Replication Stress Research Articles

Replication stress promotes cellular transformation in Drosophila epithelium

The accurate control of DNA replication is crucial for the maintenance of genomic stability and cell viability. In this study, we explore the consequences of depleting the replicative DNA Polymerase α (POLA) in the wing disc of Drosophila melanogaster. Our findings reveal that reduced POLA activity induces DNA replication stress and activates the replication checkpoint in vivo. Consistent with this, we demonstrate that dATR, a key component in DNA replication checkpoint signaling, is essential for the maintenance of tissue integrity under conditions of compromised POLA activity. We show that cells within the wing disc exhibiting reduced POLA activity arrest in the G2 phase and undergo p53-dependent apoptosis. We also reveal a critical role for DNA Ligase 4 in sustaining cell viability when POLA function is impaired. Most notably, we report the appearance of oncogenic traits in wing disc cells with diminished POLA activity when apoptosis is suppressed. In this context, the overexpression of the oncogene cdc25/string enhances the oncogenic phenotype. These results indicate that a combination of oncogenic activation, replication stress, and suppression of apoptosis is sufficient to promote the emergence of hallmarks of tumorigenesis, highlighting major implications for cancer development in humans.

Abstract B010: Exploring the synthetic lethal interactions between mRNA methyl-5-cytocine at sites of R-loops and mismatch repair deficiency in solid tumor

Abstract The RNA methyltransferase TRDMT1 generates 5-methyl Cytosine (m5C) at R-loops which promotes transcription coupled homologous recombination (TC-HR) and thus impedes cellular sensitivity to R-loop induced DNA double-strand breaks (DSBs) at transcribing regions of the genome. We performed TRDMT1 inhibitor based drug screening approach in 300 cancer cell lines and identifies key factors associated with DNA mismatch repair (MMR) pathway especially in solid tumors. The R-loop accumulation correlated to severe replication stress and generated more transcription replication conflicts (TRCs) in TRDMT1 and MMR deficient background. Our study revealed synthetic lethal interactions between TRDMT1 and MMR which can provide a promising future strategy for improving treatment of solid tumors. Citation Format: Li Lan. Exploring the synthetic lethal interactions between mRNA methyl-5-cytocine at sites of R-loops and mismatch repair deficiency in solid tumor [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B010.

The Biology in the Pattern: Metastatic organotropism and clinical outcome depend on DNA damage response and immune interactions in pancreatic cancer.

Metastatic cancer remains a leading cause of cancer-related mortality, with a 5-year survival rate of just 8% for pancreatic ductal adenocarcinoma (PDAC). Among patients with metastatic PDAC, those with liver metastases experience significantly worse outcomes compared to the rare cases of isolated lung metastases. Recent findings by Link and colleagues reveal that these distinct metastatic patterns reflect underlying biological differences beyond established molecular subtypes. Specifically, the authors curated a primary organotropism (pORG) gene signature that is enriched in the liver cohorts. In detail they found that high-pORG/liver-avid tumors are characterized by high replication stress, enriched DNA repair pathways, and an immunosuppressive microenvironment, whereas low-pORG/lung-avid tumors display stronger immune infiltration, higher T cell density and reduce the richness of TCR repertoire, and better survival outcomes. These insights suggest that the clinical pattern of metastasis provides meaningful information about tumor biology and prognosis, complementing current subtype classifications in PDAC.

Replication stress: an early key event in ochratoxin a genotoxicity?

Abstract Ochratoxin A (OTA), a widespread food contaminant and potent renal carcinogen in rodents, is weakly genotoxic in mammalian cells. The mechanisms underlying OTA-induced genetic damage are still poorly understood. In its recent risk assessment, the European food safety authority (EFSA) considered that the specific spectrum of mutations and chromosomal damage induced by OTA may derive from unresolved replication stress. The aim of the present work was to experimentally test the hypothesis that OTA interferes with DNA replication and to characterize the cellular response to OTA-mediated replication stress. Using the DNA fiber assay to study replication fork dynamics at single molecule resolution, a small but statistically significant global delay in replication fork progression was observed in human kidney (HK-2) cells exposed to OTA at ≥ 10 µM. OTA-mediated interference with DNA replication was confirmed by a concentration-related decrease in incorporation of the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) into newly replicating DNA in HK-2 cells arrested in late G1/S via double thymidine block and treated with OTA during S phase. Western blot and immunofluorescence analyses revealed a significant concentration-related increase in γH2AX in cells exposed to OTA. Co-localization of γH2AX foci with 5-chloro-2′-deoxyuridine (CldU) incorporated into cells during S phase and increased γH2AX labeling along newly replicating chromatin fibers visualized using the extended chromatin fiber assay support a replication-coupled mechanism of OTA-induced DNA damage. Experiments with cells synchronized in late G1/S or late G2 demonstrated that exposure of cells to OTA during S phase, but not mitosis, leads to a significant concentration-related increase in H2AX, providing further evidence that OTA may act primarily during S phase of the cell cycle. However, OTA did not appear to efficiently activate ATR-Chk1 and ATM-Chk2 DNA damage response pathways, suggesting that cells with under-replicated DNA or unresolved DNA damage may escape checkpoint control and may continue into mitosis, with potentially deleterious consequences for genomic integrity. Overall, results from this study provide first experimental evidence for perturbation of the S phase replisome machinery by OTA and point toward replication stress as an early key event in OTA genotoxicity.

Ingenane Diterpenoids from Euphorbia peplus as Potential New CHK1 Inhibitors That Sensitize Cancer Cells to Chemotherapy.

Phosphorylation of checkpoint kinase 1 at Ser-345 (p-CHK1(S345)) mediates the replication stress response in cancer cells, leading to chemotherapy resistance. Therefore, finding inhibitors of p-CHK1(S345) is currently a promising strategy to prevent acquired drug resistance. In this study, 14 ingenane diterpenoids (1-14), involving two undescribed compounds possessing an unprecedented exocyclic double bond Δ6(20), were identified from Euphorbia peplus. The inhibitory effects of the isolated compounds on p-CHK1(S345) and their structure-activity relationship (SAR) were investigated. Compounds 7 and 8 presented the strongest inhibitory effects, abrogated cell cycle arrest, and caused the accumulation of DNA damage, improving the sensitivity of cancer cells to chemotherapeutic drugs. An in vivo assay confirmed the enhancement of 8 on the anticancer effect of topotecan via blocking of p-CHK1(S345). Mechanistically, 8 increased CHK1 ubiquitination to inhibit p-CHK1(S345) via activation of protein kinase C (PKC). PKC activation was first found to enhance CHK1 ubiquitination to block p-CHK1(S345). Above all, this finding not only indicates that compound 8 could be developed as a novel CHK1 inhibitor but also reveals a previously unrecognized role of PKC in regulating cancer chemotherapy sensitivity.

Replication stress, microcephalic primordial dwarfism, and compromised immunity in ATRIP deficient patients.

Ataxia telangiectasia and Rad3-related (ATR) kinase and its interacting protein ATRIP orchestrate the replication stress response. Homozygous splice variants in the ATRIP gene, resulting in ATRIP deficiency, were identified in two patients of independent ancestry with microcephaly, primordial dwarfism, and recurrent infections. The c.829+5G>T patient exhibited lymphopenia, poor vaccine responses, autoimmune features with hemolytic anemia, and neutropenia. Immunophenotyping revealed reduced CD16+/CD56dim NK cells and absent naïve T cells, MAIT cells, and iNKT cells. Lymphocytic defects were characterized by TCR oligoclonality, abnormal class switch recombination, and impaired T cell proliferation. ATRIP deficiency resulted in low-grade ATR activation but impaired CHK1 phosphorylation under genotoxic stress. ATRIP-deficient cells inadequately regulated DNA replication, leading to chromosomal instability, compromised cell cycle control, and impaired cell viability. CRISPR-SelectTIME confirmed reduced cell fitness for both variants. This study establishes ATRIP deficiency as a monogenic cause of microcephalic primordial dwarfism, highlights ATRIP's critical role in protecting immune cells from replication stress, and offers new insights into its canonical functions.

Chromatin assembly factor 1 subunit A promotes TLS pathway by recruiting E3 ubiquitin ligase RAD18 in cancer cells

The translesion DNA synthesis (TLS) pathway mediated by proliferating cell nuclear antigen (PCNA) monoubiquitination is an essential mechanism by which cancer cells bypass DNA damage caused by DNA damage to maintain genomic stability and cell survival. Chromatin assembly factor 1 subunit A (CHAF1A) traditionally promotes histone assembly during DNA replication. Here, we revealed that CHAF1A is a novel regulator of the TLS pathway in cancer cells. CHAF1A promotes restart and elongation of the replication fork under DNA replication stress. Mechanistically, the C-terminal domain of CHAF1A directly interacts with E3 ubiquitin ligase RAD18, enhancing RAD18 binding on the stalled replication fork. CHAF1A facilitates PCNA K164 monoubiquitination mediated by RAD18, thereby promoting the recruitment of Y-family DNA polymerases and enhancing cancer cell resistance to DNA damage. In addition, CHAF1A-mediated RAD18 recruitment and PCNA monoubiquitination are independent of the CHAF1A-PCNA interaction and its histone assembly function. Taken together, these findings improve our understanding of the mechanisms that regulate the TLS pathway and provide insights into the relationship between CHAF1A and DNA replication stress in cancer cells.

DDX37 and DDX50 Maintain Genome Stability by Preventing Transcription-dependent R-loop Formation.

DDX37 and DDX50 Maintain Genome Stability by Preventing Transcription-dependent R-loop Formation.

Deficiency of ATF2 retards senescence induced by replication stress and pamidronate in mouse jaw bone marrow stem cells.

Deficiency of ATF2 retards senescence induced by replication stress and pamidronate in mouse jaw bone marrow stem cells.

Reversing regulatory safeguards: Targeting the ATR pathway to overcome PARP inhibitor resistance.

The development of poly (ADP-ribose) polymerase inhibitors (PARPis) is widely considered a therapeutic milestone in the management of BRCA1/2-deficient malignancies. Since a growing number of cancer treatment guidelines include PARPis, the inevitably emerging PARPi resistance becomes a serious limitation that must be addressed. Targeting the DNA damage response signaling kinase, ATR (ataxia telangiectasia and rad3-related serine/threonine kinase), activated in response to PARPi-induced replication stress, represents a promising approach in fighting PARPi-resistant cancers. The success of this combination therapy in preclinical models has inspired efforts to translate its potential through extensive clinical research and clinical trials. However, the available clinical evidence suggests that PARPi/ATRi combinations have yet to reach their anticipated therapeutic potential. In this review, we summarize work elucidating mechanisms underpinning the effectiveness of ATRi in fighting PARPi resistance and review translational studies reporting efficacy in different types of cancer. Finally, we discuss potential biomarkers of patient selection for customized combinations of PARPi/ATRi treatments.

Biomolecular Condensates in Telomere Maintenance of ALT Cancer Cells.

Biomolecular Condensates in Telomere Maintenance of ALT Cancer Cells.

MYC amplification sensitizes TNBC to CHK1 inhibitors.

MYC amplification sensitizes TNBC to CHK1 inhibitors.

INDUCTION OF EARLY PULMONARY SENESCENCE IN EXPERIMENTAL SEPSIS.

Background: Sepsis continues to pose a significant threat to human life and represents a substantial financial burden. In addition to replicative stress resulting from telomeric loss, recent studies have identified multiple factors contributing to cell cycle arrest. Furthermore, our understanding of pathways associated with cellular senescence, such as CD47-mediated suppression of efferocytosis, has expanded. However, beyond in vitro experiments, the impact of cell stress during complex systemic illnesses, including sepsis, remains poorly understood. Consequently, we conducted an investigation into molecular alterations related to senescence-associated pulmonary mechanisms during experimental nonpulmonary sepsis. Methods: Male C57BL/6JRj mice were anesthetized and subjected to either control conditions (sham) or cecal ligation and puncture (CLP) to induce sepsis. Twenty-four hours or 7 d after CLP, animals were killed, and blood, bronchoalveolar fluids, and lungs were harvested and analyzed for morphological and biochemical changes. Results: Histological damage in pulmonary tissue, as well as increases in plasma levels of surfactant protein D, indicated development of alveolar-focused acute lung injury after CLP. Additionally, we observed a significant upregulation of the CD47-QPCTL-SHP-1 axis in lungs of septic mice. Whereas the expression of p16, a marker primarily indicating manifested forms of senescence, was decreased after CLP, the early marker of cellular senescence, p21, was increased in the lungs during sepsis. Later, at 7 d after CLP, pulmonary expression of CD47 and QPCTL-1 was decreased, whereas SHP-1 was significantly enhanced. Conclusion: Our findings suggest an activation of senescent-associated pathways during experimental sepsis. However, expanding the experiments to other organ systems and in vivo long-term models are necessary to further evaluate the sustained mechanisms and immunopathophysiological consequences of cellular senescence triggered by septic organ injury.

Mono-ubiquitination of TopBP1 by PHRF1 enhances ATR activation and genomic stability.

The TopBP1-ATR axis is critical for maintaining genomic stability during DNA replication stress, yet the precise regulation of TopBP1 in replication stress responses remains poorly understood. In this study, we identified PHD and Ring Finger Domains 1 (PHRF1) as an important ATR activator through its interaction with TopBP1. Our analysis revealed a correlation between PHRF1 and genomic stability in cancer patients. Mechanistically, PHRF1 is recruited to DNA lesions in a manner dependent on its PHD domain and histone methylation. Subsequently, PHRF1 mono-ubiquitinates TopBP1 at lysine 73, which enhances the TopBP1-ATR interaction and activates ATR. Depletion of PHRF1 disrupts ATR activation and sensitizes cells to replication stress-inducing agents. Furthermore, conditional knockout of Phrf1 in mice leads to early lethality and impaired ATR-Chk1 axis signaling. Collectively, our findings establish PHRF1 as a novel E3 ligase for TopBP1, coordinating the replication stress response by enhancing TopBP1-ATR signaling.

Targeting Aurora A to Overcome Cisplatin Resistance in Head and Neck Cancer.

Cisplatin-based chemotherapy is a cornerstone treatment for advanced recurrent head and neck squamous cell carcinoma (HNSCC). However, the effectiveness of the treatment is often hindered by intrinsic and acquired resistance and associated toxicity, highlighting a pressing unmet clinical need. Here, our compound screening identified Aurora kinase inhibitors, particularly those targeting Aurora A kinase, as potential agents to sensitize resistant HNSCC cells to cisplatin. While Aurora kinases are well-established regulators of mitosis, their precise role in cisplatin resistance is largely unknown, given that cisplatin confers toxicity primarily in cells undergoing DNA replication. We confirmed that depletion of Aurora A or its activators enhanced cisplatin response in resistant HNSCC cells. Analyses of a comprehensive database and locally treated HNSCC patient samples revealed compelling associations between Aurora A overexpression/activation and cisplatin resistance, tumor recurrence, and poor patient survival. Pharmacologic inhibition of Aurora A effectively synergized with cisplatin treatment in cellular assays and a syngeneic mouse tumor model of HNSCC. Mechanistically, Aurora A inhibition enhanced apoptosis induction after cisplatin treatment, particularly in S-phase cells; induced replication stress; and suppressed the repair of cisplatin-induced DNA crosslinking. Taken together, our findings shed light on important functions of Aurora A kinase beyond mitotic regulation. The multifaceted roles of Aurora A suggest its potential as a prime anticancer drug target. Given the ongoing investigations into numerous Aurora inhibitors for cancer therapy, exploring their clinical applications in HNSCC, especially in combination with platinum drugs, may hold significant promise.

Stochastic variation in the FOXM1 transcription program mediates replication stress tolerance.

Stochastic variation in the FOXM1 transcription program mediates replication stress tolerance.

Abstract A118: Targeting Replication Stress Promotes Anti-tumour Immune Responses that are Suppressed by Tumour-associated Myeloid cells

Abstract Replication stress is a common feature of solid cancers, and drugs targeting replication stress such as Checkpoint kinase 1 inhibitors (CHK1i) have demonstrated significant preclinical activity especially in combination with replication stress promoting chemotherapies. However, this has not translated into an effective clinical treatment, primarily due to high normal tissue toxicity. We have previously demonstrated that a combination of CHK1i with a subclinical dose of hydroxyurea selectively targets a range of tumour types, importantly with little normal tissue toxicity to even chemo-sensitive tissues. The CHK1i combination also promotes a pro-inflammatory response and immunogenic cell death. In vivo, this drug combination induces tumour regression which is dependent on an adaptive immune response. Here we report the immune responses triggered by this combination in mouse models and demonstrate that this response is suppressed by tumour-associated myeloid cells. Syngeneic mouse melanoma and ovarian cancer models were treated in vivo with the combination of CHK1i SRA737 and low dose hydroxyurea. The tumour immune microenvironment and peripheral immune cell responses were assessed by single cell expression analysis and immune cell marker multiparameter flow cytometry. In the panel of cancer models tested in syngeneic mice, the CHK1i combination, controlled tumour growth. Immune responses were elicited by the CHK1i combination in all tumours but found different types of immune cell responses in the different models and cancers investigated. The combination enhanced immune responses independent of the initial tumour immune microenvironment, including in tumours that were immunologically “cold”. The common features of the immune responses in all the models are increased cytolytic activity and reduced immune suppression in the tumour microenvironment. The responses were dependent on CD8+ T cells. Myeloid cells in the tumour microenvironment were immunosuppressive, expressing high levels of PD-L1 in response to treatment. This could be reduced by depletion with CSF-1R antibody or reducing tumour CSF-1 expression. This demonstrates the CHK1i combination is highly selective with minimal normal tissue toxicity, does not adversely affect immune responses, and can trigger an effective anti-tumour immune response in range of tumour settings, including current treatment resistant tumours. Reducing tumour associated myeloid number or activity was associated with enhanced anti-tumour immune responses. This work suggests that the myeloid component of tumours may significantly alter treatment responses by suppressing anti-tumour immune activity and that targeting this population may enhance the durability of the anti-tumour immune response even in immunologically cold tumours. Citation Format: Zhen Zeng, Ritu Bhatt, Anastasia Gandini, Jazmina Gonzalez Cruz, Martina Proctor, Katharine Irvine, Riccardo Dolcetti, James Wells, Brian Gabrielli. Targeting Replication Stress Promotes Anti-tumour Immune Responses that are Suppressed by Tumour-associated Myeloid cells [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A118.

Abstract A044: LP-184, a Novel Acylfulvene, Sensitizes Immuno-refractory Triple-Negative Breast Cancers (TNBCs) To Anti-PD1 Therapy by Affecting the Tumor Microenvironment

Abstract Drugs that induce DNA double strand breaks (DSBs) can activate the cGAS/STING pathway, leading to immune responses. Lantern Pharma is advancing a novel acylfulvene small molecule therapeutic, LP-184, a DNA damaging alkylating agent that creates DNA DSBs. LP-184 is a prodrug requiring bioactivation by the enzyme prostaglandin reductase 1 (PTGR1). RNAseq analysis of cancer cells exposed to LP-184 revealed activation of the STING pathway. A link between replication stress response (RSR) defect signature and enhancement of anti-PD1 activity in mouse models implicated certain DNA Damage Repair (DDR) targeting agents in induction of RSR defects and modulation of response to immuno-oncology (IO) agents. Assessing the effect of LP-184 on potentiating anti-PD1-driven immune checkpoint blockade in triple-negative breast cancer (TNBC) in a syngeneic mouse model showed that LP-184 combined with an anti-PD1 agent elicited a greater anti-tumor response than monotherapies in mouse TNBC tumors that are non-hypermutated and IO resistant. Investigation of effects of LP-184 on immune factor modulation in the tumor microenvironment (TME) with and without co-treatment with anti-PD-1 agent by fluorescence-activated cell sorting demonstrated that LP-184 plus anti-PD1 led to a larger T cell population in the TME along with higher activity of T cells. Flow cytometry based cell cycle analysis in TNBC cells revealed that LP-184 treatment created RSR defects comparable to that induced by CHK1 inhibitor Prexasertib, a promising agent in TNBC, often characterized by high levels of genomic instability and concomitant addiction to the RSR pathway involving ATR/CHK1. Results suggest that LP-184 can synergize with an anti-PD1 agent in a TNBC mouse model potentially via reshaping the TME by decreasing M2 macrophages, increasing T cell infiltration, and enhancing individual T cell function when combined with ICB therapy. Potential mechanisms of LP-184-driven M2 macrophage reduction could include decreased M2 macrophage polarization via tumor cell death or specific targeting of this PTGR1-expressing immunosuppressive cell type. LP-184, currently in a monotherapy Phase 1 dose finding/optimization safety study, thus exhibits potential to sensitize tumors non-responsive to anti-PD-1 therapy. Furthermore, single-cell RNA sequencing was performed on tumor samples from mice treated with control, LP-184, anti-PD1, and their combination. Clustering and differential expression analysis uncovered treatment-responsive cell populations and genes. Enrichment analysis highlighted key pathways affected by each treatment, focusing on immune-related pathways, cytokines, and chemokines. This proved to be a powerful approach that provides insights into immune-tumor cell interactions modulated by LP-184 and anti-PD1 within the TME. Next steps include testing whether LP-184 treatment elevates cytoplasmic ssDNA or dsRNA, activates the STING pathway, induces immunogenic cell death, and increases the expression of immunostimulatory cytokines. Citation Format: Yong Du, Aditya Kulkarni, Jianli Zhou, Hui Dai, Kishor Bhatia, Shiaw-Yih Lin. LP-184, a Novel Acylfulvene, Sensitizes Immuno-refractory Triple-Negative Breast Cancers (TNBCs) To Anti-PD1 Therapy by Affecting the Tumor Microenvironment [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A044.

Abstract A113: The CD73 Inhibitor, AB680 Enhances Chemotherapy-Induced STING Activation and T Cell-Mediated Tumor Immunity in Pancreatic Ductal Adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive tumor immune microenvironment (TIME) where combinations of chemotherapy and immune checkpoint blockade (ICB) have shown limited efficacy. Standard of care FOLFIRINOX (FFX) chemotherapy promotes replication stress in tumor cells triggering synthesis of 2’-3’ cGAMP, an intercellular mediator of STING activation in nearby leukocytes. STING activation induces Type 1 Interferon (IFN) production and a cascade of anti-tumor immune responses. In parallel, dying tumor cells release ATP that is rapidly converted to immunosuppressive adenosine (Ado) by extracellular ectonucleotidases, widely expressed throughout the PDAC TIME. Accordingly, Ado-related gene expression is associated with worse overall survival in PDAC (P < 0.0002). Using a mouse model of orthotopic PDAC, we demonstrated that FFX was only effective when combined with AB680, a drug in clinical trials that blocks the extracellular ectonucleotidase responsible for Ado production (CD73). FFX+AB680 significantly decreased tumor mass (P < 0.01), reduced liver metastases, and eliminated lung metastases (P < 0.01) compared to treatment with FFX alone. Flow cytometry of tumor-infiltrating leukocytes (TILs) showed that FFX+AB680 enhanced CD8+ T cell activation, reduced CD8+ T cell exhaustion, and increased M1 macrophages. Depleting CD4+ or CD8+ T cells reversed FFX+AB680 efficacy, and CD4+ T cell depletion alone reduced intratumoral CD8+ T cell activation (P < 0.05). scRNA-seq of CD45⁺ TILs confirmed that FFX+AB680, but not FFX alone, increased the frequencies of effector T cells and M1 macrophages and upregulated Type I IFN gene signatures (P < 0.0001). Cell Chat analysis identified significantly stronger ligand-receptor interactions directed from macrophages to T cells after treatment with FFX+AB680 compared to FFX alone. Using murine bone-marrow derived macrophages, we demonstrated that adenosine-receptor agonism could prevent Ifnb1 expression by limiting phosphoactivation of Irf3 downstream of STING activation. These suppressive effects are reversed by Ado receptor blockade, highlighting a regulatory interplay between Ado metabolism and STING signaling that influences immune responses in the PDAC TIME. Ado inhibition of STING signaling was also demonstrated with human PBMCs and an ex vivo living tissue model of PDAC patient tumors. Our data support a model whereby adenosine, generated as a consequence of chemotherapy, can limit tumor-suppressive IFN production by interrupting STING signaling in macrophages in the PDAC TIME. These data reveal CD73 activity as a critical therapeutic vulnerability in PDAC that can improve responses to FFX. An ongoing UCLA clinical trial combining FFX with AB680 dramatically reduces serum adenosine, precludes tumor involvement in lymph nodes and vasculature, and yields exceptionally rare near complete/complete pathologic responses in 50% of patients. Citation Format: Alykhan Premji, Jason Link, Khalid Rashid, Weihang Zhao, Evan Abt, Michael Srienc, Serena Zheng, Luyi Li, Zev Wainberg, Caius Radu, Timothy Donahue. The CD73 Inhibitor, AB680 Enhances Chemotherapy-Induced STING Activation and T Cell-Mediated Tumor Immunity in Pancreatic Ductal Adenocarcinoma [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A113.

Succinate Dehydrogenase loss causes cascading metabolic effects that impair pyrimidine biosynthesis.

Impaired availability of the amino acid aspartate can be a metabolic constraint of cell proliferation in diverse biological contexts. However, the kinetics of aspartate depletion, and its ramifications on downstream metabolism and cell proliferation, remain poorly understood. Here, we deploy the aspartate biosensor jAspSnFR3 with live cell imaging to resolve temporal relationships between aspartate and cell proliferation from genetic, pharmacological, and nutritional manipulations. In cells with impaired aspartate acquisition from mitochondrial complex I inhibition or constrained uptake in aspartate auxotrophs, we find that the proliferation defects lag changes in aspartate levels and only manifest once aspartate levels fall below a critical threshold, supporting the functional link between aspartate levels and cell proliferation in these contexts. In another context of aspartate synthesis inhibition, impairing succinate dehydrogenase (SDH), we find a more complex metabolic interaction, with initial aspartate depletion followed by a rebound of aspartate levels over time. We find that this aspartate rebound effect results from SDH inhibition disproportionately impairing pyrimidine synthesis by inhibiting aspartate transcarbamoylase (ATCase) through the dual effect of diminishing aspartate substrate availability while accumulating succinate, which functions as a competitive inhibitor of aspartate utilization. Finally, we uncover that the nucleotide imbalance from SDH inhibition causes replication stress and introduces a vulnerability to ATR kinase inhibition. Altogether, these findings identify a mechanistic role for succinate in modulating nucleotide synthesis and demonstrate how cascading metabolic interactions can unfold to impact cell function.