cell death. Together, these data reveal that CDK7 inhibitors primarily inhibit TNBC growth by causing mitotic dysfunction and potentiating genomic instability by inducing micro- and multi-nucleation. In addition, they suggest that suppressing LIN9 expression by inhibiting CDK7 may lead to a selective approach for targeting proliferation of TNBC and improving patient outcomes from this disease. Citation Format: Webb BM, Sahni JM, Keri RA. Cyclin dependent kinase 7 (CDK7) inhibition with THZ1 induces mitotic failure and increases genomic instability in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-06.

Background: Genomic instability is one of tumor characteristics. Enhancing the DNA damage load or impairing the ability of DNA damage repair has been therapeutics of cancer. Breast cancer type 1 susceptibility gene (BRCA1) has been studied a lot about the DNA damage and repair in breast cancer and ovarian cancer. BRCA1 mutation increases the risk of developing breast cancer by an eighth to tenth. The C terminal domain of BRCA1 can associates with three proteins Abraxas, Bach1 and CtIP in a phosphorylation-dependent manner forming mutually exclusive complexes, namely BRCA1-A, B, and C complexes. The BRCA1-A complex is necessary in DNA damage repair, and study implicates the complex play roles in chemotherapy resistance. Methods: We explored tandem affinity purification mass-spectrometry (TAP-MAS) technique to identify potential subunits associated with NBA1(one component of BRCA1-A complex). Then we made the genome-editing CRISPR-Cas9 sgRNA library into lentivirus to infect U2OS cells. And 5 Gy dose of Ionizing radiation (IR) was used to induce DNA damage on the cell. After 14 days cultivation, we extracted the DNA from the cells, performed polymerase chain reaction (PCR) and analyzed the correlation between potential genes and DNA damage-repair passage by bioinformatics methods. We generated 200 breast cancer patient tissue samples in our cancer center and performed immunostaining assay. Results: By the TAP-MAS technique of NBA1 tagged with HA and Flag, we found 93 potential subunits except those known subunits in BRCA1-A complex. Combined with CRISPR-Cas9 sgRNA library screening, we scored the relativity of DNA damage and repair passageway of identified 93 potential subunits. We found that nucleoside-triphosphatase, cancer-related (NTPCR) as a new potential subunit of BRCA1-A complex (P value=0.0034) had the highest score. Endogenous and exogenous co-IP validated NTPCR physically associates with subunits within BRCA1-A complex. Besides, we found that NTPCR associated with the same domain of NBA1 as the other subunits in BRCA1-A complex. Immunohistochemistry of patient tissue samples indicated that high levels of NTPCR expression was correlated with poor prognosis in multivariate analysis (HR: 4.990; 95%CI: 1.433-17.378; p value: 0.012). Conclusion: NTPCR is a new subunit in BRCA1-A complex. And high expression of NTPCR is a negative prognostic factor in breast cancer patients. Citation Format: Zhao H, Jiang H, Sun W, Shao Z, Hu X. Combined tandem affinity purification mass-spectrometry technique with genome-editing CRISPR-Cas9 knockout screening to identify potential subunits in the BRCA1 complex [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-06-10.

We are constantly exposed to a variety of both external and internal DNA damaging agents, such as UV light from the sun and reactive oxygen species created as by-products of aerobic respiration. As a result, our DNA accumulates thousands of instances of damage per cell per day. DNA damage response (DDR) pathways, which include DNA repair and cell-cycle checkpoints, are responsible for the repair of DNA damage and are critical for protecting against mutagenesis and maintaining genome integrity. DNA double-stranded breaks (DSBs) are the most deleterious type of DNA damage and are repaired by one of two pathways: Non-homologous end-joining (NHEJ), an error-prone mechanism of repair active throughout the entire cell cycle, or homologous recombination (HR), considered to be an 'error-free' method for DSB repair that occurs in the S and G2 phases of the cell cycle. Deficiencies in NHEJ or HR can result in genomic instability via genomic incorporation of chromosomal aberrations, which can ultimately lead to an increased risk of cancer. However, in many cases, the mechanisms by which defects in these pathways lead to an increased risk of developing cancer is unknown, making preventative care and treatment of resulting cancers more difficult. Breast Cancer 1 (BRCA1), an established tumor suppressor, is a protein necessary for the proper repair of DNA DSBs through the HR pathway. Defects in BRCA1, whether genetically inherited or spontaneously developed, have been linked to different types of cancer in both men and women, including breast, ovarian, and pancreatic cancer. Yet, the regulation of BRCA1 in HR is not well understood and thus highlights a major a gap in our understanding of how deficiencies in HR contribute to the development of cancer. Our lab has discovered that SIRT2, a class III NAD+ dependent histone deacetylase and putative human tumor suppressor, plays a crucial role in the DDR and repair of DNA DSBs. We have shown that depletion of SIRT2 impairs HR and increases cell sensitivity to ionizing radiation in a deacetylase-dependent manner. A mass spectrometry analysis showed SIRT2 interacts with several proteins involved in DDR, including BRCA1. We validated the interaction between SIRT2 and BRCA1 and found SIRT2 deacetylates BRCA1 both in vitro and in cells. Depletion of SIRT2 and subsequent deacetylation of BRCA1 decreases BRCA1 protein levels in cells, impairing HR. Our results show SIRT2 is a novel regulator of BRCA1 and is critical for the repair of DNA DSBs through HR. These findings provide invaluable insights into how to exploit the interplay between SIRT2 and BRCA1 as a novel therapeutic approach for the prevention and treatment of cancer. Citation Format: Minten EV, Zhang H, Li C, Head PE, Yu DS. Regulation of BRCA1 by SIRT2 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-06-01.

The histone demethylase KDM4B is frequently overexpressed in various cancer types, and previous studies have indicated that the primary oncogenic function of KDM4B is its ability to demethylate H3K9me3 in different tumors, resulting in altered gene expression and genome instability. A genome-wide analysis to evaluate the effect of KDM4B on the global or local H3K9me3 level has not been performed. In this study, we assess whole-genome H3K9me3 distribution in cancer cells and find that H3K9me3 is largely enriched in long interspersed nuclear element-1 (LINE-1). A significant proportion of KDM4B-dependent H3K9me3 was located in evolutionarily young LINE-1 elements, which likely retain retrotransposition activity. Ectopic expression of KDM4B promoted LINE-1 expression, while depletion of KDM4B reduced it. Furthermore, KDM4B overexpression enhanced LINE-1 retrotransposition efficacy, copy number, and associated DNA damage, presumably via the histone demethylase activity of KDM4B. Breast cancer cell lines expressing high levels of KDM4B also exhibited increased LINE-1 expression and copy number compared with other cell lines. Pharmacologic inhibition of KDM4B significantly reduced LINE-1 expression and DNA damage in breast cancer cells with excessive KDM4B. Our study not only identifies KDM4B as a novel regulator of LINE-1, but it also suggests an unexpected oncogenic role for KDM4B overexpression in tumorigenesis, providing clues for the development of new cancer prevention strategies and therapies. Significance: The histone demethylase KDM4B promotes tumorigenesis by inducing retrotransposition and DNA damage.

Osteosarcoma is the most common primary bone malignancy, and the lung is the most frequent site of metastasis. The limited understanding of the tumoral heterogeneity and evolutionary process of genomic alterations in pulmonary metastatic osteosarcoma impedes development of novel therapeutic strategies. Here we systematically illustrate the genomic disparities between primary tumors and corresponding pulmonary metastatic tumors by multiregional whole-exome and whole-genome sequencing in 86 tumor regions from 10 patients with osteosarcoma. Metastatic tumors exhibited a significantly higher mutational burden and genomic instability compared with primary tumors, possibly due to accumulation of mutations caused by a greater number of alterations in DNA damage response genes in metastatic tumors. Integrated analysis of the architecture and relationships of subclones revealed a dynamic mutational process and diverse dissemination patterns of osteosarcoma during pulmonary metastasis (6/10 with linear and 4/10 with parallel evolutionary patterns). All patients demonstrated more significant intertumoral rather than intratumoral heterogeneity between primary tumors and metastatic tumors. Mutated genes were enriched in the PI3K–Akt pathway at both the early and late stages of tumor evolution and in the MAPK pathway at the metastatic stage. Conversely, metastatic tumors showed improved immunogenicity, including higher neoantigen load, elevated PD-L1 expression, and tumor-infiltrating lymphocytes than the corresponding primary tumors. Our study is the first to report the dynamic evolutionary process and temporospatial tumor heterogeneity of pulmonary metastatic osteosarcoma, providing new insights for diagnosis and potential therapeutic strategies for pulmonary metastasis. Significance: High-throughput sequencing of primary and metastatic osteosarcoma provides new insights into the diagnosis of and potential clinical therapeutic strategies for pulmonary metastasis.

Chromothripsis is a form of genome instability by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosomes. Widely assumed to be an early event in tumor development, this phenomenon plays a prominent role in tumor onset. In this study, an analysis of chromothripsis in 252 human breast cancers from two patient cohorts (149 metastatic breast cancers, 63 untreated primary tumors, 29 local relapses, and 11 longitudinal pairs) using whole-genome and whole-exome sequencing reveals that chromothripsis affects a substantial proportion of human breast cancers, with a prevalence over 60% in a cohort of metastatic cases and 25% in a cohort comprising predominantly luminal breast cancers. In the vast majority of cases, multiple chromosomes per tumor were affected, with most chromothriptic events on chromosomes 11 and 17 including, among other significantly altered drivers, CCND1, ERBB2, CDK12, and BRCA1. Importantly, chromothripsis generated recurrent fusions that drove tumor development. Chromothripsis-related rearrangements were linked with univocal mutational signatures, with clusters of point mutations due to kataegis in close proximity to the genomic breakpoints and with the activation of specific signaling pathways. Analyzing the temporal order of events in tumors with and without chromothripsis as well as longitudinal analysis of chromothriptic patterns in tumor pairs offered important insights into the role of chromothriptic chromosomes in tumor evolution. Significance: These findings identify chromothripsis as a major driving event in human breast cancer.

Heterozygous mutations in the BRCA1 gene predispose women to breast and ovarian cancer, while biallelic BRCA1 mutations are a cause of Fanconi anemia (FA), a rare genetic disorder characterized by developmental abnormalities, early-onset bone marrow failure, increased risk of cancers, and hypersensitivity to DNA-crosslinking agents. BRCA1 is critical for homologous recombination of DNA double-strand breaks (DSB). Through its coiled-coil domain, BRCA1 interacts with an essential partner, PALB2, recruiting BRCA2 and RAD51 to sites of DNA damage. Missense mutations within the coiled-coil domain of BRCA1 (e.g., L1407P) that affect the interaction with PALB2 have been reported in familial breast cancer. We hypothesized that if PALB2 regulates or mediates BRCA1 tumor suppressor function, ablation of the BRCA1–PALB2 interaction may also elicit genomic instability and tumor susceptibility. We generated mice defective for the Brca1–Palb2 interaction (Brca1 L1363P in mice) and established MEF cells from these mice. Brca1L1363P/L1363P MEF exhibited hypersensitivity to DNA-damaging agents and failed to recruit Rad51 to DSB. Brca1L1363P/L1363P mice were viable but exhibited various FA symptoms including growth retardation, hyperpigmentation, skeletal abnormalities, and male/female infertility. Furthermore, all Brca1L1363P/L1363P mice exhibited macrocytosis and died due to bone marrow failure or lymphoblastic lymphoma/leukemia with activating Notch1 mutations. These phenotypes closely recapitulate clinical features observed in patients with FA. Collectively, this model effectively demonstrates the significance of the BRCA1–PALB2 interaction in genome integrity and provides an FA model to investigate hematopoietic stem cells for mechanisms underlying progressive failure of hematopoiesis and associated development of leukemia/lymphoma, and other FA phenotypes. Significance: A new Brca1 mouse model for Fanconi anemia (FA) complementation group S provides a system in which to study phenotypes observed in human FA patients including bone marrow failure. See related commentary by Her and Bunting, p. 4044

The BRCA1–PALB2–BRCA2 axis plays an essential role in DNA homologous recombination repair, defect in which drives genome instability and cancer development. How cells with defects in this pathway respond to DNA damage in vivo and how tumors develop from these cells remain poorly defined. Here, we analyzed several aspects of the DNA damage response in multiple tissues of Palb2-mutant mice in which the interaction between PALB2 and BRCA1 is disengaged. Without any challenge, the mutant mice showed increased endogenous DNA damage. Following ionizing radiation, the mutant mice displayed higher levels of DNA breaks and stronger induction of p53 and p21, but continued DNA synthesis, reduced apoptosis, and accelerated tumor development. The differences in p21 induction, DNA synthesis, and apoptosis between wild-type and mutant mice were substantially more pronounced in the mammary gland than in the intestine, suggesting a potential contributing factor to the increased risk and the tissue specificity of BRCA/PALB2-associated tumor development. Moreover, the mutant mice showed higher levels of reactive oxygen species and constitutive activation of NF-κB, an antiapoptotic transcription factor inducible by both DNA damage and oxidative stress. Treatment of the mutant mice with an inhibitor of NF-κB reactivated apoptosis and delayed tumor development following radiation. Thus, our results also suggest a prosurvival and pro-oncogenic role of NF-κB in PALB2-mutant cells. Significance: This study explores novel tumor suppression mechanisms of the BRCA1-PALB2 DNA damage response pathway and implicates NF-κB activation as a protumorogenic event and possible therapeutic target. Cancer Res; 78(14); 3969–81. ©2018 AACR.

Loss of the short arm of chromosome 3 (3p) occurs early in >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiquitous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor suppressors as early drivers of tumorigenesis. We previously reported methyltransferase SETD2, which trimethylates H3 histones on lysine 36 (H3K36me3) and is located in the 3p deletion, to also trimethylate microtubules on lysine 40 (αTubK40me3) during mitosis, with αTubK40me3 required for genomic stability. We now show that monoallelic, Setd2-deficient cells retaining H3K36me3, but not αTubK40me3, exhibit a dramatic increase in mitotic defects and micronuclei count, with increased viability compared with biallelic loss. In SETD2-inactivated human kidney cells, rescue with a pathogenic SETD2 mutant deficient for microtubule (αTubK40me3), but not histone (H3K36me3) methylation, replicated this phenotype. Genomic instability (micronuclei) was also a hallmark of patient-derived cells from ccRCC. These data show that the SETD2 tumor suppressor displays a haploinsufficiency phenotype disproportionately impacting microtubule methylation and serves as an early driver of genomic instability. Significance: Loss of a single allele of a chromatin modifier plays a role in promoting oncogenesis, underscoring the growing relevance of tumor suppressor haploinsufficiency in tumorigenesis. Cancer Res; 78(12); 3135–46. ©2018 AACR.

Multinucleation is found in more than one third of tumors and is linked to increased tolerance for mutation, resistance to chemotherapy, and invasive potential. The integrity of the genome depends on proper execution of the cell cycle, which can be altered through mechanotransduction pathways as the tumor microenvironment stiffens during tumorigenesis. Here, we show that signaling downstream of matrix metalloproteinase-3 (MMP3) or TGFβ, known inducers of epithelial–mesenchymal transition (EMT), also promotes multinucleation in stiff microenvironments through Snail-dependent expression of the filament-forming protein septin-6, resulting in midbody persistence, abscission failure, and multinucleation. Consistently, we observed elevated expression of Snail and septin-6 as well as multinucleation in a human patient sample of metaplastic carcinoma of the breast, a rare classification characterized by deposition of collagen fibers and active EMT. In contrast, a soft microenvironment protected mammary epithelial cells from becoming multinucleated by preventing Snail-induced upregulation of septin-6. Our data suggest that tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Significance: These findings reveal tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Cancer Res; 78(9); 2277–89. ©2018 AACR.

We are reconstructing the dynamics of tumour growth, treatment response, and origins of resistance of the Breast International Group (BIG) 1-98 study to determine the evolutionary paths of non-responders. We established the somatic alteration landscape of primary tumours collected at surgery from BIG 1-98 patients using targeted DNA sequencing of 287 genes of FFPE samples from 538 patients. Combined analysis of genetic and clinical data indicates increased genomic instability; TP53 mutations and copy number variations of 11q13 and 8p11 are associated with poor prognosis. We developed an in silico simulation framework to investigate if the non-responder outcome is due to (over)-treatment or intrinsic to evolutionary selection prior to diagnosis. To reconstruct the evolutionary history of tumours, we created a computational multitype branching process that tracks the expansion of diverse clonal lineages as they acquire driver and passenger mutations that alter their proliferation and mutation rates. To account for the heterogeneity between patients, we created a fitting procedure based on the Cramer-von Misses statistic to find the likelihood of parameters of our computational model that recreate the mutational landscape and clinicopathological factors observed in each patient. Once the fitting procedure is done, we simulate the course of treatment following the study arm scheme accounting for the cell-cycle action mechanism of Tamoxifen and Letrozole. Using our tool, we are characterising the range of tumour development scenarios covering different degrees of aggressiveness and genomic instability. Our computational model allows simulation of diverse adjuvant-schemes to predict optimised treatment regimes for validation in patient-derived tumour xenograft mouse models. Citation Format: Lara-Gonzalez LL, Loi S, Ferrari D, Papenfuss A, Goode DL. Reconstructing the evolutionary paths of BIG 1-98 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-08-01.

Background: to determine the distribution and evolution of mutational processes in metastatic breast cancers (mBC), together with their clinical relevance Methods: Whole exome sequencing (Hi-Seq, Illumina) and determination of copy number alterations (CNA) (CGH array / SNP6.0) were performed in 240 and 692 metastatic breast cancers respectively. Mutational processes were defined according to Alexandrov (Nature, 2013). Homologous Recombination Deficiency (HRD) was determined by genome wide assessment of loss-of-heteroygosity (LOH) on SNP6.0 (n = 210). Finally, genomic instability was assessed by the % of genome altered assessed by CGH / SNP6.0 Results: Whole exome sequencing showed that HR+/Her2- metastatic breast cancer presented an increased contribution of APOBEC-related signatures, as compared to early breast cancer (TCGA) (58% of the mutations vs 31%, p < 0.0001). Twelve percent of the HR+/Her2- mBC acquired an hypermutator genotype ( > 200 non-synonymous mutations). This acquisition of an hypermutator genotype was confirmed in five paired primary-metastatic samples. An operational APOBEC-related signature 13 was associated with a poor outcome in a multivariate analysis (HR: 1.75, 95%CI: 1.1-2.7, p = 0.017). High LOH score (HRD) was observed in 30% of HR+/Her2- mBC as compared to 13% of early HR+/Her2- early BC (p < 0.0001). The opposite was observed in TNBC (43% in mTNBC versus 58% in early TNBC ,p = 0.032). High LOH score was associated with a trend for poor outcome in HR+/Her2- mBC (multivariate 1.67, 95%CI: 0.949-2.951, p = 0.075).The % of genome altered was associated with a poor outcome in multivariate analyses both in the overall and HR+/Her2- mBC (HR / 10 increase:1.144, 95%CI:1.038-1.261, p = 0.007 and HR:1.18, 95%CI:1.037-1.344, p = 0.012 respectively). Copy number analyses identified 143 genes that are more frequently amplified as compared to early breast cancers (FDR < 0.01) Conclusions: metastatic HR+/Her2- metastatic breast cancer present an increased in APOBEC-related mutational burden and in LOH score as compared to early breast cancers. APOBEC-related signature 13 and genome instability are associated with a poor outcome and could be used in the future to better stratify metastatic breast cancer patients. Citation Format: Patsouris A, Filleron T, Jacquet A, Goncalves A, Bonnefoi H, Letourneau C, Bachelot T, Jimenez M, Andre F. Mutational processes, genome evolution and outcome in metastatic breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD8-12.

Genomic instability is a hallmark of cancer which promotes oncogenic mutations in pancreatic ductal adenocarcinoma (PDAC), leading to more aggressive tumors with greater potential for drug resistance. Deregulation of the master transcription factor, MYC, is found in virtually all PDAC tumors and promotes genomic alterations by increasing replication stress, augmenting DNA repair pathways, and promoting cell survival. To resolve replication stress, stalled forks are trafficked to the nuclear pores where the necessary machinery accumulates. Activated MYC is spatially reorganized to the nuclear pores as well, a mechanism that is exacerbated in cancer. Whether MYC activity at the nuclear pores contributes to resolving replication stress in PDAC remains to be known. To investigate this mechanism, we are creating genomic and proteomic tools to determine MYC’s function at the nuclear pores during replication stress induced by olaparib treatment in PDAC cells. We found that olaparib significantly increases MYC accumulation at the nuclear pore in low-passage cell lines we established from PDAC patient tumors. Furthermore, we showed that MYC interacts with the nuclear pore resident SUMO protease, SENP1, which plays a key role in resolving stalled forks. Our preliminary data suggest a role for MYC resolving replication stress at nuclear pores, and could indicate MYC activity as a therapeutic target in olaparib-resistant PDAC. Citation Format: Gabriel M. Cohn, Colin J. Daniel, Daniel F. Liefwalker, Rosalie C. Sears. Studying MYC's contribution to replication stress at the nuclear pore [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-081.

Cytidine deaminase (CDA) converts cytidine and deoxycytidine into uridine and deoxyuridine within the pyrimidine salvage pathway for DNA and RNA synthesis. In this regard, loss of CDA provokes genomic instability in Bloom Syndrome, and CDA overexpression is associated with tumor resistance to chemotherapy with pyrimidine analogs. However, the precise role of CDA per se in cancer has been totally underexplored so far. Patient cohort analysis demonstrate that CDA is overexpressed in PDAC, a disease with no cure with increasing incidence, and associated with a worse prognosis in patient. Functional studies demonstrate that CDA is essential to PDAC cell proliferation and tumor growth. We found that CDA expression is associated with gene set enrichment in DNA replication signature, in both PDAC tumors and experimental models. Using enforced expression, genetic or pharmacologic targeting, we demonstrate that CDA promotes DNA replication, localizes to the replication fork and increases replication fork fitness. These effects are strictly dependent on CDA deaminase activity. Hence, we found that CDA controls replication stress as CDA expression is inversely correlated with the level of DNA breaks during S-phase, and that CDA targeting is associated with gene set enrichment in replication stress signature and CHK1 protein activation. Next, we demonstrate that CDA preserves genomic stability of PDAC cells, as CDA expression is inversely correlated with micronuclei formation and DNA damage transfer to daughter cells. We next explored PDAC cell lines from the Cancer Cell Lines Encyclopedia and found that CDA expression is associated with resistance to drug targeting DNA synthesis. Functional studies demonstrate that overexpressing CDA, and not CDA catalytically inactive mutant, protects PDAC cell lines from camptothecin, a topoisomerase inhibitor, while targeting CDA sensitizes PDAC cells to treatment by camptothecin and oxaliplatin, that forms platinum-DNA adducts. To gain further insights into the clinical potential of such finding, we targeted CDA expression in primary cells from patient with PDAC and found that CDA is essential to primary cell growth, and that CDA targeting sensitizes primary cells to treatment by oxaliplatin. Taken together, our results reveal for the first time that CDA controls DNA replication, replication stress level and genomic stability of PDAC cells, and that this new role of CDA is involved in tumor resistance to drugs inducing DNA damages. Thus, our work stems for new strategies based on CDA targeting to defeat PDAC resistance to treatment. Citation Format: Audrey Lumeau, Nicolas Bery, Cyril Ribeyre, Samad Elkaoutari, Guillaume Labrousse, Miguel Madrid-Mencia, Vera Pancaldi, Marie-Jeanne Pillaire, Valérie Bergoglio, Nelson Dusseti, Jean-Sébastien Hoffmann, Louis Buscail, Malik Lutzmann, Pierre Cordelier. Cytidine deaminase protects pancreatic cancer cells from replicative stress and drive response to DNA-targeting drugs [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-043.

Genomic instability and immune evasion are hallmarks of cancer. Apoptotic cancer stem cells can evade cell death by undergoing cellular transformation by constructing “blebbishields” from apoptotic bodies. In this study, we report a novel linkage between genomic instability and phagocytosis evasion that is coordinated by the blebbishield emergency program. Blebbishield emergency program evaded genomic instability checkpoint, expressed genomic instability–associated genes at distinct phases of cellular transformation, exhibited chromosomal instability, and promoted increase in nuclear size. Blebbishields fused with immune cells to evade phagocytosis, and the resultant hybrid cells exhibited increased migration, tumorigenesis, metastasis, red blood cell recruitment to tumors, and induced hepatosplenomegaly with signatures of genomic instability, blebbishield emergency program, and phagocytosis evasion to offer poor prognosis. Overall, our data demonstrate that the blebbishield emergency program drives evasion of chromosomal instability and phagocytosis checkpoints by apoptotic cancer stem cells. Cancer Res; 77(22); 6144–56. ©2017 AACR.

Pancreatic ductal adenocarcinomas (PDAC) harbor recurrent functional mutations of the master DNA damage response kinase ATM, which has been shown to accelerate tumorigenesis and epithelial–mesenchymal transition. To study how ATM deficiency affects genome integrity in this setting, we evaluated the molecular and functional effects of conditional Atm deletion in a mouse model of PDAC. ATM deficiency was associated with increased mitotic defects, recurrent genomic rearrangements, and deregulated DNA integrity checkpoints, reminiscent of human PDAC. We hypothesized that altered genome integrity might allow synthetic lethality-based options for targeted therapeutic intervention. Supporting this possibility, we found that the PARP inhibitor olaparib or ATR inhibitors reduced the viability of PDAC cells in vitro and in vivo associated with a genotype-selective increase in apoptosis. Overall, our results offered a preclinical mechanistic rationale for the use of PARP and ATR inhibitors to improve treatment of ATM-mutant PDAC. Cancer Res; 77(20); 5576–90. ©2017 AACR.

Sister chromatids are held together by cohesin, a tripartite ring with a peripheral SA1/2 subunit, where SA1 is required for telomere cohesion and SA2 for centromere cohesion. The STAG2 gene encoding SA2 is often inactivated in human cancer, but not in in a manner associated with aneuploidy. Thus, how these tumors maintain chromosomal cohesion and how STAG2 loss contributes to tumorigenesis remain open questions. Here we show that, despite a loss in centromere cohesion, sister chromatids in STAG2 mutant tumor cells maintain cohesion in mitosis at chromosome arms and telomeres. Telomere maintenance in STAG2 mutant tumor cells occurred by either telomere recombination or telomerase activation mechanisms. Notably, these cells were refractory to telomerase inhibitors, indicating recombination can provide an alternative means of telomere maintenance. STAG2 silencing in normal human cells that lack telomerase led to increased recombination at telomeres, delayed telomere shortening, and postponed senescence onset. Insofar as telomere shortening and replicative senescence prevent genomic instability and cancer by limiting the number of cell divisions, our findings suggest that extending the lifespan of normal human cells due to inactivation of STAG2 could promote tumorigenesis by extending the period during which tumor-driving mutations occur. Cancer Res; 77(20); 5530–42. ©2017 AACR.

Defects in genes crucial for the process of DNA repair by homology-directed DNA repair (HDR), such as BRCA1 and BRCA2, are well-known contributors to cancer pathogenesis as well as an Achilles' heel that can be exploited therapeutically. BRCA1/2-deficient cells are exquisitely sensitive to agents that stall replication forks, such as PARP inhibitors and platinating drugs, presumably due to the inability to repair double-stranded breaks that form as a consequence of replication fork collapse. BRCA1/2 also promote tolerance to DNA replication stress by protecting replication forks from nucleolytic degradation. Both biological endpoints involve the deposition of RAD51 onto single-stranded DNA (ssDNA) for homology searching and strand exchange during HDR repair, as well as protection of newly synthesized DNA from nucleolytic degradation (i.e., replication fork protection). In this issue of Cancer Research, Panzarino and colleagues performed multiple separation-of-function studies and identify the lesion most associated with intolerance to replication stress in BRCA1/2-deficient cells is persistent ssDNA gaps in newly synthesized DNA, resulting from a failure to restrain DNA replication. Mechanisms that suppress gap formation are closely associated with chemoresistance, and the authors’ findings challenge the paradigm that lack of HR repair or fork protection underlie the phenotype known as BRCAness. See related article by Panzarino et al., p. 1388

HLA LOH is an immune escape mechanism subject to microenvironmental selection pressures in tumor evolution. In previous study, the prevalence of HLA LOH in Chinese cancer patients has been characterized. However, the genomic features of patients harboring HLA LOH remains unknown. Methods The sequencing data of 1021 cancer genes from 1504 advanced patients across 11 diverse cancer types was analyzed to investigate the internal relationship between HLA LOH and genomic alteration. Exon 2, exon 3, and bilateral introns of HLA-A/B/C genes were well covered in the 1021-gene panel. HLA LOH was analyzed using the LOHHLA algorithm. Results Consistent with previous study, we found that the occurrence of HLA LOH is related to genomic instability. In colorectal cancer, the HLA LOH ratio of MSS samples was significantly higher than that of MSI-H samples. Among the remaining 10 cancer types, the trend was consistent (Table 1). These results revealed that HLA LOH is a potential immune escape mechanism for MSS patients. In NSCLC and BRCA, HLA LOH is associated with an elevated TMB (p Conclusions and Discussion Statistical results revealed that the occurrence of HLA LOH was related to genomic instability, including high TMB level and alterations in oncogenic signaling pathways, which may facilitate tumor formation. Overall, these findings may provide useful information for clinical practice and immunotherapy. The correlation between MSI stage and HLA LOHCancer TypesMSIHLA LOHHLA stablePercent with HLA LOHP valueColorectal cancerMSS617146.2%0.025MSI-H31416.7%Remaining 10 cancer typesMSS48549949.3%0.031MSI-H51525.0% Citation Format: Jian Zhao, Jie Hu, Xiuqin Zhang, Xuan Gao, Fan Tong, Xiaorui Fu, Yuting Yi, Yanfang Guan, Xuefeng Xia, Jian'an Huang. The genomic features of Chinese cancer patients harboring HLA LOH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1896.

Background: Clinical benefit of PARP inhibitors or Platinum-based chemotherapy has been observed and well proved in BRCA mutation patients. However, there are other mechanisms to induce recombination repair deficient (HRD), such as BRCA methylation, ATM expression loss; HRR gene mutation in addition to BRCA mutation. Whether these complicated HRD events or phenotype could be measured by a unified model to accurately identify HRD positive patients is important. Methods: In this study, we use support vector machine method to build genomic scar (GS) model to predict HRD events, which takes different types of chromosomal copy number into consideration to evaluate genomic instability. The accuracy of GS model was tested by different NGS platform from WGS to two captured SNP Panel. GS status association with Cisplatin-based chemotherapy was also preliminarily validated. Results: GS model displayed more than 95% accuracy to detect BRCA-ness events and is also able to enrich different types of HRR dysfunction, such as HRR-related mutation or BRCA1 promoter methylation. Moreover, GS model can identify patient benefits from Cisplatin-based chemotherapy, and GS score (GSS) high group had longer progression free survival (PFS) than GSS low group (11 vs 8.5 months, HR=0.5, P=0.05). Meanwhile, GSS showed the high concordance among different NGS platform including WGS; two captured SNP Panels, which implied the high robustness of GS model. Conclusion: GS was a robust model to predict HRD and had the clinical potential to enrich patients who will response to PARP inhibitors and/or Platinum-based chemotherapy. Citation Format: Hao Wen, Shuang Yang, Xuejun Chen, Ying Weng, Changbin Zhu, Li Ruan, Hua Dong, Xiaohua Wu. Genomic scar score: a robust model to predict recombination repair deficient based on genomic instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2044.

Metabolic reprogramming and genomic instability are two hallmarks of cancer known to drive tumorigenesis. While each has been investigated individually, there is a growing interest in their interplay during early cancer development. High expression of the cystine/glutamate antiporter SLC7A11 (xCT) is frequently observed in lung cancer. Our laboratory previously demonstrated that overexpressing xCT in normal bronchial airway epithelial cells induced metabolic reprogramming and caused cells to display precancerous properties, such as increased proliferation and colony formation in soft agar. In this study, we further investigated the effects of xCT overexpression in the airway epithelium with the goal of elucidating the role of xCT in influencing cancer-driving hallmarks and the interplay of these hallmarks during early tumorigenesis. We hypothesized that xCT overexpression-induced metabolic reprogramming leads to epigenetic changes through the perturbation of epigenetic-related metabolite levels. Furthermore, we posited that this altered epigenome changes the expression levels of key genes involved in genome stability maintenance, ultimately inducing genomic instability. Utilizing a LC-MS/MS-based metabolomic analysis, we observed significantly altered levels of 224 different metabolites in our xCT overexpressing cells, including metabolites involved in epigenetic processes such as S-adenosylhomocysteine and acetyl-CoA. We found that xCT overexpression alters several global histone modifications, increasing H3K9me3 and H3K9ac by 3.2 (P = 0.02) and 3.9-fold (P = 0.02) respectively. We also showed that xCT overexpression increased global DNA methylation by 56% (P = 0.04). Additionally, RNA-seq revealed significant alterations (logFC>2 or Citation Format: Dalton Hill, Jamshedur Rahman, Christien Kluwe, Jamey Young, Emily Hodges, Pierre P. Massion. xCT overexpression induces metabolic reprogramming, epigenetic alterations, and DNA damage in precancerous airway epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2350.

Genomic instability is a hallmark of most cancers that enables tumor cells to adapt and evolve, but also creates vulnerabilities that can be therapeutically exploited. Cancer therapies that target the DNA Damage Response (DDR) have proven to be efficacious across a broad range of cancer types, but often present a narrow therapeutic window and limited duration of response due to the development of resistance. Through mining our proprietary CRISPRomics® Oncology dataset we identified the ubiquitin protease, USP1, as an attractive target with activity in ovarian and triple negative breast cancers (TNBC). USP1 facilitates DNA repair via its role regulating the Fanconi anemia complex and translesion synthesis. We developed a series of potent, selective USP1 inhibitors to investigate the therapeutic potential of targeting USP1 in tumor settings dependent on those DNA repair processes. When profiled across a broad range of cancer cell line models, the USP1-inhibitor KSQ-4279 showed anti-proliferative effects in a subset of cell lines, often characterized by the presence of homologous recombination deficiencies (HRD), including mutations in BRCA1/2. To better understand the mechanism of action of KSQ-4279, we employed large scale functional genomic screens using KSQ-4279, olaparib, and cisplatin, in combination with a DNA-repair focused CRISPR library, to identify genetic determinants of sensitivity and anticipate modes of drug resistance. In addition to recovering genetic associations between KSQ-4279 and genes that modify USP1's direct substrates, our screens identified genetic interactions of KSQ-4279 with multiple DNA repair pathways, including genes involved in ubiquitin-mediated signaling events that regulate the response to DNA damage and replication stress. These studies revealed that the profile of resistance to KSQ-4279 is distinct from, and in some cases complementary to, other DNA damaging agents, and provide a mechanistic rationale for the use of KSQ-4279 in combination with other agents that target DNA repair. Consistent with the largely non-overlapping resistance profile of USP1 and PARP inhibitors, we found that the combination of KSQ-4279 with olaparib was able to induce strong and durable regressions across a number of ovarian and TNBC PDX models. Citation Format: Sol Shenker, Hugh Gannon, Alyssa Carlson, Paula Grasberger, Pamela Sullivan, Chris Middleton, Anne Dodson, Caroline Bullock, Michael McGuire, Erica Tobin, Kerstin Sinkevicius, Mike Schlabach, Frank Stegmeier, Louise Cadzow, Andrew Wylie. Functional genomic characterization of the USP1 inhibitor KSQ-4279 reveals a distinct mechanism of action and resistance profile relative to other DDR targeting drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1337.

Background: CDX models have emerged as tractable systems to explore mechanisms involved in metastatic progression and tumor-initiating properties of CTCs. However, their development is challenging due to low prevalence of CTCs in patient blood. We report the genomic and functional characterization of 4 NSCLC CDX models and their derived cell lines. We focus on genome and chromosomal instability (CIN) mechanisms operating in the CDX-derived cell lines and decipher relevant therapeutic targets. Methods: CTCs were enriched from 30mL blood samples of 58 advanced NSCLC patients and implanted subcutaneously into Nod/Scid-IL2Rγ-/- (NSG) mice. Tumors were palpable within a median of 108 days. Among the 4 CDX models established, 3 CDX-derived cell lines (GR-CDXL1, GR-CDXL3 and GR-CDXL4) were obtained. Cell lines were characterized by immunofluorescence (IF), immunohistochemistry and whole-exome sequencing (WES). CIN and DNA damage response (DDR) activity were evaluated using IF and western blot. Tumorigenic potential of CDX-derived cell lines was assessed in the chick embryo chorioallantoic membrane (CAM) and NSG mice engrafted intravenously. IC50 was assessed using CellTiter-Glo®. Results: All CDX models had an epithelial phenotype. CDX and cell lines recapitulated the corresponding tumor histological features (available for 3 patients). WES revealed multiple copy number alterations (CNAs) in driver genes implicated in diverse genome instability mechanisms. GR-CDXL1 presented BRCA2 mutation and FANCA promoter deletion, while AKT gain was detected in GR-CDXL3 in addition to whole genome doubling. This led us to investigate the DDR in the cell lines. Homologous recombination deficiency and unrepaired DNA damage post-mitosis were observed in GR-CDXL1. GR-CDXL3 presented numerical CIN and centrosome clustering. GR-CDXL4 showed high levels of DNA damage and CIN. CNA and functional analysis provided a biological rationale for the selection of drug candidates in pharmacological testings on the CDX-derived cell lines. The assays mirrored patients' response to chemotherapy and revealed olaparib efficiency in GR-CDXL1 and GR-CDXL4 cells, which was validated in the CAM. GR-CDXL3 cells were sensitive to phosphoinositide 3-kinase-α inhibitor alpelisib, suggesting tumor dependence on PI3K/AKT pathway. Cell lines were tumorigenic in the CAM and mice. Notably, GR-CDXL3 seeded multiple metastases, which is concordant with its distinguished CIN characteristics and WGD. In ovo and in vivo validation of candidate therapeutic strategies is ongoing and will be presented. Conclusion: This study reveals distinct genome and CIN patterns operating in our CDX-derived cell lines that may play a critical role in CTC seeding capacity. Our CDX models offer new tools for designing therapeutic strategies targeting metastatic progression in NSCLC. Citation Format: Tala Tayoun, Vincent Faugeroux, Marianne Oulhen, Emma Pailler, Olivier Deas, Laura Mezquita, Laura Brulle-Soumare, Stephano Cairo, Jean-Yves Scoazec, Virginie Marty, Agathe Aberlenc, Maud NgoCamus, Claudio Nicotra, David Planchard, Patricia Kannouche, Benjamin Besse, Jean-Gabriel Judde, Patrycja Pawlikowska, Françoise Farace. Patterns and dynamics of genome instability drive metastatic activity in non-small cell lung cancer (NSCLC) circulating tumor cell (CTC)-derived xenograft (CDX) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 595.

Unchecked growth of cancer cells places simultaneous demands on DNA replication and transcription. Targeting the transcription-replication conflict (TRC), a major source of genome instability, could have important therapeutic implications. However, testing this hypothesis is difficult without agents directly targeting TRC. Here, we report a small molecule ligand (AOH1996) of the replisome component PCNA, which stabilizes interaction between PCNA and the large subunit (rpb1) of RNA polymerase II through the APIM motif, resulting in proteasome-dependent degradation of rpb1 and lethal DNA damages. Orally administrable, AOH1996 suppresses tumor growth without discernable toxicity at more than 10 time the compound's effective dose. In addition, AOH1996 works synergistically with known DNA damage agents to kill cancer cells. These results reveal TRC as cancer-selective vulnerability and demonstrated the therapeutic potential of AOH1996. Citation Format: Long Gu, Caroline Li, Robert Lingeman, Robert J. Hickey, Linda H. Malkas. Pharmacological targeting of transcription-replication conflict leads to anti-cancer efficacy with minimal side effects in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1269.

As DNA is frequently subject to a wide array of molecularly distinct forms of damage, life has evolved multiple DNA repair and associated processes, collectively termed the DNA-damage response (DDR). The importance of DNA repair and other DDR mechanisms is underlined by their deregulation or loss causing cancer and various human genetic disorders. Work in my laboratory aims to decipher DDR mechanisms, particularly those triggered by DNA double-strand breaks. In my talk, I will begin by briefly summarising past work by me and my collaborators that led to the world's first registered DDR enzyme inhibitor drug - the PARP inhibitor olaparib/Lynparza. I will then describe some of our recent work using CRISPR-Cas9 genetic screens to discover new DDR factors/regulators, identify potential new avenues for anti-cancer therapy, and help us understand how cancer cells can evolve resistance to DNA-damaging chemotherapies, PARP inhibitors or other DDR enzyme inhibitors. Citation Format: Stephen P. Jackson. Exploiting genome instability for new cancer therapies: PARP inhibitors and beyond [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr PL06-03.

Background:The enzyme Poly(ADP-ribose) glycohydrolase (PARG) plays an important role during the DNA damage repair process through hydrolysis of poly(ADP-ribose)(PAR) chains and accounts for approximately 90% of dePARylation activity within the cell. PAR chain synthesis is achieved by poly(ADP-ribose) polymerases (PARPs), which use donor NAD+ molecules to link repeating ADP-ribose units. The resulting PAR chains serve as a platform to recruit DNA repair proteins that are critical for single-strand break (SSB) repair. Following DNA repair PARG hydrolyzes the linkages to break down PAR chains, completing the cycle. Recent studies also found that dePARylation is necessary for the translocation of DNA repair proteins such as XRCC1 to SSB suggesting a more direct role for PARG in DNA damage repair. PARP inhibitors (PARPi), demonstrate synthetic lethality in cells with impaired homologous recombination (HR)-mediated DNA repair. Clinical studies have shown that responses to PARPi are often accompanied by high rates of resistance, generating a need for additional therapies for patients with HR-deficient tumors. The ability of PARG inhibition (PARGi) to exacerbate replication deficiencies makes it a promising therapeutic target for a broad range of cancer types with genomic instability. We therefore sought out to evaluate the effects of PARGi in HR-deficient ovarian and breast cancer models. Methods: To understand the effects of PARGi in cell growth and survival, a panel of HR-deficient cell lines and cell line derived xenografts (CDX) were exposed to PARGi alone or in combination with DNA damage response inhibitors (DDRi). In these studies, PAR chain accumulation was assessed as a marker of target engagement and PARPi was used to assess on target effects in vitro. Results: Using cellular proliferation assays and xenograft models, we find that PARGi increases the cellular levels of PAR and significantly decreases the viability of HR-deficient cancer cell lines. Furthermore, inhibition of cell proliferation by PARGi is antagonized by PARPi, which is consistent with an on-target cellular mechanism of action (MOA). In conclusion, PARGi induces significant accumulation of PAR chains and decreases cell proliferation both in vitro and in vivo in HR-deficient tumor cells. Ongoing exploratory studies will further delineate the molecular underpinning of the relationship between HR-deficiency and PARGi. Citation Format: Monah Abed, Diana Munoz, Firoz Jaipuri, Nandini Ravindran, Vidya Seshadri, Marya Liimatta, Claire Neilan, Xin Linghu, An Nguyen, Marie-Claire Wagle, Zineb Mounir. Synthetic lethality of PARG inhibition in tumors with homologous recombination deficiencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2043.

Esophageal cancer is the 8th most common cancer worldwide and has the 6th highest mortality rate of all cancers. The 5-year survival rate following esophageal adenocarcinoma (EAC) diagnosis is dismal at less than 15 percent, indicating a dire need for improved therapeutic strategies and early detection. EAC develops stepwise following exposure to chronic gastric reflux: From pre-malignant Barrett's metaplasia, through stages of low- and high-grade dysplasia until developing into invasive cancer. Mutation or loss of common tumor suppressor genes TP53 and SMAD4 act as markers for cancer progression, occurring in high-grade dysplastic tissue and invasive EAC, respectively. Our novel in vivo tumorigenesis model demonstrates progression of Barrett's metaplasia to EAC, in which SMAD4-deficient Barrett's metaplasia cells form tumors in immunodeficient mice after a period of latency and in a dose-dependent manner. This delayed tumor growth onset suggests further drivers are required for oncogenesis, and these SMAD4-deficient cells and tumors display a greater degree of genomic instability than wildtype-SMAD4 controls. A genome-wide CRISPR-Cas9 knockout screen unveiled a synthetic lethal relationship between SMAD4-deficiency and cell cycle checkpoint inhibition, suggesting a role for SMAD4 in maintaining genomic stability and a potential novel therapeutic avenue for SMAD4-deficient EAC. Additionally, a concurrent in vivo CRISPR-Cas9 tumorigenesis screen produced tumors 4-fold faster than the previous model and identified regulators of mTOR signaling as co-operative drivers of tumorigenesis in EAC. Wildtype-SMAD4 cells failed to generate tumors despite undergoing the same genetic perturbations, indicating a potential gatekeeping effect of SMAD4 in mTOR-mediated EAC tumorigenesis. In sum, loss of SMAD4 acts as a double-edged sword, increasing genomic instability and thereby rendering EAC cells sensitive to cell cycle checkpoint inhibition, whilst simultaneously co-operating with modulated mTOR signaling to promote tumorigenesis in EAC xenograft models. Citation Format: Julia V. Milne, Jovana R. Gotovac, Kenji M. Fujihara, Cuong P. Duong, Wayne A. Phillips, Nicholas J. Clemons. SMAD4 as a potential gatekeeper for genomic instability and mTOR-mediated tumorigenesis in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2671.

Structural variations (SV), a hallmark of genomic instability in cancer can either activate oncogenes or inactivate tumor suppressor genes. SVs tend to be recurrent and have been associated with several cancer types. Next-generation sequencing (NGS) is mostly blind to large SVs, lacking sensitivity with false positive rates up to 89% in SV detection. Long-read, single-molecule sequencing platforms can address larger variations as they typically generate read lengths of tens of thousands of bases and have helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. However, the throughput and coverage offered by whole genome long read sequencing makes it infeasible to conduct large-scale genomic studies.Targeted sequencing significantly improves accuracy and coverage by offering the depth necessary to detect rare alleles in a heterogenous population of cells. Using the nanopore Cas9-targeted sequencing (nCATS), a PCR-free enrichment system from Oxford Nanopore Technologies, we targeted 10 prominent cancer genes in MCF 10A and SK-BR-3 breast cell lines. However, we observed that the number of reads generated for targets longer than 30kb were not sufficient to accurately call SVs. To further enhance this approach, we developed an Affinity-based Cas-9-Mediated Enrichment (ACME) step, that uses HisTag DynabeadsTM to pulldown non-target reads. With ACME we achieved ~ 75-fold enrichment of the BRCA2 region and 35-65x coverage of this ~90 kb target on our panel. We observed an increase in enrichment and coverage of other genes on the panel as well, with enrichment as high as 5000-fold for some genes. While ACME is a biochemical approach, recently, adaptive sequencing or “Read Until”, a computational approach that ‘rejects' non-target reads has been used by a couple of groups for target enrichment. Using UNCALLED, a variation of Read Until, we observed an ~10x coverage of our targets. Recognizing the potential and limitations of both, biochemical and computational approaches, our efforts now are directed towards a combined approach where we expect to achieve a minimum enrichment of 200-fold across all targets. With a 200-fold enrichment of a 3.5Mb target, we should get between 200x - 400x coverage of the target, which should be high enough to detect SVs in as low as 10% of the cells in the sample. This would support large scale SV analysis to help define the landscape of such variants in the population and identify regions of therapeutic or diagnostic interest. Citation Format: Shruti V. Iyer, Melissa Kramer, Sara Goodwin, W. Richard McCombie. Understanding genetic variation in cancer using nanopore targeted sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB201.

Coronavirus disease-19 (COVID-19). Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel coronavirus resulting in a severe threat to safety and public health. Genetic analyses suggest that SARS-COV-2 belongs to the Betacoronavirus group and Middle East respiratory syndrome coronavirus (MERS-CoV). Like most viruses, some are oncogenic, and genetic instability is involved in the initiating factor for tumor progression that is known to lead to malignancy, resulting in disease sequelae. Genomic instability is characterized by the increased rate of acquisition of alterations in the mammalian genome. These changes encompass a diverse set of biological end points including gene mutation and amplification, cellular transformation, clonal heterogeneity and delayed reproductive cell death. The loss of stability of the genome is becoming accepted as one of the most important aspects of viral induced carcinogenesis, and the numerous genetic changes associated with cancer cell implicate genomic stability as contributing to the neoplastic phenotype. The aim of this study is to analyze the influence of COVID-19 cancer related diseases and determine the epigenetic-genomic profiles in these diseases. In our experimental procedures, we will use oral epithelium cells as a model for this cancer study. We proposed a novel approach to study COVID-19-induced genetic changes with oral epithelium cells. We used these cells with COVID-19 co treatment to determine mechanisms that lead cells into crisis. Preliminary data using oral viral infected cells show that the viral infection can trigger certain gatekeeper proteins like p53 and pRB to be degraded and forcing cells to go into crises, and telomerase is turned on. In this study we will determine if COVID-19 transfected cells can follow the process using the Hayflick model, which show cells that are in crises, and some may progress to cancer. In summary, preliminary data show that viral infected cells that are none cancerous and are telomerase positive, may use a similar mechanism to send cells into crisis that turn telomerase on resulting in cellular malignancy. Citation Format: Eva McGhee, Mengtao LI, Yi-Ling Lin, Nefertari Carr, Chinelo Njubigbo, Rohun Sadeghi, Brooke Su-Velez, Julian Handler, Adin Handler, Judith Okoro, Sebhat Afework, Jay Vadgama. The hallmark of COVID-19 cancer disparities: Cellular sequelae [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 722.

Understanding the mechanistic basis for transition to late stage aggressive disease is vital for both assigning patient risk status in the localised setting and also identifying novel treatment strategies to prevent progression. Sub-regions of intratumoral hypoxia are found in all solid tumours and are associated with many biologic drivers of tumour progression. Crucially, more recent findings show the co-presence of hypoxia and genomic instability can confer a uniquely adverse prognosis in prostate cancer. In-depth informatic and functional studies suggests a role for hypoxia in co-operating with oncogenic drivers (e.g. loss of PTEN) and suppressing DNA repair capacity. To delineate a direct relationship between hypoxia and PTEN loss, we interrogated the impact of chronic hypoxia (1% O2, 0.2% O2; 72 h) on PTEN oncogenic signalling and chromosome instability. Quantitative immunofluorescence of centrosome aberrations, using antibodies to phospho-Histone H3 and Centrin-1, show significant increases in centriole numbers (p < 0.05) in mitotic cells under chronic hypoxic conditions and an increase in the incidence of micronuclei (p < 0.005) specifically in chronic hypoxic cells with PTEN loss. Hypoxic suppression of homologous recombination represents a "contextual lethal" vulnerability in hypoxic prostate tumours which could extend the application of existing DNA repair targeting agents such as poly-ADP ribose polymerase inhibitors. Additional mechanistic studies using hTERT-immortalized, primary prostate epithelial cells isogenic for c-MYC, BRCA2, or TP53/RB (SV40-transduced) 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) in selected clones by up to 30% in TP53/RB null cells. This finding is now being quantitated with a clonal evolution model and genotype-phenotype associations are being validated using functional transformation and invasion assays. Further investigation is now required to assess this relationship on the background of existing genomic alterations relevant to prostate and other cancers and also characterise the role of hypoxia in driving early metastatic spread. Supported by core funding from Cancer Research UK Manchester Institute and grants within the Cancer Research UK Manchester RadNet Unit, NIHR Manchester Biomedical Research Centre and a Prostate Cancer UK-Movember Centre of Excellence award. Citation Format: Robert G. Bristow. Genomic instability and hypoxia as co-drivers of prostate cancer aggression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY37-01.

Tumor mutational burden (TMB), defined as the number of somatic mutations per Megabase (Mb) in a neoplastic specimen measured by next-generation sequencing (NGS), is shown to increase its rate in association with higher genomic instability. Neoadjuvant chemoradiotherapy (NACRT) in locally advanced rectal adenocarcinoma (LARC) may be more active in genomically unstable tumors. However, response to NACRT is highly variable and pathological complete response is observed in only 15-25% of cases. Currently, the association of TMB with response to NACRT in LARC is unknown. The aim of this study was to investigate the role of TMB as a potential predictive biomarker of response in NACRT-treated LARC. We collected a set of pre-treatment biopsies from LARC cases, clinical stage II or III, undergoing NACRT with capecitabine combined with radiotherapy (N = 22). After DNA extraction and quality and concentration evaluation, we performed mutational profiling and assessed TMB through multigene sequencing of 409 cancer-related genes by NGS (OncomineTM Tumor Mutation Load Assay, Thermo Fisher Scientific). The most common mutations were, as expected, in KRAS, TP53 and PIK3CA. We compared our results with those of The Cancer Genome Atlas (TCGA) database and proportion was similar, with no statistically significant differences. We found three cases with TMB over 10 mutations/Mb, a threshold above which neoantigen formation occurs with higher likelihood and response to NACRT may be more efficient due to the stimulation of immune system through radiotherapy. Two cases harbored deleterious mutations in genes involved in DNA damage repair (PMS2 and NBN). Furthermore, cases with mutations in the transcription factor FBXW7 (N = 4/22) and in the receptor tyrosine kinase ERBB3 (N = 3/22) had significantly higher TMB than non-mutated ones (p-value = 0.0090 and 0.0107 respectively, false discovery rate < 0.1 for both genes). We compared TMB in major/complete responders vs. minor/non-responders as evaluated by post-surgical pathological examination (responders if Dworak tumor regression grading score, TRG, was equal to 3-4 and non-responders if TRG was equal to 0-1) and investigated the possible association of higher TMB with better response to NACRT. In our cohort we found a trend for higher TMB in responder vs. non-responder cases (5.08 mutations/Mb, IQR 3.40-9.29 vs. 4.25 mutations/Mb, IQR 2.54-5.5) (p-value = 0.05, one-sided). In conclusion, our study shows that higher TMB is correlated with a better response to NACRT in LARC and can be found in cases presenting with defects in DNA damage repair as well as enriched for specific gene mutations, notably in the receptor tyrosine kinase ERBB3. Citation Format: Martina Dameri, Gabriella Cirmena, Anna Garuti, Lorenzo Ferrando, Maurizio Gallo, Shirin Djahandideh Sheijani, Ester Cecchella, Edoardo Isnaldi, Roberto Murialdo, Valentina Barbero, Lucia Tixi, Federica Grillo, Luca Mastracci, Ciro Marrone, Andrea Massobrio, Stefano Scabini, Alberto Ballestrero, Gabriele Zoppoli. High tumor mutational burden subtends better response in locally advanced rectal adenocarcinoma undergoing neoadjuvant chemoradiotherapy and is associated with mutations in DNA damage repair genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB032.

Hypomethylation of repetitive sequences is a common feature reported in cancer, particularly of retrotransposon elements. Retrotransposon elements can be deleterious due to their potential as insertional mutagens, so cells have deployed several control mechanisms to silence them through methylation of DNA. These methylation pathways are different between somatic and germline cells. In germline cells, the piRNA pathway is the main process responsible for silencing the retrotransposon LINE-1. GTSF1 is germline cell specific and an important part of this pathway. Along with PIWI proteins in the piRNA pathway, GTSF1 processes small RNA molecules and identifies active retrotransposons, recruiting the DNA methylation machinery to those sites. Aberrant expression of GTSF1 has been reported in cancer, particularly on lymphomas. We performed a Bayesian Analysis and Markov Chain Monte Carlo Method with GTSF1 levels of expression reported on The Cancer Genome Atlas (TCGA), which confirmed high levels of mRNA in lymphomas. Nonetheless, the role of GTSF1 in retrotransposon control in cancer and in carcinogenesis remains unknown. To address this issue, we have performed shRNA mediated knockdown in head and neck squamous cell carcinoma and cutaneous T-cell lymphoma cell lines, and we have overexpressed the cDNA of GTSF1 in immortalized keratinocytes. We evaluated the impact of the knockdown and overexpression on cell biology, retrotransposons, and genomic instability. Overall, GTSF1 expression seems to affect LINE-1 retrotransposon protein expression and function. Also, we are assessing the impact on genomic instability and methylation profiles. The aberrant expression of this gene poses an excellent clinical opportunity for targeted therapy development. Its germline cell restricted expression will potentially result in less secondary effects than current available therapies. Citation Format: Amelia Martínez Villarreal, Jennifer Gantchev, Ivan Litvinov. The role of GTSF1 as a regulator of retrotransposons and its impact on carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2415.

While our knowledge of the evolutionary features of primary tumors has grown exponentially over the last few years our understanding of evolution of metastases is more limited and many open questions remain, regarding the timing of metastatic dissemination, the acquisition of metastatic competence and the underpinning of the variety of metastatic phenotypes observed in the clinic from latent metastases to solitary and oligo-metastases to widespread metastases. We have leveraged unique translational protocols that allow interrogation of tumor evolution from the earliest stages of diseases to death to address some of these questions. In the context of renal cell cancers we show that the mode of evolution at the primary tumor site determines the tempo and distribution of systemic metastases; that the features that distinguish metastasis-competent clones included genome instability and aneuploidy; and that metastatic competence often emerges in the centre of the primary tumor. In the context of melanoma we observe a wide spectrum of metastatic seeding that includes organ-specific metastatic clones, monophyletic and polyphyletic seeding and polyclonal mixing at metastatic sites. We observe progressive increase in aneuploidy and occurrence of whole genome doubling as melanoma metastases progress and resist treatment suggesting this as a a mechanism of immune-evasion and treatment resistance. Citation Format: Samra Turajlic. Understanding evolution of metastatic disease in renal cancer and melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY02-02.

Background: Previous study indicated that >80% of osteosarcomas (OSs) have genomic instability characteristics similar to BRCA1/2-deficient cancers. Such BRCA-like traits could manifest a deficiency in the DNA damage repair (DDR) pathway. Patients with alterations in DDR pathway was reported sensitive to platinum-based chemotherapy and PARP inhibitors. In order to seek more precision therapeutic opportunities, we explored the molecular profile of Chinese OS patients to identify whether the analogous phenomena exists as reported. Material and Methods: 44 archival Chinese OS patients from 3D Medicines and a public dataset including 35 OS patients (download from cBioPortal), were collected and analyzed next-generation sequencing (NGS) results on tumor tissues for gene mutations, copy number alterations (CNAs) and fusions in 33 cancer-associated DDR genes. Simultaneously, 44 Chinese OS patients' tumor analysis generated homologous recombination deficiency (HRD) scores (defined as the unweighted sum of LOH, TAI and LST scores) through the 733 gene panel sequencing. Results: Among 44 Chinese OS patients, 18 (40.9%) of OS had DDR pathway gene somatic alterations, and TP53 (31.8%) were the most frequently mapped gene. Other DDR somatic alterations were identified in BRCA2 (6.8%), BARD1 (4.5%), BRCA1 (2.3%), PMS2 (2.3%), FANCA (2.3%) and CHEK1 (2.3%). It was worth mention that all gene alterations were copy number reduction except TP53. Collectively, the frequencies of HRR and mismatch repair (MMR) alterations were 15.9% and 2.3%, respectively. HRD score were available in 18 patients, the median was 40.5 (range, 0-81). As to public dataset, 6 (17.1%) patients harbored somatic alterations in DDR genes. The variations were BRCA2 (5.7%), ATR (2.9%), BARD1 (2.9%), FANCC (2.9%), MLH1 (2.9%), MSH6 (2.9%) and PMS2 (2.9%). The HRR mutations frequency was 11% and MMR was 8.6%. Conclusions: This is the first large-scale comprehensive genomic instability characteristics analysis for Chinese patients with OS. Chinese patients and public dataset patients covers many common features. The DDR gene alteration frequency and HRD score are higher in OS patients, the association of variations in DDR genes and HRD score could be considered substitutable in predicting platinum-based therapy and PARP inhibitor response in clinical practice. Citation Format: Yonggang Wang, Hongtao Li, Jing Xia, Shiqing Chen, Yuezong Bai. Comprehensive genomic instability characteristics analysis in Chinese patients with osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2050.

BACKGROUND. Fanconi Anemia (FA) is a rare disease characterized by congenital abnormalities, bone marrow failure and cancer susceptibility. The pivotal role of the FA pathway is DNA interstrand crosslink (ICL) repair, where FANCD2-FANCI complex monoubiquitination is the key regulatory step. Recent studies have linked defects in the FA pathway to R-loop metabolism and implicated R-loops as an endogenous source of genomic instability that contributes to the FA phenotype, although the mechanism remains elusive. Splicing factors, such as SRSF1 (ASF/SF2), have also been linked to R-loops, cancer, and myelodysplastic syndrome (MDS), so we hypothesized a link between SRSF1 and FA. METHODS. Cells: FA-D2 mutant (PD20) and corrected (D2), HeLa. siRNA transfections were used to reduce SRSF1 and FANCD2 protein levels. Survival assays were performed to determine cell sensitivity to DNA damaging agents. In vitro immunoprecipitation was performed using SRSF1 purified from bacteria and FANCD2-FANCI purified from insect cells. R-loops were studied using the Damage At RNA Transcription (DART) assay and S9.6 immunofluorescence. RNA FISH was used to measure mRNA export. RESULTS. In order to study the involvement of splicing factors in the FA pathway, we knocked down SRSF1 by using siRNA. SRSF1 knockdown leads to impaired FANCD2 monoubiquitination after DNA damage as well as diminished cell survival, thus creating a FA-like phenotype. In addition, immunoprecipitation in cells and in vitro showed direct interaction between FANCD2 and SRSF1. We found that FANCD2 and SRSF1 knockdown leads to increased R-loops visualized by S9.6 immunofluorescence. Besides, DART assay revealed colocalization of SRSF1 and FANCD2 with R-loops in highly transcribed genomic sites, known to be R-loop hotspots. Additionally, we found that SRSF1 interacts with the mRNA export factor NXF1 in a FANCD2-dependent fashion and through RNA. Interestingly, SRSF1-NXF1 interaction is enhanced upon DNA damage. Finally, we found that FANCD2-deficient cells present defects in mRNA export, suggesting that SRSF1 and FANCD2 could cooperate in the prevention of R-loop formation by exporting accumulated RNA in the nucleus. CONCLUSION. Our findings suggest a novel non-canonical mechanism of FANCD2 via regulation of SRSF1 in R-loop prevention via facilitation of mRNA export, thus preventing genomic instability. Citation Format: Anne Olazabal-Herrero, Allison M. Green, Xiaoyong Chen, Patrick Sung, Pillai M. Manoj, Gary M. Kupfer. Binding of FANCD2 to SRSF1 splicing factor prevents genomic instability through R-loop regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2046.

Background: Tumors with infiltrating lymphocytes (TIL) demonstrate a better prognosis particularly in TNBC and HER2 positive breast cancer. Two competing hypothesis predict contrasting relationships of TILs and genomic heterogeneity. On one hand, a strong immune response may lead to “pruning” of intratumor heterogeneity by eliminating immunogenic clones resulting in a near equilibrium, hence better prognosis, while cancers that escape the surveillance may evolve towards greater clonal heterogeneity and genomic complexity. In some cancers, the predicted neoantigens are less frequent than expected by chance also suggesting immune mediated elimination of neoplastic clones (Rooney et al. 2015). Studies also showed an inverse association between immune cell infiltration and intratumor clonal heterogeneity (Morris et al. 2016). On the other hand, cancers with greater genomic instability and mutational burden will have larger clonal heterogeneity and therefore more neoantigens and greater immune infiltration. Indeed, a positive correlation between overall mutation load and immune activity in the tumor microenvironment was observed in pooled data across a broad range of cancer types (Brown et al. 2014, Rooney et al. 2015, Schumacher and Schreiber 2015). Methods: We assessed these two competing hypothesis and examined the relationship between genomic complexity and immune gene expression in different breast cancer subtypes. We used previously described immune metagene expression (DNA microarray n=655) as measures of immune infiltration in the TCGA data set (RNA-Seq n=1215). We compared somatic mutations, mutation count, neoantigen load, clonal heterogeneity metrics and the distribution of mutations in 119 canonical cancer genes and 12 cancer pathways between good and poor prognosis TNBC (n=208) corresponding to high and low immune infiltration. Results:A positive but weak correlation between mutation count and immune metagene expression was observed when all breast cancer subtypes were analyzed together (P=0.08). This was driven by the generally higher mutation count and immune infiltration in TNBC. When TNBC was analyzed separately, good prognosis TNBC with high immune infiltration had lower total mutation count (P=0.021) and predicted neo-antigen count (P=0.035). Clonal heterogeneity was also lower in good prognosis TNBC (P=0.001). There was a strong inverse relationship of dispersion in mutation variant allele frequencies and immune metagene expression. CASP8 was the top enriched mutation in TNBC with high immune infiltration (P=0.007 with no adjustment for multiple testing). Conclusions:High immune infiltration is associated with reduced intratumor heterogeneity in TNBC suggesting immune sculpting of the tumor and a near equilibrium between the cancer and immune surveillance. Surgical resection of the primary tumor may tilt the balance towards the immune system resulting in the better prognosis of high-TIL TNBC. TNBC with low immune infiltration has greater clonal heterogeneity and mutation load and may represent the consequence of escape from immune surveillance. Mutation of CASP8 may be one way to evade tumor cell killing in high-TIL TNBC as previously noted. Citation Format: Karn T, Jiang T, Hatzis C, Sänger N, El-Balat A, Holtrich U, Becker S, Bianchini G, Pusztai L. Immune sculpting of the triple negative breast cancer genome [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-07.

There remains great interest in practical strategies to limit the elevated risks of familial breast and ovarian cancers driven by BRCA1 mutation. Here, we report that limiting the production of reactive oxygen species (ROS) is sufficient to reduce DNA lesions and delay tumorigenesis in a murine model of BRCA1-deficient breast cancer. We documented a large amount of endogenous estrogen oxidative metabolites in the mammary gland of the model, which induced DNA adducts and apurinic/apyrimidinic sites associated with DNA double-strand breaks and genomic instability. Repressing estrogen oxidation via antioxidant treatments reduced oxidative DNA lesions and delayed the onset of mammary tumors. Overall our work suggests an answer to the long-standing question of why germline BRCA1 mutations cause tissue-specific tumors, in showing how tissue-specific, ROS-induced DNA lesions create a nongenetic force to promote mammary tumors in BRCA1-deficient mice. Our findings create a rationale for evaluating suitable antioxidant modalities as a chemopreventive strategy for familial breast cancer. Cancer Res; 77(2); 448–58. ©2016 AACR.