Proximity Ligation Research Articles

Abstract 1488: DNA polymerase kappa slows replication fork speed by promoting fork reversal in glioblastoma

Abstract Replication fork stalling in response to replication stress triggers the activation of replication stressresponse (RSR) pathways to facilitate repair and restart replication processes, including fork reversal andtranslesion DNA synthesis (TLS). During fork reversal, the replication fork undergoes a structural rearrangementto form a four-stranded structure, known as a “chicken foot, ” which helps regulate fork speed by recruitment ofdifferent fork reversal factors, including HLTF, and SMARCAL1. Optimal replication conditions ensure a relativelyconstant and rapid fork progression rate, which is essential for timely genome duplication. TLS requiresspecialized DNA polymerases that assist fork progression by promoting direct bypass of replication blocks. Inglioblastoma, the most aggressive and resistant form of brain cancer, the TLS polymerase kappa (Pol κ) isoverexpressed. This overexpression causes resistance to standard therapeutics and is associated with poorprognosis. A major goal of our study is to understand how Pol κ protects glioma cells from endogenous oncogenicstressors and the DNA-damaging effects of chemotherapeutics. Results from Eoff lab indicate that Pol κ slowsreplication fork speed in glioma cells (T98G, U118-MG) without significantly affecting non-glioma cells (HAP-1, U2OS, RPE). We hypothesized that Pol κ slows replication fork speed by promoting fork reversal in glioma cells.To test this hypothesis, we used a proximity ligation assay (PLA) to verify the colocalization of fork reversalfactors SMARACL1, and HLTF to sites of DNA synthesis in glioma cells. PLA measures the proximity betweenproteins and newly synthesized DNA labeled with nucleotide analog 5-ethynyl-2′-deoxyuridine (EdU), indicatingtheir potential interaction or colocalization, as evidenced by the appearance of fluorescent foci. EdU-SMARCAL1, or EdU-HLTF PLA foci were increased in T98G WT without a significant increase in POLK KO T98G cellsfollowing treatment with 50 nM CPT with no considerable increase in POLK KO T98G cells. These resultssuggest that Pol κ has a role in promoting the colocalization of fork reversal proteins to sites of DNA synthesisin T98G (glioma) cells. To support this conclusion and to study how this affects fork speed, we used DNA fiberspreading (DFS) to determine fork speed with/without depletion of the fork reversal factor SMARCAL1 and HLTF.Depletion of SMARCAL1 and HLTF produced longer IdU tracts in T98G WT cells, indicating faster fork speed.The fork acceleration phenotype in POLK-KO T98G cells remained unaffected by the knock-down ofSMARCAL1, suggesting that these proteins work in the same pathway to regulate fork dynamics in glioblastomacells. These results are the first to establish a link between Pol κ and fork reversal, helping to explain how glioma-specific mechanisms tolerate high rates of DNA damage in the tumor microenvironment. Citation Format: Reham S. Sewilam, Megan R. Reed, Robert L. Eoff. DNA polymerase kappa slows replication fork speed by promoting fork reversal in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1488.

Abstract 2952: The oncomucin MUC4 EGF domains: A new paradigm to target ErbB/HER and RTK receptors in pancreatic adenocarcinoma using inhibitory small molecules

Abstract Pancreatic adenocarcinoma is one of the deadliest cancers with an increasing incidence. It is mainly due to absence of diagnosis and no effective treatments, and a strong resistance to therapies. It is thus crucial for this devastating cancer to find new therapeutic alternatives. In this aim, we work on the interaction of the mucin MUC4 with receptor tyrosine kinases (RTK), and the targeting of MUC4 using small inhibitory molecules. MUC4, a large glycoprotein overexpressed at the surface of pancreatic cancer cells, is associated with poor prognosis of the tumor, increase of proliferation and migration as well as apoptosis and resistance to therapies. Moreover, we have shown that MUC4EGF domains interact with and upregulates the activity of ErbB2/HER2, this complex being a strong promoter of tumorigenesis. Use of small inhibitory molecules would circumvent the steric hindrance played by MUC4 glycosidic chains. We have now, using molecular dynamics and peptide library screening, identified a specific MUC4EGF domain inhibitory peptide (i) capable of inhibiting MUC4-ErbB2 complex formation and activity, (ii) having anti-proliferative activity and (ii) resensitizing pancreatic cancer cells to Trastuzumab and Pertuzumab ErbB2-targeted therapies.During these studies, we also observed that MUC4 interaction with ErbB2 led to the activation of the other members of the ErbB/HER family as well as other RTKs showing structural similarities with the ErbB/HER family. Our hypothesis is thus that MUC4 may play the role of an oncogenic interaction platform at the surface of pancreatic cancer cells. The interactions between MUC4 and the ErbB/HER family, Met, Axl, EpCAM, and IGF-1R were studied and characterized using Proximity Ligation Assay, MicroScale Thermophoresis, co-immunoprecipitation, GST pull-down. Our results show that MUC4 interacts directly via its EGF domains with and activate ErbB1,3,4, Met, Axl, IGF-1R and EpCAM. These interactions (i) are independent of ErbB2 expression and (ii) activate downstream oncogenic signaling pathways to maintain proliferation and migration capacities of pancreatic cancer cells. At this time, other drugs (tyrosine kinase inhibitors, mAbs) are currently being screened in association with our MUC4 inhibitory peptide to find the best therapeutic combination possible. Taken together, these results provide new paradigms on the inhibitory small molecule-based therapeutics targeting of ErbB/HER and RTK receptors as partners of the oncomucin MUC4 in pancreatic cancer but also in all epithelial cancers overexpressing these complexes. Citation Format: Tristan Girgin, Nicolas Stoup, Elsa Faillon, Maxime Liberelle, Nicolas Lebegue, Isabelle Van Seuningen. The oncomucin MUC4 EGF domains: A new paradigm to target ErbB/HER and RTK receptors in pancreatic adenocarcinoma using inhibitory small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2952.

Abstract 392: Pyrimethamine inhibits NRF2Mut in esophageal squamous cell carcinoma as a molecular glue

Abstract Esophageal squamous cell carcinoma (ESCC) is a deadly disease and one of the most aggressive cancers of the gastrointestinal tract. As a master transcription factor regulating the stress response, nuclear factor erythroid 2-related factor 2 (NRF2) is often mutated and becomes hyperactive, and thus causes chemo-radioresistance and poor survival in human ESCC. In our previous study, we have identified pyrimethamine (PYR) as an NRF2 inhibitor using a small molecule screening, and further validated it as an inhibitor of NRF2 expression in NRF2Mut ESCC cells in vitro and Nrf2E79Q-driven esophageal phenotype in mice. As a result, PYR has moved into a Phase I window of opportunity trial (NCT05678348). This study is aimed to investigate the molecular and phenotypic consequences of NRF2 hyperactivation in ESCC, and elucidate the mechanism of action of PYR. We first generated isogenic human ESCC cells through CRISPR-Cas9 knockout for comparison with its parental cells. RNAseq and Western blotting showed that NRF2Mut overexpression downreulated the expression of squamous differential markers and upregulated the expression of proliferation markers, while NRF2WT overexpression showed different patterns. Flow cytometry and live cell imaging further showed that NRF2 activation modulated cell cycle distribution and proliferation, but not apoptosis of human ESCC cells. PYR increased chemosensitivity and radiosensitivity in NRF2Mut ESCC cells. Co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA) suggested that PYR promoted NRF2-KEAP1 binding as a molecular glue in NRF2Mut ESCC cells, but not in NRF2WT ESCC cells. In summary, NRF2 hyperactivation differentially modulated squamous differentiation and cell proliferation in NRF2Mut and NRF2WT ESCC cells. PYR enhanced chemo-radiosensitivity by acting as a molecular glue specifically in NRF2Mut ESCC cells, supporting PYR as a novel therapy for NRF2Mut ESCC. Citation Format: Zhaohui Xiong, Chorlada Paiboonrungruang, Boopathi Subramaniyan, Candice Bui-Linh, Yahui Li, Huan Li, Francis Spitz, Xiaoxin Chen. Pyrimethamine inhibits NRF2Mut in esophageal squamous cell carcinoma as a molecular glue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 392.

Abstract 5663: High-content screening platform for comprehensive profiling in drug discovery

A robust, multi-modal high-content screening (HCS) platform enables fast and quantitative assessment of cellular phenotypes over a variety of research applications. Our integrated system combines innovative imaging technologies, automated analysis, and cell profiling capabilities to drive drug discovery and biological research programs. Selvita HCS platform features multiparametric cellular phenotyping: simultaneous measurement of morphological, functional, and molecular cellular properties. The data is acquired by automated fluorescence microscopy: high-resolution imaging with multiplexed fluorescent markers. The images are then analyzed, as needed, by standard or machine learning-assisted image analysis. Advanced algorithms are used to accurately extract cellular features. This enables building various kinetic and endpoint assays: cell viability, apoptosis, proliferation, and subcellular localization studies, to name a few. The platform allows multiple application domains, including therapeutic target validation, mechanism of action studies, pharmacological screening and toxicity assessment. Validated across a variety of models under different experimental conditions, our HCS platform generates high-throughput data with high inter-assay consistency. The customizable protocols facilitate adaptable research support across programs in oncology, neuroscience, immunology, and metabolic disease. Examples of successful cases include: tissue analysis with automated identification of areas of interest and subsequent high-resolution rescanning, combined FISH / immunofluorescence studies in target validation, 100+ K compound screening campaign based on reporter cell line and protein-protein interaction study in DNA repair complex assembly, using proximity ligation assay. Future development will focus on expanding the machine learning capabilities, multiplexing potential, and integration of advanced computational biology tools to aid in the improvement of predictive cellular modeling. Citation Format: Maciej B. Olszewski, Natalia Gostynska, Klaudia Lesniak, Alicja Martyniak, Magdalena Kieltyka. High-content screening platform for comprehensive profiling in drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5663.

Abstract 3847: APE1 mediates angiogenesis by stabilizing SMAD3 and blocking its ubiquitin induced degradation in esophageal adenocarcinoma

Abstract Background: The incidence of esophageal adenocarcinoma (EAC) is rapidly rising in the United States and Western countries. Unfortunately, EAC has one of the lowest survival rates due to limited treatment options. Identifying disease drivers through functional studies is crucial for improving treatment options. This study aimed to explore the role of APE1 in regulating SMAD3 and promoting EAC progression. Methods and Results: Analysis of publicly available datasets revealed that APE1 and SMAD3 are upregulated and positively correlated in EAC compared to normal samples. Immunofluorescence staining of esophageal cancer progression tissue arrays confirmed significantly higher levels of APE1 and SMAD3 in EAC than in normal tissues. Western blot analysis showed that silencing APE1 reduced SMAD3 nuclear expression, which was also confirmed by immunofluorescence staining showing a loss of nuclear SMAD3 accumulation following APE1 knockdown. Immunoprecipitation and proximity ligation assays indicated a direct interaction between APE1 and SMAD3 in the nucleus. Further analysis revealed that APE1 binds to the C-terminal MH2 domain of SMAD3, protecting it from ubiquitin-mediated proteasomal degradation by preventing its interaction with the RING finger protein, ROC1. Notably, inhibiting APE1’s redox or endonuclease activity with pharmacological inhibitors or mutants disrupted APE1-SMAD3 binding, suggesting a conformational regulation. Interestingly, knocking down APE1 reduced angiogenesis in tube formation assays and 3D co-cultures; however, this effect was reversed by overexpressing SMAD3, suggesting that APE1 regulates angiogenesis through SMAD3. Conclusion: The findings establish the role of APE1 in regulating SMAD3 in EAC and suggest a potential therapeutic approach for treating EAC patients. Citation Format: Farah Ballout, Heng Lu, Nadeem Bhat, Lei Chen, Dunfa Peng, Zheng Chen, Alexander Zaika, Oliver McDonald, Wael El Rifai. APE1 mediates angiogenesis by stabilizing SMAD3 and blocking its ubiquitin induced degradation in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3847.

Abstract 4491: HSP70 leverages STUB1 to modulate N-Myc protein turnover in lethal prostate cancer

Abstract Background: Imbalances in protein homeostasis (proteostasis) are associated with a wide range of diseases, including neurodegeneration and tumorigenesis. Prostate cancer has proven to be a key model for understanding how proteostasis regulates tumor progression. Neuroendocrine prostate cancer (NEPC) is the most aggressive type of prostate cancer with no effective treatment. The amplification of N-Myc (encoded by MYCN) is a key modification of NEPC. However, it has proven challenging to identify therapeutic strategies that reduce N-Myc transcriptional activity or levels owing to the presence of intrinsically disordered functional domains and lack of enzymatically active sites. In this study, we sought to take a different approach and identify the molecular chaperones involved in N-Myc proteostasis. Methods: N-Myc was knocked down by siRNA. Cell proliferation and N-Myc expression were assessed using cell viability assays and Western blotting, respectively. Rapid immunoprecipitation pull-down assays followed by label-free mass spectrometry were used to identify N-Myc-binding proteins. Co-immunoprecipitation (Co-IP) and Proximity Ligation Assay (PLA) were performed to detect the interaction between HSP70 and STUB1 with N-Myc. In vitro ubiquitination assays were conducted to detect the ubiquitination of N-Myc. Site-directed mutagenesis (SDM) was used to construct N-Myc, STUB1, and ubiquitination deletion or point mutation plasmids. Cycloheximide (CHX) chase assays were employed to examine the half-life of N-Myc. Results: Knockdown of N-Myc by siRNA significantly inhibited the growth of H660, UCDCaP-CR, and CWR22Rv1 cells. A total of 779 proteins were identified as N-Myc-binding proteins. Among them, several HSP70 family proteins, such as HSPA1B (HSP70), HSPA6, and HSPA8 (HSC70), were the most enriched. The HSP70/STUB1 complex interacted with N-Myc and regulated N-Myc protein turnover. This interaction appeared to occur at a degron ‘SELILKR’ in N-Myc. The K416 and K419 of N-Myc seem to be sites for ubiquitination by STUB1, where it added K11-linked polyubiquitin chains for further proteasome degradation. Conclusions: The HSP70 interacts with N-Myc at a conserved binding motif and regulates N-Myc protein turnover through STUB1. Source of Funding: This work was supported in part by grants from National Institutes of Health R37CA249108 (C.L.), R01CA251253 (C.L.), R21CA277171 (C.L.), Department of Defense HT9425-23-1-0144 (C.L.), HT9425-23-1-0325 (C.L.) Citation Format: Pengfei Xu, Joy C. Yang, Bo Chen, Christopher Nip, Shu Ning, Allen C. Gao, Chengfei Liu. HSP70 leverages STUB1 to modulate N-Myc protein turnover in lethal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4491.

Abstract 716: Discovery of biomarkers of brain metastasis using Genomic Proximity Mapping (GPM) on formalin-fixed paraffin-embedded head and neck squamous cell carcinomas

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) encompass a heterogenous classification of solid tumors that account for ∼5% of the total new cancer cases globally. HNSCC presents as metastatic disease at estimated rates of 4-25%, most commonly in bone, liver and lung. Brain metastasis (BM) is uncommon and is particularly deadly. There is a need to better understand biomarkers that may presage brain metastasis in hopes of identifying those patients requiring aggressive treatment to head-off metastatic disease. Furthermore, identification of common biomarkers may elucidate therapeutic strategies to treat this disease. To compliment ongoing targeted sequencing efforts, we have applied high-resolution cytogenomic analysis using the genomic proximity mapping (GPM) platform, CytoTerra, to investigate chromosomal abnormalities associated with BM HNSCC. Methods: GPM uses proximity ligation and short-read sequencing to detect alterations in genomic structure. This technology is compatible with formalin-fixed paraffin-embedded tissue samples because it uses formalin fixation to capture genomic proximity information. 15 µm molecular curls were used as inputs for library preparation from 2 primary tumors of BM HNSCC and 3 samples of non-BM HNSCC. Chromosomal abnormalities were detected using the cloud-based CytoTerra analytic platform which utilizes a suite of AI-based and bioinformatic tools. Results: GPM libraries with sufficient QC statistics were generated from all five samples. Evaluation of HNSCC genomes revealed a wide array of structural alterations commonly observed in cancer, including balanced and unbalanced translocations, inversions, chromothripsis, deletions, duplications, and copy-neutral loss of heterozygosity. Notably structural rearrangements involving the JAK1 and EGFR loci were observed in BM HNSCC samples. In addition we identified a novel NRG1::FAM110B fusion in one case of BM HNSCC. None of these rearrangement involved the disruption of the coding sequence of the gene and therefore would escape detection through standard panel sequencing. Non-BM HNSCC showed similar classes of chromosomal abnormalities and included a deletion of KDM6A. Variants identified in serial sampling of neighboring HNSCC lesions in non-BM HNSCC demonstrated a high degree of overlap, demonstrating reproducibility. Uniquely identified variants allow inferences on tumor evolution between the two lesions. Conclusion: GPM with the CytoTerra next generation cytogenomic platform successfully identified chromosome abnormalities in FFPE HNSCC samples. BM HNSCC samples demonstrated structural alterations in known and targetable oncogenes suggesting that CytoTerra can be a useful tool for novel biomarker discovery in solid tumors, including HNSCC. Citation Format: Emily Reister, Maika Malig, Mary Wood, Alexander Muratov, Ivan Liachko, Stephen Eacker, Ida Deichaite. Discovery of biomarkers of brain metastasis using Genomic Proximity Mapping (GPM) on formalin-fixed paraffin-embedded head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 716.

Abstract 5254: Multi-plexed spatial interaction analysis of the tumor microenvironment in head and neck cancers

Abstract Head and neck cancers are the 7th most common cancer type globally accounting for over 900, 000 cases annually. With increasing numbers of systemic and targeted therapies, including immune checkpoint inhibitors (ICI), it has become increasingly important to understand the complex tumour microenvironment (TME) milieu dictating clinical responses to therapy. In this study, we profiled 40 whole slide, head and neck cancer tissues from patients prior to ICI therapy and profiled the TME for spatial interactions using the NaveniPlex in situ proximity ligation assay (isPLA, Navinci ). Using the Leica BOND RX, formalin-fixed, paraffin-embedded (FFPE) tissue sections were prepared by baking, deparaffinization, and epitope unmasking. Staining was then carried out using the NaveniPlex isPLA technology on the Leica BOND RX instrument. To assess interactions between PD1/PD-L1, CD8/MHCI, and CD3+ T cells, three pairs of Navenibodies were employed simultaneously. Briefly, the NaveniPlex isPLA protocol involves tissue blocking, followed by incubation with primary Navenibodies, ligation, and rolling circle amplification (RCA). Each Navenibody pair generates a distinct RCA product. The RCA products for PD1/PD-L1, CD8/MHCI, and CD3+ T cells were visualized using oligonucleotides conjugated to Atto647N, Atto550, and AlexaFluor488, respectively.Our spatial interaction analysis for PD1/PDL1, CD8/MHCI and CD3+ T-cells showed distinct TME interactions associated with responders and non-responders to ICI therapy. Interactions mapped with cellular phenotypes revealed the cellular neighbourhoods and communities at which these interactions may be useful prognostically. Taken together, this study provides a methodological framework to profile pre-ICI treatment tissues to identify spatial interactions associated with clinical endpoints. Citation Format: Vahid Yaghoubi Naei, Sara Bodbin, Habib Sadeghirad, Rafael Tubelleza, James Monkman, Agata Wicher, Subham Basu, Arutha Kulasinghe. Multi-plexed spatial interaction analysis of the tumor microenvironment in head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5254.

Abstract 2909: Targeting Polθ suppresses CCNE1-amplified cancers via transcription-replication conflicts mediated DNA damage and RAD51 toxicity

Abstract Background: CCNE1 amplification is prevalent in various cancers, including ovarian, endometrial, and breast cancers, and is associated with elevated cyclin E levels, chemotherapy resistance, and poor prognosis. Despite its clinical relevance, no approved therapeutic strategies currently target CCNE1-amplified tumors. DNA polymerase θ (Polθ) has emerged as a promising therapeutic target in DNA repair-deficient cancers, such as HRD tumors. However, its function in oncogene-amplified cancers characterized by high replication stress and endogenous DNA damage remains underexplored. Methods & Results: Public dataset analyses revealed a positive correlation between Polθ and CCNE1 expression across various tumor types in both clinical samples and cancer cell lines. Elevated Polθ expression was linked to worse survival outcomes exclusively in CCNE1-high tumors. Using a doxycycline (Dox)-inducible CCNE1 overexpression HeLa cell model and a panel of CCNE1-amplified, CCNE1-gain, and CCNE1-low cell lines, we demonstrated that CCNE1 overexpression upregulated Polθ expression, enhanced MMEJ repair activity, and increased sensitivity to Polθ inhibitors. Polθ inhibition, achieved through chemical inhibitors or shRNA knockdown, significantly impaired cell viability and exacerbated DNA damage, replication stress, and genomic instability in CCNE1-overexpressing cells. RNA-seq analysis revealed that CCNE1 overexpression promoted replication and transcription initiation events, while immunofluorescence and proximity ligation assays confirmed transcription-replication conflicts (TRCs) as a key outcome of CCNE1 overexpression. Polθ inhibition exacerbated TRC-associated DNA damage, both at the end of early S phase and after a round of cell cycle. In CCNE1-high cells, Polθ inhibition resulted in the accumulation of large RAD51 foci. Unlike normal CCNE1-high cells, where RAD51 foci resolved during cell cycle progression, Polθ inhibition caused persistent foci, indicating failed homologous recombination (HR) repair. Colocalization studies implicated genome G4 sites in this process, suggesting that Polθ compensates for stalled RAD51-mediated repair at G4-prone regions. Conclusions: Polθ inhibition suppresses CCNE1-amplified tumors by amplifying replication stress, DNA damage, and genomic instability. We propose that Polθ supports tumor cell survival in CCNE1-amplified contexts by resolving TRC-associated DNA damage and toxic HR intermediates. Additionally, CCNE1-induced TRCs primarily occur at G4-prone regions, where Polθ mitigates RAD51 repair failure, underscoring its critical role in maintaining genomic stability under oncogene-induced replication stress. Citation Format: Chuancong Zhu, Yu Xu, Xuejiao Zhao, Gordon B. Mills, Ding Ma, Qinglei Gao, Yong Fang. Targeting Polθ suppresses CCNE1-amplified cancers via transcription-replication conflicts mediated DNA damage and RAD51 toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2909.

Abstract 750: Exploring PD1/PDL1 interactions and macrophage-tumor barriers in head and neck cancer: A spatial approach to immunotherapy response

Abstract With nearly a million new cases expected, the global death rate from mucosal head and neck cancer is projected to reach around 50% in 2024. In the U.S., patients with localized tumors have a five-year survival rate of 50-60%. While immune checkpoint inhibitors (ICIs) have shown promise in extending survival, significant challenges remain, particularly the limited effectiveness of PD-1/PD-L1 blockade therapies. Since many studies have shown that PD-L1 protein expression alone may not be a reliable predictor of response to ICI therapy, understanding the spatial context of PD-1/PD-L1 interactions might be crucial for identifying immune evasion mechanisms and predicting responses to ICIs in head and neck cancer. To address this, we aimed to take a more comprehensive approach by mapping PD-1/PD-L1 interactions across n=35 mucosal head and neck squamous cell carcinoma (HNSCC) tissue samples collected before ICI treatment, using a combination of high-plex spatial proteomics and the in situ Navinci Proximity Ligation Assay (isPLA). Formalin-fixed, paraffin-embedded (FFPE) tissue samples were stained using multiplex immunofluorescence and the Navinci Diagnostics isPLA assay to detect PD-1/PD-L1 interactions. After incubation with primary anti-PD1 and anti-PD-L1 antibodies, secondary probes tagged with oligonucleotides were applied, enabling PLA to highlight the interactions through a ligation step and an amplification process to enhance the visibility of the protein interactions. Finally, a detection solution was applied to visualize the amplification product at the sites of PD1/PD-L1 interaction.We identified distinct spatial patterns of PD-1/PD-L1 interactions at the tumor-stroma interface, particularly in progressive disease (PD) patients, who exhibited dense layers of isPLA+ macrophages, CD3e T cells, and tumor cells, suggesting an immunosuppressive barrier in this region. In contrast, complete response (CR) patients showed rare or absent barriers, with enriched immune cell aggregates, including B cells and T cells, near the tumor boundary. These findings were corroborated through neighborhood clustering analysis, revealing significant associations between isPLA+ regions and poor prognosis.The spatially resolved nature of our analysis underscores the critical role of PD-1/PD-L1 interactions in shaping the tumor microenvironment (TME) and highlights their potential as predictive biomarkers for ICI therapy efficacy. Unlike methods that detect mere colocalization, isPLA technology enables us to detect true PD-1/PD-L1 interactions, which are more likely to affect immune suppression directly. Our study also emphasizes the need for further exploration of macrophage and T cell subtypes, as well as PD-1/PD-L1 interactions across tumor-stroma boundaries in various cancers to improve immunotherapy strategies. Citation Format: Vahid Yaghoubi Naei, Rafael Tubelleza, James Monkman, Habib Sadeghirad, Meg L. Donovan, Tony Blick, Agata Wicher, Sara Bodbin, Subham Basu, Robert Stad, Catherine Barnett, Ken O’Byrne, Rahul Ladwa, Caroline Cooper, Majid Ebrahimi Warkiani, Brett GM Hughes, Arutha Kulasinghe. Exploring PD1/PDL1 interactions and macrophage-tumor barriers in head and neck cancer: A spatial approach to immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 750.

Abstract 2084: Development of a novel tri-omic spatial biology assay that provides in-depth immune profiling of the tumor immune microenvironment

Spatial biology has advanced our understanding of cancer biology and shed light on the immense transcriptomic and proteomic heterogeneity that exists within the tumor-immune microenvironment (TiME). To date, most analytical and diagnostic approaches only provide one type of biological readout per tissue section, and the generation of true multi-omic data has remained challenging. We developed a workflow that allows the integration of multiple omic modalities on a single section. The workflow is centered around a new signal removal strategy, EpicIF™ technology, which effectively and rapidly removes fluorophores while leaving tissue and probe chemistries unaffected. Using this workflow on the CellScape™ platform, we iteratively performed spatial proteomic, transcriptomic, and interactomic assays all on the same tissue sections. Formalin-fixed, paraffin-embedded tissue sections were subjected to standard histological processing and then incubated with primary antibodies against PD1 and PD-L1, which were then treated with oligonucleotide-modified secondary probes designed for an in situ Proximity Ligation Assay (isPLA) to detect PD1 and PD-L1 interactions. Next, the detection of RNA targets was accomplished by adding 12 HCR™ HiFi RNA-ISH probes, which were visualized by addition of target-specific fluorescently labeled HCR™ Gold amplifiers (RNA-FISH). Lastly, spatial proteomic labeling was conducted with VistaPlex™ multiplex immunofluorescence (mIF) panels targeting 30+ biomarkers. We deployed this tri-omic assay on samples of primary CNS lymphoma (PCNSL), a rare and aggressive form of B-cell lymphoma that remains an area of unmet clinical need, with over half of diagnosed patients succumbing to the disease. Unlike diffuse large b-cell lymphoma (DLBCL), its morphologically identical systemic counterpart, PCNSL shows sensitivity to PD1 checkpoint inhibition. We investigated if our assay could describe unique interactions between the PD1-PDL1 axis and the lymphoma TiME in PCNSL (n=24), in comparison to DLBCL (n=152) cases. High-resolution imaging with the CellScape platform allowed for precise detection and quantification of isPLA, RNA-FISH and mIF signals on the same slide. The combined assay showed differential biomarker expression and interaction profiles associated with immune-regulatory processes in the interrogated tumor samples, revealing remarkable inter- and intra-tumoral heterogeneity. This spatial multi-omics approach allows for a more comprehensive insight into the complex interplay of immune and non-immune cell populations in the TiME. Citation Format: Arne Christians, Jannik Boog, Daniel Jimenez Sanchez, Sara Bodbin, Agata Wicher, Subham Basu, Aneesh Acharya, Randy Chen, Hong Liang, Shruti Shridar, Tan Char Loo, Ng Siok Bian, Michelle Poon, Anand Jeyasekharan, Oliver Braubach. Development of a novel tri-omic spatial biology assay that provides in-depth immune profiling of the tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2084.

Abstract 5767: Multiplex in situ proximity ligation assay for functional studies of proteins and their interactions in patient samples

Abstract Detecting protein-protein interactions (PPIs) and their localization is essential for the understanding of protein function and enables deep-diving into processes such as signal transduction, synaptic signaling, regulation of gene expression in cancer, identification of diagnostic markers, patient stratification and more. In situ proximity ligation assay (isPLA) is a well-established tool for the study of PPIs in cell and tissue samples. It detects proteins located within 40 nm of each other via antibodies conjugated to oligonucleotides that generate amplified fluorescent or chromogenic signal. The method can be automated and/or combined with multiplexed immunofluorescence (mIF) staining of individual proteins to visualize tissue context e.g., the tumor microenvironment or the immune landscape. However, current isPLA technology has been limited to the detection of a single PPI at a time. We hereby present a new multiplexed mix-and-match approach to isPLA, which overcomes this limitation. We developed a 9-plex assay based on the Naveni® technological solution, suitable for the simultaneous detection of up to nine PPIs or single proteins (here collectively referred to as markers) of the user’s choice. Every marker is detected by two directly conjugated primary antibodies (Navenibodies) that participate in the isPLA reaction provided the proximity criterion is fulfilled, leading to specific and strong signal. Additional cycles incorporating other technologies such as mIF or HCR-FISH are also compatible with the method. For isPLA markers, users can choose from a set list of possibilities, including various signal transduction networks such as the RAS/MAPK pathway, the PD1/PD-L1 and PD1/PD-L2 receptor-ligand interactions, various B- and T-cell markers, cell adhesion proteins and more. Thanks to this flexible approach, it was possible to create a tailored 9-plex study, investigating interactions involving and downstream of PD1, as well as activation of nodes in the RAS/MAPK pathway. ERK activation downstream of KRAS mutations has been shown to increase PD-L1 expression in several cancers (e.g., lung and colorectal cancer), which in turn helps tumor cells evade immune detection by binding to PD1 on T-cells, inhibiting T-cell activity and promoting immune tolerance to the tumor. Our data could help indicate which patients would be the best candidates for immune checkpoint or MAPK inhibition therapy. In summary, our multiplex isPLA approach is a sensitive and specific method that visualizes low and high abundant targets in tissues and adds value by uncovering protein interplay in complex signaling networks with implications in translational research. Citation Format: Hampus Elofsson, Desirée Edén, Olof Hahne, Linda Arngården, Frida Tallqvist, Carl-Magnus Clausson, Axel Klaesson, Annica Önell, Sara Bodbin, Caroline Gallant, Agata Zieba Wicher. Multiplex in situ proximity ligation assay for functional studies of proteins and their interactions in patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5767.

Bipedal DNA walker integrated resonance energy transfer to construct a sensitive electrochemiluminescence biosensor for serotonin detection.

Bipedal DNA walker integrated resonance energy transfer to construct a sensitive electrochemiluminescence biosensor for serotonin detection.

CD70 recruitment to the immunological synapse is dependent on CD20 in B cells

CD20 is a four-transmembrane protein expressed at the surface of B cells from late pro-B cells to memory B cells, with the exception of plasma cells. Its expression pattern makes it an attractive therapeutic target for different B cell malignancies and autoimmune diseases. Despite the clinical success of CD20-targeting antibodies, the biology of the CD20 protein is still not well understood. We investigated CD20 binding partners in the membrane of human B cells using immunoprecipitation followed by mass spectrometry analysis. We identified a molecular interaction between CD70 and CD20, and confirmed this using proximity ligation assays. CD20-CD70 spatiotemporal colocalization was validated at the plasma membrane of B cells using high-resolution microscopy. Cell surface expression of CD70 was found to be enhanced upon CD20 overexpression, suggesting a role for CD20 in stabilizing CD70 at the B cell membrane. Moreover, we observed impaired B-T cell synapse formation and defective recruitment of CD70 to the immunological synapse in the absence of CD20. Impaired synapse formation was confirmed by deleting CD20 in primary B cells, and analysis of B cells from a CD20-deficient patient. Finally, CD20-deletion resulted in diminished T cell activation and cytokine secretion. Together, this study demonstrates that CD20 interacts with CD70 at the B cell membrane, and that CD20 is required for immune synapse formation between B and T cells and consequent T cell activation.

Technical Considerations for Detecting Protein-Protein Interactions Using Proximity Ligation Assay.

Proximity ligation assay has been widely used to detect protein-protein interaction in cells and tissues. While with great sensitivity, its specificity was often neglected. Here, we report the existence of varying levels of false positives observed with this assay, most likely due to its high sensitivity. We also provide suggestions to minimize false positives for more accurate detection of protein-protein interactions, especially for membrane proteins. These suggestions include co-staining target proteins, using various negative controls and suitable antibodies, avoiding detergents if possible, and validating interactions with complementary methods.

Analysis of rad-51 separation of function allele suggests divergence of the SDSA and dHJ pathways prior to RAD-51 filament disassembly

Abstract DNA double-strand breaks (DSBs) are formed in meiosis, so their repair in the homologous recombination (HR) pathway will lead to crossover formation, which is essential for successful chromosomes segregation. HR contains two sub pathways: synthesis dependent strand annealing (SDSA) that creates non-crossover, and double Holliday junction (dHJ) that generates crossovers. RAD-51 is a protein essential to the formation of all products of HR, as it assembles on the processed DSB, allowing the invasion of the ssDNA into a region of homology. RAD-51 is removed by RAD-54.L after invasion to allow for repair to occur. Here we investigate a separation of function allele of rad-51, rad-51::FLAG, as compared to two other RAD-51 alleles: rad-51::degron and GFP::rad-51. rad-51::FLAG displays slowed repair kinetics, resulting in an accumulation of RAD-51 foci. rad-51::FLAG worms also activate the DSB checkpoint, but to a less extant than that of rad-51 null mutants. In a proximity ligation assay, RAD-54.L and RAD-51 show enriched colocalization in rad-51::FLAG germlines (but not in rad-51::degron), consistent with stalling at the strand invasion step in HR. The defects in RAD-51 disassembly in rad-51::FLAG mutants lead to formation of chromosomal fragments, similar in their magnitude to ones observed in rad-51 or rad-54.L null mutants. However, rad-51::FLAG mutants, (unlike a rad-51 null, GFP::rad-51 or rad-54.L null mutants), displayed no defects in the formation of crossover designated sites (via GFP::COSA-1 localization). Given that rad-51::FLAG worms show checkpoint activation and chromosomal fragments, these results suggest that crossover repair concludes normally, while the non-crossover pathway is perturbed. This is strikingly different from rad-51::degron and GFP::rad-51 strains, which are proficient or deficient in both pathways, respectively. These results suggest that non-crossovers vs crossovers have distinct recombination intermediates and diverge prior to RAD-51 disassembly.

Seeding-competent early tau multimers are associated with cell type-specific transcriptional signatures

The initial molecular alterations of Alzheimer’s disease (AD) are unknown. Established AD is characterized by profound structural and transcriptional alterations in the human brain, with the hallmark neuropathological features being beta-amyloid (Aβ) accumulation in senile plaques and hyperphosphorylated fibrillar tau in neurofibrillary tangles (NFTs). Previous evidence indicates that tau multimerization into small aggregates is one of the earliest molecular alterations, anticipating the accumulation of hyperphosphorylated tau in NFTs. In this study, we investigated the seeding capacity of these early small tau multimers and the transcriptional changes associated with them, aiming to unveil early pathogenic processes in AD-type tau pathology. Early tau multimers visualized with tau proximity ligation assay (tau-PLA) in the post-mortem temporal cortex demonstrated high seeding activity detected by real-time quaking-induced conversion (RT-QuIC) assay and induction of aggregates in a tau biosensor cell line. Using single-nucleus transcriptomics, we showed that brain tissue harboring seeding-competent early tau multimers, but without significant NFT pathology, is associated with substantial gene expression alterations across diverse cell types when compared to control tissue lacking either multimers or NFTs. Differentially expressed genes, such as APP, MAPT, and PSEN1, exhibited significant enrichment of AD heritability in up-regulated genes within excitatory neurons, astrocytes, and oligodendrocytes. Pseudotime analysis exposed a positive correlation between the progression of tau pathology and the expression of genes marking reactive astrocytes. In summary, our results support the hypothesis that seeding-competent tau multimerization may initiate AD-type tau pathology cascades before the accumulation of tau in NFTs. This research contributes valuable insights into the early molecular events associated with AD, with implications for future diagnostic and therapeutic strategies.

Robust visualization of membrane protein by aptamer mediated proximity ligation assay and Förster resonance energy transfer.

Robust visualization of membrane protein by aptamer mediated proximity ligation assay and Förster resonance energy transfer.

VAPA mediates lipid exchange between Leishmania amazonensis and host macrophages.

Leishmania is a vacuolar pathogen that replicates within parasitophorous vacuoles inside host phagocytes. To promote its replication, Leishmania relies on a panoply of strategies to acquire macromolecules such as lipids from host macrophages. In this study, we have evaluated the role of VAPA, an endoplasmic reticulum-resident membrane protein involved in inter-organellar lipid transport, in macrophages infected with L. amazonensis. Following infection of bone marrow-derived macrophages with metacyclic L. amazonensis promastigotes, we observed that VAPA gradually associates with communal parasitophorous vacuoles. Knockdown of VAPA prevented the replication of L. amazonensis, which was accompanied by an impaired parasitophorous vacuole expansion. Using fluorescent ceramide, we established that VAPA is required for the transport of sphingolipids to the parasitophorous vacuoles and for its acquisition by L. amazonensis amastigotes. Proximity-ligation assays revealed that L. amazonensis hijacks VAPA by disrupting its interactions with the host cell lipid transfer proteins CERT and ORP1L. Finally, we found that VAPA is essential for the transfer of the Leishmania virulence glycolipid lipophosphoglycan from the parasitophorous vacuoles to the host cell endoplasmic reticulum. We propose that VAPA contributes to the ability of L. amazonensis to colonize macrophages by mediating bi-directional transfer of lipids essential for parasite replication and virulence between the parasitophorous vacuoles and the host cell endoplasmic reticulum.

MIRO1 Is Required for Dynamic Increases in Mitochondria-ER Contact Sites and Mitochondrial ATP During the Cell Cycle.

Mitochondria-ER contact sites (MERCS) are vital for mitochondrial dynamics, lipid exchange, Ca2+ homeostasis, and energy metabolism. We examined whether mitochondrial metabolism changes during the cell cycle depend on MERCS dynamics and are regulated by the outer mitochondrial protein mitochondrial rho GTPase 1 (MIRO1). Wound healing was assessed in mice with fibroblast-specific deletion of MIRO1. Wild-type and MIRO1-/- fibroblasts and vascular smooth muscle cells were evaluated for proliferation, cell cycle progression, number of MERCS, distance, and protein composition throughout the cell cycle. Restoration of MIRO1 mutants was used to test the role of MIRO1 domains; Ca2+ transients and mitochondrial metabolism were evaluated using biochemical, immunodetection, and fluorescence techniques. MERCS increased in number during G1/S compared with during G0, which was accompanied by a notable rise in protein-protein interactions involving VDAC1 and IP3R as well as GRP75 and MIRO1 by proximity-ligation assays. Split-GFP ER/mitochondrial contacts of 40 nm also increased. Mitochondrial Ca2+ concentration ([Ca2+]), membrane potential, and ATP levels correlated with the formation of MERCS during the cell cycle. MIRO1 deficiency blocked G1/S progression and the cell-cycle-dependent formation of MERCS and altered ER Ca2+ release and mitochondrial Ca2+ uptake. MIRO1 mutants lacking the Ca2+-sensitive EF hands or the transmembrane domain did not rescue cell proliferation or the formation of MERCS. MIRO1 controls an increase in the number of MERCS during cell cycle progression and increases mitochondrial [Ca2+], driving metabolic activity and proliferation through its EF hands.