Replication Stress Research Articles

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

Chemo-Phosphoproteomic Profiling with ATR Inhibitors Berzosertib and Gartisertib Uncovers New Biomarkers and DNA Damage Response Regulators

The ATR kinase protects cells against DNA damage and replication stress and represents a promising anti-cancer drug target. The ATR inhibitors (ATRi) berzosertib and gartisertib are both in clinical trials for the treatment of advanced solid tumors as monotherapy or in combination with genotoxic agents. We carried out quantitative phospho-proteomic screening for ATR biomarkers that are highly sensitive to berzosertib and gartisertib, using an optimized mass spectrometry pipeline. Screening identified a range of novel ATR-dependent phosphorylation events, which were grouped into three broad classes: (i) targets whose phosphorylation is highly sensitive to ATRi and which could be the next generation of ATR biomarkers; (ii) proteins with known genome maintenance roles not previously known to be regulated by ATR; (iii) novel targets whose cellular roles are unclear. Class iii targets represent candidate DNA damage response proteins and, with this in mind, proteins in this class were subjected to secondary screening for recruitment to DNA damage sites. We show that one of the proteins recruited, SCAF1, interacts with RNAPII in a phospho-dependent manner and recruitment requires PARP activity and interaction with RNAPII. We also show that SCAF1 deficiency partly rescues RAD51 loading in cells lacking the BRCA1 tumor suppressor. Taken together these data reveal potential new ATR biomarkers and new genome maintenance factors.

FANCD2 counteracts O6-methylguanine-induced mismatch repair-dependent apoptosis.

Sn1-type alkylating agents methylate the oxygen atom on guanine bases thereby producing O6-methylguanine. This modified base could pair with thymine and cytosine, resulting in the formation of O6-methylguanine/thymine mismatch during DNA replication, recognized by the mismatch repair (MMR) complex, which then initiates the DNA damage response and subsequent apoptotic processes. In our investigation of the molecular mechanisms underlying MMR-dependent apoptosis, we observed FANCD2 modification upon the activity of alkylating agent N-methyl-N-nitrosourea (MNU). This observation led us to hypothesize a relevant role for FANCD2 in the apoptosis induction process. We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci. Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.

DBF4, not DRF1, is the crucial regulator of CDC7 kinase at replication forks.

CDC7 kinase is crucial for DNA replication initiation and is involved in fork processing and replication stress response. Human CDC7 requires the binding of either DBF4 or DRF1 for its activity. However, it is unclear whether the two regulatory subunits target CDC7 to a specific set of substrates, thus having different biological functions, or if they act redundantly. Using genome editing technology, we generated isogenic cell lines deficient in either DBF4 or DRF1: these cells are viable but present signs of genomic instability, indicating that both can independently support CDC7 for bulk DNA replication. Nonetheless, DBF4-deficient cells show altered replication efficiency, partial deficiency in MCM helicase phosphorylation, and alterations in the replication timing of discrete genomic regions. Notably, we find that CDC7 function at replication forks is entirely dependent on DBF4 and not on DRF1. Thus, DBF4 is the primary regulator of CDC7 activity, mediating most of its functions in unperturbed DNA replication and upon replication interference.

CHK1 inhibitor induced PARylation by targeting PARG causes excessive replication and metabolic stress and overcomes chemoresistance in ovarian cancer

Chemoresistance contributes to the majority of deaths in women with ovarian cancer (OC). Altered DNA repair and metabolic signaling is implicated in mediating therapeutic resistance. DNA damage checkpoint kinase 1 (CHK1) integrates cell cycle and DNA repair in replicating cells, and its inhibition causes replication stress, repair deficiency and cell cycle dysregulation. We observed elevated Poly-ADP-ribosylation (PAR) of proteins (PARylation) and subsequent decrease in cellular NAD+ levels in OC cells treated with the CHK1 inhibitor prexasertib, indicating activation of NAD+ dependent DNA repair enzymes poly-ADP-ribose polymerases (PARP1/2). While multiple PARP inhibitors are in clinical use in treating OC, tumor resistance to these drugs is highly imminent. We reasoned that inhibition of dePARylation by targeting Poly (ADP-ribose) glycohydrolase (PARG) would disrupt metabolic and DNA repair crosstalk to overcome chemoresistance. Although PARG inhibition (PARGi) trapped PARylation of the proteins and activated CHK1, it did not cause any significant OC cell death. However, OC cells deficient in CHK1 were hypersensitive to PARGi, suggesting a role for metabolic and DNA repair crosstalk in protection of OC cells. Correspondingly, OC cells treated with a combination of CHK1 and PARG inhibitors exhibited excessive replication stress-mediated DNA lesions, cell cycle dysregulation, and mitotic catastrophe compared to individual drugs. Interestingly, increased PARylation observed in combination treatment resulted in depletion of NAD+ levels. These decreased NAD+ levels were also paralleled with reduced aldehyde dehydrogenase (ALDH) activity, which requires NAD+ to maintain cancer stem cells. Furthermore, prexasertib and PARGi combinations exhibited synergistic cell death in OC cells, including an isogenic chemoresistant cell line and 3D organoid models of primary patient-derived OC cell lines. Collectively, our data highlight a novel crosstalk between metabolism and DNA repair involving replication stress and NAD+-dependent PARylation, and suggest a novel combination therapy of CHK1 and PARG inhibitors to overcome chemoresistance in OC.

Abstract A015: Exploiting endogenous replication stress with a novel targeted therapy in Ewing sarcoma

Abstract Ewing sarcoma (EwS) is a rare and aggressive disease that typically affects the bone of children and young adults. Although there are moderate 5-year survival rates for primary disease, there is a high rate of recurrence and a lack of targeted therapies in this setting. Furthermore, pediatric exposure to chemotherapies leads to an increased risk of long-term complications, including secondary cancers, cardiomyopathy, and infertility. Therefore, there is a desperate need for targeted therapies which improve therapeutic outcomes and reduce exposure to chemotherapy in these patients. Like many pediatric cancers, EwS has a low mutational burden. Instead, it is driven by chromosomal translocation between a FET family member, EWSR1, and an ETS family member, usually FLI1. The EWSR1-FLI1 fusion protein acts as an aberrant and oncogenic transcription factor, disrupting cell cycle control, cell migration, and proliferation. ETS family members in these fusions retain their ability to bind to the SWI/SNF chromatin remodeling complex, further dysregulating DNA methylation and gene expression. Although targeting of the EWSR1 fusion has not yet been successful, these elevated levels of replication stress (RS) present a unique element of vulnerability within the cell. To this end, our lab has previously identified replication stress response (RSR) inhibitors, DDKi and WEE1i, which can be utilized to exploit the characteristic ability of EwS to maintain high levels of replication stress (RS) and push EwS cells into mitotic catastrophe in vitro. Here, we evaluate the efficacy of the replication stress response inhibitors (RSRi) TAK931 (DDKi) and MK1775 (WEE1i) with chemotherapy regimens commonly utilized in the relapsed/refractory setting in vivo. Using cell line derived xenografts in NSG mice, we show that DDKi and WEE1i with irinotecan limits tumor growth significantly more than existing therapy (temozolomide + irinotecan). Dosing schedules are optimized through dosing de-escalation and alternative-day dosing strategies. We further demonstrate the ability of our RSRi combination to effectively limit tumor growth over time in vivo even when irinotecan dosage is decreased. We go on to investigate the RSRi combination in the presence of ultra-low dose irinotecan. Finally, we utilize reduced representation bisulfite sequencing to examine the epigenetic effects of these treatment regimens. Overall, the exploitation of high endogenous replication stress in EwS by targeted RSRi presents a promising potential therapeutic option for this pediatric patient population. Citation Format: Emily Isenhart, Ajay Gupta, Bryan Gillard, Kristopher Attwood, Joyce Ohm. Exploiting endogenous replication stress with a novel targeted therapy in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A015.

Abstract B003: Identification of novel genes that regulate aneuploidy tolerance by attenuating aneuploidy-induced stresses

Abstract Aneuploidy, an imbalanced number of chromosomes or chromosome arms, is the most common genetic aberration in cancer and is associated with a worse prognosis and advanced disease. However, in non-transformed cells, aneuploidy has a substantial fitness cost, leading to cell cycle arrest and cell death. This fitness cost is the result of several cellular stresses associated with aneuploidy, including proteotoxic, metabolic, mitotic and replication stress. Therefore, for aneuploidy to become beneficial, tolerance mechanisms that enable cancer cells to cope with aneuploidy-induced cellular stresses must come into play. Such mechanisms may uncover novel synthetic lethalities of aneuploid cancer cells. To this end, we systematically dissected the cellular mechanisms that facilitate aneuploidy tolerance. We combined large-scale analyses of gene expression, as well as genetic and pharmacological dependency datasets from human tumors, cancer cell lines and non-transformed cells, to identify genes and pathways associated with aneuploidy tolerance. We identified 55 genes associated (with high confidence) with aneuploidy tolerance. Gene set enrichment analysis (GSEA) revealed an enrichment for ribosome biogenesis and translation, DNA repair, replication stress and the p53 pathway. Interestingly, these pathways were all previously implicated with the aforementioned aneuploidy-induced cellular stresses. We plan to conduct a dependency mini-screen to validate the top ‘hits’ from our comprehensive bioinformatic analysis, and to explore the role of validated candidates in conferring aneuploidy tolerance. One of the top ‘hits’ of our exploratory analysis was a novel gene with an unclear cellular function. We experimentally confirmed that this gene was over-expressed in aneuploid cells, across several isogenic systems of matched diploid-aneuploid cell lines. Aneuploidy induction using MPS1 inhibitors led to its significant upregulation, demonstrating that its increased expression is a general feature of aneuploid cells. Mechanistically, we found that the novel gene had a role in regulating chromosome segregation, as its knockdown increased the rate of mitotic aberrations – as well as the sensitivity to mitotic checkpoint inhibition – resulting in increased chromosomal instability. We now study the molecular basis for the effect of this novel gene on aneuploidy tolerance. Specifically, we knocked it down in aneuploid cells, and over-expressed it in diploid cells, and we currently explore how these manipulations affect the above-mentioned aneuploidy tolerance pathways, namely replication stress, ribosome biogenesis, DNA damage repair and tp53 activation. In summary, we revealed a novel gene that is a key regulator of aneuploidy tolerance in cancer cells, likely due to its direct involvement in pathways required for the cellular coping with aneuploidy-induced stresses. Citation Format: Yonatan Eliezer, Adi V Tarrab, Tal Ben-Yishay, Hajime Okada, Tom Winkler, Uri Ben-David. Identification of novel genes that regulate aneuploidy tolerance by attenuating aneuploidy-induced stresses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B003.

Abstract IA008: Exploiting tumor selective vulnerabilities with structure based drug design

Abstract It is well appreciated that the integrity of both pluripotent and terminally differentiated cellular genomes is under constant pressure from damage provoked by cell-intrinsic and environmental factors. As such, defects in the cellular machinery preserving genomic integrity lead to the accumulation of variants that can promote cancer, but they also confer tumor-cell specific vulnerabilities that present synthetic-lethal therapeutic opportunities. The deepening mechanistic appreciation of tumor-selective pressure on DNA replication-fork fidelity, namely oncogene-induced replication stress, has provoked great interest in the prosecution of drug targets within the cellular machinery that otherwise supports replication fork stability. Poly (ADP-Ribose) glycohydrolase (PARG) is an enzyme with a unique role in the resolution of DNA damage repair and DNA replication fork restart through hydrolysis of Poly (ADP-ribose) (PAR) chains. IDE161 is a potent, selective small molecule inhibitor of PARG that is being developed as an anti-cancer therapeutic for patients with advanced or metastatic cancer harboring defects in homologous recombination repair. In culture, the accumulation of PAR chains upon PARG inhibition causes delayed repair of DNA breaks, replication stress and mitotic catastrophe in settings with collateral liabilities in DNA damage repair and/or replication fork stability. Genome-wide CRISPR-mediated fitness screens across multiple genetic backgrounds revealed strong synthetic lethal interactions of IDE161 with otherwise non-essential components of cellular machinery supporting base excision repair, homologous recombination repair and replication fork stability. In addition, evaluation of IDE161 antiproliferative activity across a panel of 350 molecularly characterized cancer cell lines returned a response profile that was only partially overlapping with that observed with PARPi and was enriched for models harboring vulnerabilities associated with replication stress and/or defects in DNA repair. Notably, many homologous recombination deficient (HRD) cell models display differential dependency on PARG activity vs PARP1/2 activity, suggesting PARG inhibition as a potential therapeutic strategy distinct from PARP inhibitors. In vivo assessment of IDE161 anti-tumor activity in CDX and PDX models mirrored these mechanistic relationships. A number of HRD PDX models displayed a superior response to IDE161 versus PARPi, including robust regressions selectively observed with IDE161 versus niraparib in BRCA2 altered ER+, HER2-breast cancers. Thus, IDE161 has potential as novel targeted therapy that exploits the synthetic lethal relationship between PARG and genomic instability, leading to selective anti-proliferative activity in tumors harboring defects in DNA repair and replication fidelity. Citation Format: Reeja Maskey, Diana Munoz, Megan Conway, Arjun Rao, Vidhya Nagarajan, Ivan Shabalin, Kelly Trego, Jonathan Ryan, Peter Teriete, Christian Frey, Josh Taygerly, Paul Barsanti, Claire Neilan, Jasgit Sachdev, Michael White. Exploiting tumor selective vulnerabilities with structure based drug design [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA008.

Abstract IA010: Targeting genome instability in cancer: Translating synthetic lethal biology into the clinic

Abstract Membrane-associated tyrosine- and threonine-specific Cdc2-inhibitory kinase (PKMYT1) is a cell cycle regulatory kinase that inhibits CDK1/CyclinB activity, delaying entry into mitosis in tumor cells experiencing replication stress (RS). RS is frequently induced by genetic alterations that drive premature transition from G1 to S phase, promoting genome instability and creating a synthetic lethal (SL) relationship between these specific alterations and PKMYT1 inhibition. This relationship has been demonstrated preclinically and clinically with CCNE1 amplification as well as FBXW7 and PPP2R1A deleterious mutations. These genetic alterations are common across a range of tumors including ovarian, endometrial and colorectal cancers. On this basis, we developed lunresertib (RP-6306), a first-in-class, potent and selective oral PKMYT1 inhibitor currently being investigated in phase I clinical trials (NCT04855656, NCT05147272, NCT05147350). Here we discuss the translation of pre-clinical data to the clinical setting as both monotherapy and mechanistic combinations approaches. Citation Format: Michael Zinda. Targeting genome instability in cancer: Translating synthetic lethal biology into the clinic [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA010.

KBM-mediated interactions with KU80 promote cellular resistance to DNA replication stress in CHO cells

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.

MCM8 interacts with DDX5 to promote R-loop resolution

MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the replication stress response during mitosis, but its function in mitotic germ cells remains elusive. Here we found that disabling MCM8 in mice resulted in proliferation defects of primordial germ cells (PGCs) and ultimately impaired fertility. We further demonstrated that MCM8 interacted with two known helicases DDX5 and DHX9, and loss of MCM8 led to R-loop accumulation by reducing the retention of these helicases at R-loops, thus inducing genome instability. Cells expressing premature ovarian insufficiency-causative mutants of MCM8 with decreased interaction with DDX5 displayed increased R-loop levels. These results show MCM8 interacts with R-loop-resolving factors to prevent R-loop-induced DNA damage, which may contribute to the maintenance of genome integrity of PGCs and reproductive reserve establishment. Our findings thus reveal an essential role for MCM8 in PGC development and improve our understanding of reproductive aging caused by genome instability in mitotic germ cells.

Abstract B020: Novel WRN helicase inhibitors selectively target microsatellite unstable cancer cells

Abstract Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours, and WRN inhibitors are in development. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth In vitro and In vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA-repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic-lethal targeting of WRN in MSI cancer and tools to dissect WRN biology. Citation Format: Gabriele Picco, Yanhua Rao, Angham Al Saedi, Yang Lee, Shriram Bhosle, Carmen Herranz-Ors, Samantha Walker, Kieron May, Matthew Coelho, Jon Houseley, Benjamin Schwartz, Mathew J. Garnett. Novel WRN helicase inhibitors selectively target microsatellite unstable cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B020.

Abstract PR006: DNA repair inhibition via targeting ATR activates cGAS/STING signaling, promotes anti-tumor immunity, and is a synthetic lethal strategy to augment immunotherapy response in preclinical models and clinical samples of small cell lung cancer

Abstract Introduction: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Clinical trials of immune checkpoint blockade (ICB) combined with chemotherapy delivered only very modest benefits. In this study, we investigated the effect of targeting ataxia telangiectasia and rad3-related (ATR), the primary activator of the replication stress response, in SCLC. Clinical samples from treatment naïve and treated SCLC patients were analyzed by single cell and bulk RNA sequencing to ascertain the intratumoral transcriptional heterogeneity and the surrounding tumor microenvironment (TME). Methods: In this study, we investigated the effect of ATR inhibition either alone or in combination with PD-L1 blockade in multiple immunocompetent mouse models of SCLC. The downstream effects of ATR inhibition were assessed by bulk RNA sequencing, and multicolor flow cytometry. The SCLC clinical samples from treatment naïve and patients treated were analyzed by single cell and bulk RNA sequencing. Results: In multiple immunocompetent SCLC mouse models, ATR inhibition (ATRi) remarkably enhanced the anti-tumor effect of PD-L1 blockade. We next tested the ATR inhibition either alone or in combination with PD-L1 in the second-line regimen for SCLC. We observed that ATR inhibition in combination with PD-L1 blockade significantly reduced tumor volume and prolonged survival of aggressive mice models when compared to PD-L1 alone. Targeting ATR enhanced the expression of PD-L1, activated the cGAS/STING pathway, induced the expression of Type I and II interferon pathways, and caused significant infiltration of cytotoxic and memory/effector T-cells into tumors. Interestingly, ATRi also led to significant induction of MHC class I in SCLC in vitro and in vivo models. Single-cell RNA sequencing data provided a deep annotation of SCLC phenotypes and the surrounding immune TME. Treatment and subtype are associated with substantial phenotypic changes in the SCLC immune microenvironment, with greater T-cell dysfunction in SCLC-N than in SCLC-A. Moreover, a recurrent highly metastatic SCLC subset is associated with exhausted CD8+ T-cells and a pro-fibrotic, immunosuppressive monocyte/macrophage population, suggesting possible tumor-immune coordination to promote metastasis. Specifically, analysis of pre-and post-treatment clinical samples from a proof-of-concept study of a first-in-class ATR inhibitor, VX970 (berzosertib), and topotecan, in patients with relapsed SCLCs, validated the induction of MHC class I and interferon pathway genes, for the first time in this disease. Conclusion: We provide insight into tumor and immune biology in SCLC at single-cell resolution. Further, we show ATRi as a potentially transformative vulnerability of SCLC. Given the increasing importance of immunotherapy for the management of SCLC and that ATR inhibitors are already in clinical trials, combining ATR inhibitors with PD-L1-blockade offers an attractive strategy for the treatment of SCLC and contributes to the rapid translation of this combination into the clinic. Citation Format: Triparna Sen. DNA repair inhibition via targeting ATR activates cGAS/STING signaling, promotes anti-tumor immunity, and is a synthetic lethal strategy to augment immunotherapy response in preclinical models and clinical samples of small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR006.

Abstract IA024: RNA-modifying enzyme inhibitors as synthetic lethal cancer therapeutics

Abstract Over 100 modifications to RNA are known to occur in human cells, where they play critical roles in many aspects of normal cellular physiology, such as cell fate decisions and terminal differentiation, through effects on RNA biology such as protein and nucleic acid interactions. Our survey of human RNA-modifying enzymes suggests many are cancer essential enzymes with striking synthetic lethal profiles, including the XRN1 nuclease and DHX9 helicase. XRN1 degrades single stranded mRNA from the 5′→3′ direction and plays a key role in endogenous cellular mRNA turnover. XRN1 can also degrade double-stranded RNA (dsRNA), and as such plays a role in innate immunity. We identified XRN1 as a selective vulnerability in tumor cells with intrinsic elevation of a Type I Interferon Stimulated Gene (TISG) signature through analysis of publicly available CRISPR data across 483 tumor cell lines. XRN1 KO via CRISPR leads to robust anti-proliferative effects in TISG high cells, but not in TISG low cells, as well as upregulation of Interferon-β mRNA and induction of phosphorylated PKR. Exogenous treatment with Interferon-β to induce elevated Type I Interferon expression sensitizes TISG low cells to XRN1 KO, confirming this synthetic lethal relationship. Enzymatic and biophysical assays for XRN1 were developed and used to identify and optimize XRN1 inhibitors. We identified Compound 1, a substrate-competitive inhibitor with single-digit micromolar affinity for XRN1 which does not bind or inhibit XRN2. High-resolution crystal structures indicate that Compound 1 binds in an allosteric binding site, confirming the mechanism of inhibition and selectivity profile determined by in vitro assays. This data suggests that XRN1, through its regulation of dsRNA burden, is a compelling and druggable oncology target for TISG high tumors. DHX9 is a multifunctional DExH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops) and DNA/RNA G-quadruplexes. DHX9 interacts with and regulates many proteins, including key members of DNA damage repair pathways. We have found that DHX9 knockdown is synthetic lethal in microsatellite high (MSI-H) or defective mismatch repair (dMMR) tumor models. A suite of assays was developed to identify and optimize potent and selective inhibitors of the DHX9 helicase, which recapitulate our findings with genetic tools. Profiling of DHX9 inhibitors across a broad panel of cancer cell lines reveals that tumor cells with mutations in the DNA damage repair genes BRCA1 and/or BRCA2 are also responsive to DHX9 inhibitor treatment in vitro and in vivo. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress, leading to cell cycle arrest and apoptosis. These results suggest that DHX9 inhibitors are a novel treatment modality for patients with defective DNA damage repair pathways such as dMMR and/or BRCA mutations. Citation Format: Serena J Silver, Maureen M. Lynes, Brian A. Sparling, Jennifer Castro, Matthew H. Daniels, Sunaina Pai, Sophie A. Shen, David Brennan, Hyelee Lee, Gordon J. Lockbaum, Kevin Knockenhauer, Deepali Gotur, Simina Grigoriu, Shihua Yao, Shane Buker, Monique Laidlaw, Jie Wu, Stephen J. Blakemore, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager, Robert A. Copeland. RNA-modifying enzyme inhibitors as synthetic lethal cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA024.

A PRMT5-ZNF326 axis mediates innate immune activation upon replication stress.

DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.

Fragment-Based Discovery of Novel MUS81 Inhibitors.

MUS81 is a structure-selective endonuclease that cleaves various branched DNA structures arising from natural physiological processes such as homologous recombination and mitosis. Due to this, MUS81 is able to relieve replication stress, and its function has been reported to be critical to the survival of many cancers, particularly those with dysfunctional DNA-repair machinery. There is therefore interest in MUS81 as a cancer drug target, yet there are currently few small molecule inhibitors of this enzyme reported, and no liganded crystal structures are available to guide hit optimization. Here we report the fragment-based discovery of novel small molecule MUS81 inhibitors with sub-μM biochemical activity. These inhibitors were used to develop a novel crystal system, providing the first structural insight into the inhibition of MUS81 with small molecules.

Comprehensive single cell transcriptomics analysis of murine osteosarcoma uncovers Skp2 function in metastasis, genomic instability and immune activation and reveals additional target pathways.

Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new therapeutic target is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase responsible for ubiquitination and proteasome degradation of substrate p27, thus driving cellular proliferation. We have shown previously that knockout of Skp2 in an immunocompetent transgenic mouse model of OS improved survival, drove apoptosis, and induced tumor inflammation. Here, we applied single-cell RNA-sequencing (scRNA-seq) to study primary OS tumors derived from Osx-Cre driven conditional knockout of Rb1 and Trp53. We showed that murine OS models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. We further compared this model with OS models with functional disruption of Skp2: one with Skp2 knockout and the other with the Skp2-p27 interaction disrupted (resulting in p27 overexpression). We found reduction of T cell exhaustion and upregulation of interferon activation, along with evidence of replicative and endoplasmic reticulum-related stress in the Skp2 disruption models, and showed that interferon induction was correlated with improved survival in OS patients. Additionally, our scRNA-seq analysis uncovered decreased activities of metastasis-related gene signatures in the Skp2-disrupted OS, which we validated by observation of a strong reduction in lung metastasis in the Skp2 knockout mice. Finally, we report several potential mechanisms of escape from targeting Skp2 in OS, including upregulation of Myc targets, DNA copy number amplification and overexpression of alternative E3 ligase genes, and potential alternative lineage activation. These mechanistic insights into OS tumor biology and Skp2 function suggest novel targets for new, synergistic therapies, while the data and our comprehensive analysis may serve as a public resource for further big data-driven OS research.

Restricting the level of the proteins essential for the regulation of the initiation step of replication extends the chronological lifespan and reproductive potential in budding yeast

Aging is defined as a progressive decline in physiological integrity, leading to impaired biological function, including fertility, and rising vulnerability to death. Disorders of DNA replication often lead to replication stress and are identified as factors influencing the aging rate. In this study, we aimed to reveal how the cells that lost strict control of the formation of crucial for replication initiation a pre-initiation complex impact the cells’ physiology and aging. As strains with the lower pre-IC control (lowPICC) we used, Saccharomyces cerevisiae heterozygous strains having only one functional copy of genes, encoding essential replication proteins such as Cdc6, Dbf4, Sld3, Sld7, Sld2, and Mcm10. The lowPICC strains exhibited a significant reduction in the respective genes’ mRNA levels, causing cell cycle aberrations and doubling time extensions. Additionally, the reduced expression of the lowPICC genes led to an aberrant DNA damage response, affected cellular and mitochondrial DNA content, extended the lifespan of post-mitotic cells, and increased the yeast’s reproductive potential. Importantly, we also demonstrated a strong negative correlation between the content of cellular macromolecules (RNA, proteins, lipids, polysaccharides) and aging. The data presented here will likely contribute to the future development of therapies for treating various human diseases.

Genome-scale clustered regularly interspaced short palindromic repeats screen identifies nucleotide metabolism as an actionable therapeutic vulnerability in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common malignancy that develops in patients with ataxia-telangiectasia, a cancer-predisposing inherited syndrome characterized by inactivating germline ATM mutations. ATM is also frequently mutated in sporadic DLBCL. To investigate lymphomagenic mechanisms and lymphoma-specific dependencies underlying defective ATM, we applied ribonucleic acid (RNA)-seq and genome-scale loss-offunction clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens to systematically interrogate B-cell lymphomas arising in a novel murine model (Atm-/-nu-/-) with constitutional Atm loss, thymic aplasia but residual T-cell populations. Atm-/-nu-/-lymphomas, which phenotypically resemble either activated B-cell-like or germinal center Bcell-like DLBCL, harbor a complex karyotype, and are characterized by MYC pathway activation. In Atm-/-nu-/-lymphomas, we discovered nucleotide biosynthesis as a MYCdependent cellular vulnerability that can be targeted through the synergistic nucleotidedepleting actions of mycophenolate mofetil (MMF) and the WEE1 inhibitor, adavosertib (AZD1775). The latter is mediated through a synthetically lethal interaction between RRM2 suppression and MYC dysregulation that results in replication stress overload in Atm-/-nu-/-lymphoma cells. Validation in cell line models of human DLBCL confirmed the broad applicability of nucleotide depletion as a therapeutic strategy for MYC-driven DLBCL independent of ATM mutation status. Our findings extend current understanding of lymphomagenic mechanisms underpinning ATM loss and highlight nucleotide metabolism as a targetable therapeutic vulnerability in MYC-driven DLBCL.

Multi-step control of homologous recombination via Mec1/ATR suppresses chromosomal rearrangements

The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.