Changes In Expression Research Articles

Abstract 2404: Change in expression of microRNAs following limb amputation in canine osteosarcoma patients predicts survival time after chemotherapy

Abstract Osteosarcoma (OS) is the most common primary bone tumour in dogs and humans. There are an estimated 8, 000 new cases of canine OS per year in the United States compared to only 1, 000 in humans. The most common primary location for both species is the appendicular skeleton, with metastases forming mostly in the lungs and bones. Appendicular OS is not only more common in dogs, but also more aggressive, making dogs a valuable spontaneously metastasizing animal model of aggressive human OS. Median survival after amputation and adjuvant chemotherapy in dogs is 1 year, less than 30% survive 2 years, and an estimated 90% of dogs develop metastases after therapy. In contrast, humans have a 76% 5-year survival rate and 15-25% will develop metastases. Predicting clinical outcomes after treatment is difficult in both species, particularly for those without clinically detectable metastatic disease at diagnosis. MicroRNAs (miRNAs) are small, non-coding RNA that control gene expression and are often dysregulated in cancer. MiRNAs share entirely or nearly identical sequences between dogs and humans and are present in blood, making them good potential biomarkers. Recently, we identified multi-miRNA models which could reliably predict survival using pre-amputation canine OS plasma samples. Tumour cells can release miRNAs into the blood via active and passive mechanisms; therefore, with the primary tumour removed after amputation, miRNA expression levels in the blood should be altered. We hypothesize these changes in miRNA expression can be used to prognosticate canine OS patients better. MiRNA expression was measured using real-time quantitative PCR in matched pre- and post-amputation plasma samples from canine OS patients (n=25) at the Ontario Veterinary College. Change in expression was calculated for each miRNA as [(post-amputation) - (pre-amputation)] for each matched pair of samples. For each dog, survival was measured as overall survival and disease-free interval (DFI). Overall survival is the time from diagnosis to death and DFI is the time from diagnosis to clinically detectable metastasis. Dogs were censored if they died from non-OS events, were lost during follow-up, or were still alive. Prognostic ability was evaluated for individual miRNAs by identifying an optimal expression cutoff point using the survminer package in R and visualized using Kaplan-Meier curves. Decision trees were grown using the rpart package in R to evaluate whether multi-miRNA models offered improved prognostic ability compared to single miRNAs. We found that a model combining miRNA-144 and miRNA-23a, for both overall survival and DFI metrics, was able to identify 3 distinct groups which reflected survival time worse, on par, and better than the median survival time reported in literature. These models offer a simple prognostic test that may have clinical applications for veterinary and human OS patients. Citation Format: Heather Treleaven, Michael Edson, Latasha Ludwig, Alicia Viloria-Petit, R. Darren Wood, R. Ayesha Ali, Geoffrey A. Wood. Change in expression of microRNAs following limb amputation in canine osteosarcoma patients predicts survival time after chemotherapy [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 2404.

Abstract 5275: A dynamic equilibrium of innate immune deaminases and deregulation in hematopoietic malignancies

Inflammatory cytokine responsive apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC3) cytosine deaminases have been shown to be deregulated during cancer evolution, presenting as gene expression changes and inclusion of distinct C-to-T mutation patterns. However, the exact mechanisms by which APOBEC3 enzymes promote cancer initiation and progression remain to be investigated, especially in hematopoietic malignancies. Building on our advanced sequencing data of healthy, pre-leukemic, and myeloproliferative neoplasm patient samples, we seek to uncover normal and malignant patterns of APOBEC3C overexpression in hematopoietic cells. Overexpression of lentiviral APOBEC3C in normal cord blood, healthy bone marrow, and myeloproliferative neoplasm (MPN) patient hematopoietic stem/progenitor cells (HSPCs) allows us to study the effects of deaminase dysregulation in the hematopoietic niche. These overexpression studies show novel differential gene expression changes, repetitive element alterations, RNA hyper-editing sites, and DNA mutation signatures induced by APOBEC3 mutagenesis. In addition to APOBEC3, we are exploring the upregulation of adenosine deaminase acting on RNA1 (ADAR1), as we have previously shown that these two innate immune deaminases are synchronously upregulated in the high-risk myelofibrosis (MF) stem cell population compared to normal aged bone marrow. We have discovered a co-regulatory relationship dependent on a delicate equilibrium between APOBEC3C and ADAR1, indicating a correlative association between the dysregulation of these essential deaminases and the initiation and progression of hematopoietic malignancies as indicated by survival statistics and gene expression changes. We will use this comprehensive study of innate immune deaminase deregulation to uncover predictive biomarkers and hallmarks of malignant transformation. Citation Format: Jane Marie Isquith, Inge Van der Werf, Jessica Pham, Thomas Whisenant, Ludmil Alexandrov, Catriona Jamieson. A dynamic equilibrium of innate immune deaminases and deregulation in hematopoietic malignancies [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 5275.

Abstract 2241: Immune-modulatory therapeutic cancer vaccines against IDO1 and PD-L1 control tumor growth through target specific changes in the tumor microenvironment

Abstract Despite the established clinical efficacy of checkpoint inhibitors such as blocking antibodies against Programmed Death protein (PD)-1 and Programmed Death Ligand (PD-L)1, drug resistance remains a significant obstacle in various cancer types. Indoleamine 2, 3-dioxygenase 1 (IDO1) and PD-L1 are critical immunosuppressive drivers in the tumor microenvironment (TME) that favor cancer progression. The development of immune-modulatory vaccines against IDO1+ and PD-L1+ immune suppressive and/or tumor cells in the TME (IO102-IO103) has revealed promising clinical results1 and is now being investigated in a Phase 3 clinical trial in advanced melanoma. Using preclinical models, the present study aims to further characterize the mode of action of these vaccines and compare it with conventional checkpoint inhibitors. Anti-tumor efficacy of the vaccines was evaluated by monitoring tumor growth in syngeneic murine models after treatment with IDO1 or PD-L1 peptides in Montanide adjuvant. Tumor tissues, spleens and lymph nodes were collected and processed for flow cytometry or functional readouts. T cell activity was assessed using IFNγ Elispot assay. Gene expression analysis was performed using the Nanostring nCounter PanCancer IO360 Panel on bulk tumors from animals treated with IDO1 or PD-L1 vaccines; or anti-PD1/PD-L1. Vaccination against IDO1 and PD-L1 induced specific T-cell expansion in splenocytes, associated with reduced tumor growth. In particular, PD-L1 specific T cells were also detected in lymph nodes and tumor infiltrating lymphocytes from MC38 tumor-bearing animals. Target specific activation of T cells was confirmed ex vivo upon restimulation of cells by the detection of IFNγ and TNFα within CD8+ cells. Gene expression analysis of tumors revealed distinct immune signatures for each treatment group, either supporting cytotoxic T-cell activity (PD-L1 vaccine) or reducing the immune suppression within the TME (IDO1 vaccine). Comparison with samples from animals treated with anti-PD-1 or anti-PD-L1 showed non-overlapping gene expression changes induced by PD-L1 vaccine. Our results from murine models show that IDO1 and PD-L1 peptide vaccines each contribute to tumor growth control through the activation of target specific T cells and their recruitment at tumor site. Gene expression analysis suggests that this is achieved through changes in the expression of immune-suppressive markers and enhanced T cell activation and cytotoxic activity. The gene expression changes observed were different from the signatures of anti-PD-L1 and anti-PD1, suggesting a complementary mode of action to those therapeutics. Altogether, our data support the clinical potential of IDO1 and PD-L1 immune-modulatory vaccines to improve existing therapeutics in cancer. 1.Kjeldsen et al. Nat Med 2021 DOI:10.1038/s41591-021-01544-x Citation Format: Marion Chapellier, Anne Madsen, Rasmus Agerholm-Nielsen, Lea Svendsen, Inés Lecoq, Evelina Martinenaite, Shih-Chun Shen, Erika Sutanto-Ward, James DuHadaway, Souvik Dey, Dema Ghaban, Mads Hald Andersen, Alexander J. Muller, Ayako Wakatsuki Pedersen. Immune-modulatory therapeutic cancer vaccines against IDO1 and PD-L1 control tumor growth through target specific changes in the tumor 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 2241.

Abstract 5753: Multi-omic analysis links neighborhood disadvantage to elevated stress hormones and tumorigenic markers in ER+ breast cancer patient plasma and tumor samples

Intro: Estrogen receptor positive (ER+) breast cancer (BC) patients living in disadvantaged neighborhoods experience worse survival compared with patients in advantaged neighborhoods. Chronic stress, indicated by elevated cortisol levels, may mediate this disparity by dysregulating inflammatory responses driving BC tumorigenesis. However, the role of cortisol in ER+ BC remains underexplored. Objective: To explore associations between pre-treatment plasma cortisol, inflammatory proteins, tumoral gene expression changes, and neighborhood disadvantage in women with primary ER+ BC compared to cancer-free controls. Methods: Pretreatment plasma samples were collected from women with primary ER+ BC (n=98) and cancer-free controls (n=144). Tumor samples were obtained from a subset of cases (n=77). Samples were from three Chicago hospitals serving disadvantaged or advantaged neighborhoods, as determined by Community Needs Assessment reports. Plasma samples were analyzed via a steroid hormone metabolomics assay and Olink Proteomic’s inflammatory proteins panel. Gene expression in tumor samples was analyzed via the Breast Cancer 360 Nanostring panel. Mixed models, adjusted for age and BMI, compared: plasma samples from disadvantaged cases (n=48) vs. controls (n=114), advantaged cases (n=50) vs. controls (n=27), advantaged (n=27) vs. disadvantaged (n=117) controls, and advantaged (n=50) vs. disadvantaged (n=48) cases, and tumor samples from advantaged (n=44) vs. disadvantaged (n=33) cases. Results: Thirteen steroid hormones, 85 plasma proteins, and 759 tumor tissue genes were detected. Among cases vs. controls, cortisol-related metabolites (cortisol/cortisone ratio, 11-deoxycortisol, 17a-OH-progesterone, androstenedione) were elevated in cases (p<0.05). The cortisol/cortisone ratio remained significantly elevated in disadvantaged cases vs. controls (p<0.01) but not advantaged cases vs. controls. In disadvantaged individuals, 62 inflammatory proteins were elevated (p<0.05). Forty overlapped between controls-only and cases-only analyses, while 21 were unique to cases, including five weakly correlated with the cortisol/cortisone ratio (p<0.05). Seventy-six genes were upregulated in tumors from disadvantaged vs. advantaged cases, including those involved in cell cycle regulation, proliferation, and HIF-1 signaling. Motif analyses showed NFKB as a top motif (p<0.01), suggesting dysregulated cortisol signaling. Of the 45 downregulated genes, many were involved in anti-inflammatory pathways (p<0.05). Conclusion: BC diagnosis in disadvantaged individuals is associated with elevated cortisol, potentially contributing to elevated circulating inflammatory proteins and intratumoral gene expression changes that promote tumor progression. Citation Format: Hannah Heath, Oana C. Danciu, Garth Rauscher, Natalie Pulliam, Elona Liko-Hazizi, Sarah Friedewald, Seema Khan, Julie Kim, William Gradishar, Jonna Frasor, Kent F. Hoskins, Zeynep Madak-Erdogan. Multi-omic analysis links neighborhood disadvantage to elevated stress hormones and tumorigenic markers in ER+ breast cancer patient plasma and tumor 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 5753.

Abstract 6760: Anticancer mechanisms of quercetin revealed via a systems biology approach

Abstract Introduction: Quercetin is a dietary flavonoid found in apples, onions, and berries. Previous in vitro and in vivo studies have identified its pro-apoptotic, anti-angiogenic, antioxidant, and anti-inflammatory activities, in addition to its ability to sensitize cancer cells to traditional chemotherapies. However, there has not yet been a comprehensive evaluation of its anticancer properties on a large panel of cancer cell lines. We hypothesized that quercetin exhibits varied anticancer effects depending on the particular cell line and its tissue of origin. Methods: For cell survival assessments, 77 cancer cell lines from the NCI60 dataset were seeded into 96-well plates and incubated for 24 hours. DMSO control or quercetin was then added at 5 ten-fold concentrations to a maximum of 100 µM. 48 hours later, the cells were fixed and stained, and cell viability measured with CellTiter-Glo. Next, for transcriptomic profiling, the L1000 assay from The Broad Institute was used. For these experiments, 10 µM of quercetin was added to 9 core cell lines seeded in 384-well plates. Then, the Affymetrix GeneChip HG-U133 Plus 2.0 Array was used to generate a gene expression signature for quercetin, which was then compared with signatures from other compound and genetic perturbations. Connectivity scores were used to classify the L1000 signatures according to similarity to the gene expression changes produced by quercetin. The data for cell survival and transcriptomic profiling were extracted from PharmacoDB and Clue.io, respectively. Results: The most sensitive cell line to quercetin was the glioblastoma cell line U-87/H.Fine (IC50=5.03 µM), and the least sensitive was the renal cell carcinoma cell line TK-10 (IC50 = 5780.61 µM). Overall, quercetin was most potent against myeloid and prostate cancers and least potent against ovary and fallopian tubes cancers. As indicated by high median tau scores, quercetin administration induced gene expression changes similar to NFKB2, PTK2 and EPCAM knock-downs, which are associated with reductions in inflammation, cell proliferation, and cancer growth and progression. The cellular effects of quercetin were analogous to the administration of other anticancer compounds such as CP466722, rhamnetin, and piceatannol. CP46672 can sensitize tumors to ionizing radiation, rhamnetin acts as an antioxidant, and piceatannol is a naturally occurring anticancer agent. Conclusions: This study provides a comprehensive in vitro examination of the impact of quercetin on the viability and gene expression profiles of cancer cells of various origins. Further research on the molecular changes induced by quercetin in animal models and human subjects would help inform the applicability of quercetin in human cancers and guide its use in cancer prevention and treatment. Citation Format: Adriana Goraieb, Adin Aggarwal, Kenneth W. Yip. Anticancer mechanisms of quercetin revealed via a systems biology approach [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 6760.

Abstract 4606: Digital biological twin platform: multiomics in patient derived organoids combined with machine learning

Abstract Patient-derived organoids (PDOs) are in vitro 3D structures that mimic the complexity of tumors, including their molecular and phenotypic features. Recently, numerous studies have demonstrated their potential as preclinical model for drug screening and tailored treatment selection for patients.Non-small cell lung cancer (NSCLC) with its rapid growth and early metastasis onset, represents the most common cause of cancer-related deaths worldwide. Current treatments offer limited long-term survival, necessitating novel approaches. Immunotherapy, specifically anti-PD1 and anti-PD-L1, has transformed NSCLC treatment, but only a small percentage of patients respond. There is an unmet need to develop novel clinically relevant models of NSCLC to improve cancer patient risk stratification as well as to predict future targeted and/or immuno-therapy susceptibilities that may be met with PDOs.We have created the Digital Biological Twin lab, a platform that performs multi-omics analysis on PDOs combining advanced cellular, genomic, and molecular techniques with machine learning models, aiming to improve cancer patient risk stratification and predict future therapy susceptibilities. This platform, part of the Oncology translational research organization at GSK, will provide proof of concept data from patient samples collected in hospital biobank-linked organoid studies. In an ongoing study we are establishing PDO models from 200 NSCLC tissues collected at the Guy’s and St Thomas’ Biobank in London. The PDOs are treated with standard care, then co-cultured with pre-activated immune cells in the presence of IO drugs, and subjected to assays including exosome isolation, protein and DNA content analysis, imaging assays, flow cytometry analysis of tumor markers and immune phenotype, and transcriptomic analysis. This multi-omics approach allows for simultaneous analysis of different biological parameters after treatment to understand tumour cell interaction with immune cells and drug-induced changes. The results will be used to identify potential biomarkers of treatment response and generate hypotheses for drug resistance.Initial results in a pilot study with PDOs from NSCL and head and neck cancers have shown that several factors correlate with and can be combined in a linear elastic-net or robust Bayesian regression model that predicts resistance risk, as measured by PDO cell viability or IC50. These factors include the treatment induced change in CD45+ immune cell infiltrating, changes in tumour PD-L1 and immune PD-1 expression, changes in MYC, TIGIT and CD96 expression. More details of a genomic analysis will be presented in a companion presentation at this conference. Citation Format: Anna Pasto, Paul Barber, Halh Al-Serori, Maria Fankhaenel, Diianeira Maria Tsiridou, Shani Austin-Williams, Finnian Firth, Ruben M. Drews, Amro Ahmed-Ebbiary, Michael Finn, Mint Htun, Ellie N. McDowall, Annie-Rose Henry, Susan Ndagire, Eleni M. Karapanagiotou, Cheryl E. Gillet, Andrea Bille, Tony Ng. Digital biological twin platform: multiomics in patient derived organoids combined with machine learning [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 4606.

Abstract 4681: Multiomic insights into mechanisms of androgen receptor-driven radioresistance in triple-negative breast cancer

Background: Due to the lack of molecular-targeted therapies for triple-negative breast cancer (TNBC), there have been efforts to use multimodal strategies to enhance the efficacy of existing therapies such as radiation. The androgen receptor (AR) has been identified as a radiosensitizing target and is expressed in 25-50% of TNBC tumors. The combination of AR inhibitors, such as enzalutamide (enza), and radiation therapy (RT), leads to radiosensitization in AR+ TNBC models but the mechanism by which AR mediates radioresistance is not known, and targeting this radioresistance could provide a novel strategy to improve outcomes in this aggressive breast cancer subtype. Following RT, AR is translocated to the nucleus suggesting that AR may drive transcriptional changes in response to RT. Methods: Bulk RNA-seq with and without R1881 stimulation and RT was used to assess transcriptional changes at 24 hours. RPPA analysis evaluated protein and phophoprotein changes. AR ChIP-seq was used to identify changes in AR binding and Bru-seq to profile changes in nascent RNA. These experiments were performed in two AR+ TNBC models, MDA-MB-453 and MFM-223 cells, with and without enza and RT. Clonogenic survival assays were performed to assess for radiosensitivity. Results: RNA-seq and RPPA analysis demonstrated numerous changes in gene and protein expression after enza-, RT-, and combination treatment. Gene set enrichment analysis (GSEA) of these changes nominated alterations of MAPK pathway genes with R1881 and R1881+RT compared to controls. To assess the MAPK/ERK pathway, constitutively active ERK was overexpressed in AR+ TNBC models which induced radioresistance, confirming this relationship. As expected, AR ChIP-seq data showed enza treatment induced a significant reduction in AR peaks compared to DMSO-treated controls. Radiation induced fewer changes in AR binding than enza treatment. Bru-seq GSEA showed significant upregulation of MYC target gene sets in response to RT in control conditions. Conversely, with enza treatment, there was no significant MYC target gene set upregulation. While there were no changes in MYC expression or AR binding to known MYC enhancers, there was increased binding of AR to known MYC binding sites in control samples in response to RT. This RT-induced binding to MYC sites was not seen in the enza-treated samples suggesting that AR inhibition may dampen the RT induction of MYC signaling leading to radiosensitization. Conclusions: These findings provide new insights into the molecular underpinnings of AR-mediated radioresistance and identify MAPK and MYC pathways as potential therapeutic targets to enhance radiosensitivity in AR+ TNBC. Ongoing studies aim to translate these findings into clinically actionable strategies. This work expands our understanding of AR-mediated radioresistance which may uncover resistance mechanisms to combination AR inhibition and RT. Citation Format: Benjamin Hauk, Breanna McBean, Anna Michmerhuizen, Douglas Gurdak, Savannah Tidmore, Michelle Paulsen, Lynn Lerner, Connor Ward, Kassidy Jungles, Daniel Spratt, Lori Pierce, Mats Ljungman, Corey Speers. Multiomic insights into mechanisms of androgen receptor-driven radioresistance in triple-negative breast 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 4681.

Abstract 6267: Training a machine learning model to predict T cell cytokine secretion function using a novel single-cell flow cytometry method that measures kinetic phenotypes

Advances in high-dimensional single-cell analysis have transformed our understanding of cellular heterogeneity and its role in complex pathologies. Most datasets are acquired through single-cell RNA sequencing, which provides expression of thousands of genes on each cell at a given timepoint. The typical workflow involves identifying potential new cell types via clustering, generation of hypotheses, and followed by post-hoc experiments to test these hypotheses. Technical bottlenecks include the sparse and noisy nature of sequencing data, and the inability to link observations directly to function due to the destructive nature of sequencing. Supervised machine learning (ML) is an increasingly useful approach to generate powerful predictive models of complex processes. This approach requires both high quality and high quantity of training data. An ideal method to acquire this data would be able to follow individual cells over time while measuring changes in phenotype and function. Here, we describe a novel single-cell method using non-destructive optical barcodes that enables acquisition of dynamic high-dimensional data at scale. We apply this method to generate unprecedented predictive models of single-cell function using a human T-cell activation model. Our barcoding approach relies on laser particles (Kwok et al., Nat. Biomed. Eng. 2024) to track individual cells over time. T-cells are barcoded, activated, and measured repeatedly over time using a flow cytometer to capture kinetic changes in expression of protein biomarkers (“kinetic phenotyping”). We link these kinetic phenotypes to cytokine secretion function through a stimulation step and cytokine secretion assay. The single-cell phenotype-to-function datasets are then used to train ML models to predict cytokine secretion prior to stimulation. We observed increased expression of CD25, PD-1 and CD69 in T cells post-activation from 2h to 48h, as expected. However, distinct kinetic patterns were observed. For example, while CD25 increased largely monotonically, there was significant transient expression of PD-1. These observations were consistent across multiple donors. We employed supervised ML (Random Forrest) to train a classification model to predict cytokine secretion from single-cell kinetic phenotypes. The model was validated using 5-fold cross-validation and achieved ROC-AUC values of 0.74 to 0.89 for predicting TNFa, IFNg and IL2 secretion. For each cytokine, we identified the most predictive biomarkers (and timepoint). Finally, we used our classification model to describe T-cell heterogeneity by probability of cytokine secretion and validated our method using conventional analyses. Our approach is expected to be of great utility for predicting single-cell function in complex systems, from immunotherapy development to cancer pathogenesis. Citation Format: Sheldon J.J. Kwok, Yulia Shulga, Emane Rose Assita, Sarah Forward, Trevor Brown, Pratip Chattopadhyay. Training a machine learning model to predict T cell cytokine secretion function using a novel single-cell flow cytometry method that measures kinetic phenotypes [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 6267.

Abstract 3862: OC201 and OC202e, potential inhibitors of cancer metastasis, suppress EMT in pancreatic cancer

Abstract Objective: Pancreatic cancer is among the most aggressive malignancies, associated with a very poor prognosis due to its rapid progression and resistance to conventional therapies, underscoring the urgent need for novel treatment approaches. Epithelial-mesenchymal transition (EMT) is a key process that enhances the migration and invasion capabilities of cancer cells and is recognized as a pivotal mechanism driving malignancy in pancreatic cancer. Inhibiting EMT is therefore a critical therapeutic strategy. This study aimed to evaluate the anti-metastasis efficacy and define the mode of action of OC201 and OC202e in EMT-induced pancreatic cancer cells. Method: EMT was induced in Panc-1 and MIA PaCa-2 cells through treatment with TGF-β1 (5 ng/ml) and TNF-α (10 ng/ml). Cell migration and invasion were assessed through functional assays, while EMT-related markers (E-cadherin, ZEB1, Snail, Vimentin, and MMP-2) were analyzed via qPCR and Western blot. The cells were subsequently treated with OC201 and/or OC202e, and changes in expression of EMT-related signaling proteins were evaluated. An in vivo hepatic metastasis model was also established using MIA PaCa-2 cells to evaluate the effects of OC201 and/or OC202e treatment on metastasis following four weeks of therapy. Results: Treatment with TGF-β1 and TNF-α significantly increased migration and invasion in both Panc-1 and MIA PaCa-2 cells, accompanied by decreased E-cadherin expression and increased expression of ZEB1, Snail, and Vimentin. Treatment with either OC201 or OC202e reduced cell migration and invasion, suppressed the expression of EMT markers (ZEB1, Snail, Vimentin, and MMP-2), and increased E-cadherin expression. Both compounds inhibited the activation of key EMT signaling proteins, AKT and IκBα, with combination treatment resulting in a stronger inhibition of AKT and IκBα activation and an enhanced E-cadherin expression compared to monotherapy. OC202e alone inhibited autophagy, while OC201 showed no significant effect. In the hepatic metastasis model, both OC201 and OC202e monotherapy significantly reduced liver metastatic nodules in mice compared to control, with combination therapy yielding a more substantial reduction, confirming the efficacy in reducing metastasis to the liver. Conclusion: This study demonstrated that OC201 and OC202e effectively inhibited TGF-β1- and TNF-α-induced EMT via the AKT and NF-κB signaling pathways, highlighting their potential to target critical mechanisms underlying pancreatic cancer metastasis. Both compounds effectively suppressed EMT while maintaining cell viability, with combination therapy exhibiting superior efficacy. These findings suggest that the combined use of OC201 and OC202e may serve as a promising therapeutic strategy for pancreatic cancer. Citation Format: Youngjin Kim, Dong Sub Jung, Sewon Kim, So Jung Sung, Yenny Kim, Yi Rang Kim, Jihoon Kang. OC201 and OC202e, potential inhibitors of cancer metastasis, suppress EMT in pancreatic 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 3862.

Abstract 3480: Gene electrotransfer in cancer: different immunomodulatory profiles and cell death pathways depend on different electric pulse protocols

Abstract Gene electrotransfer (GET) is an excellent strategy for delivering therapeutic genes and vaccines into the target cell using electric pulses. In view of the insights gained during the last COVID-19 pandemic, it is widely acknowledged that genetic vaccines represent a safe and affordable option with considerable promise for addressing a range of diseases, including infectious diseases and cancer. Growing evidence from preclinical studies and clinical trials, which include both intramuscular and intratumoral immunotherapy, suggests that GET-based methods could be adapted for application in veterinary and clinical treatment protocols. At present, a key focus of research is on enhancing plasmid DNA (pDNA) electrotransfer techniques to reduce tissue damage and improve gene transfection efficiency. However, the molecular effects of electroporation (EP)-assisted DNA delivery in vivo are still not well understood. We assessed two GET protocols for the intratumoral administration of pDNA: Short and High Voltage (SHV) electric pulses or an adapted High Voltage - Low Voltage (HV-LV) pulses protocol, both of which are used for reversible electroporation. We investigated the ability of these two electric pulse conditions to trigger immune and tissue responses in murine CT26 colorectal tumors treated with intratumoral non-coding pDNA EP, instead of using an antigen or immunostimulatory agent. Immune cell infiltration in the tumors was assessed after both treatments, and a transcriptomic analysis through RNA sequencing was conducted to examine the gene expression changes induced by GET, with a particular focus on immune and cellular signaling pathways. We observed no significant differences between the two GET protocols in counteracting tumor development in the absence of immunomodulatory molecules. However, important variations in intratumoral macrophage, CD4+ T cells and even higher CD8+ T cells recruitment were reported between the two treatment methods. Transcriptomic analysis revealed that apoptosis is a predominant mode of cell death after GET by SHV pulses, whereas, GET by HV-LV pulses is associated with immunogenic necrotic pathways as well as a prominent activation of both innate and adaptive immune response. We showed that varying pulse parameters can trigger different immunomodulatory profiles within the tumor microenvironment, emphasizing the significance of choosing the right pulse settings in EP-based therapies. This analysis could provide valuable insights for selecting the most effective EP method or for developing new EP-based strategies to enhance the success of GET protocols used for vaccination or gene therapy. Citation Format: Mariangela De Robertis, Tim Bozic, Iva Santek, Flaviana Marzano, Bostjan Markelc, Domenico Alessandro Silvestris, Apollonia Tullo, Graziano Pesole, Maja Cemazar, Emanuela Signori. Gene electrotransfer in cancer: different immunomodulatory profiles and cell death pathways depend on different electric pulse protocols [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 3480.

Abstract 3006: Efficacy of novel CDK9/CDK4/6/PI3K triple inhibitors, LCI133 and LCI139, in Ewing sarcoma

Abstract Introduction: The survival rate of patients with Ewing sarcoma (ES) has seen slow improvement over the past several decades and remains dismal for those with recurrent or metastatic disease. Initial clinical trials in relapsed/refractory ES with single agent targeted therapies, or combinations of single agents, have not yet improved patient outcomes. To overcome this barrier, the Molecular Targeted Therapeutics Laboratory has developed by in silico design “first in class” small molecule multikinase inhibitors (MKIs) which simultaneously target multiple tumor-promoting pathways with a single drug at nM potency. LCI133 (CDK4/6/CDK9) and LCI139 (PI3K/CDK4/6/CDK9) potently inhibit key kinases that control mitogenic signaling, proliferation, and transcription. The purpose of this study is to evaluate the potency, efficacy, and toxicity of LCI133 and LCI139 in ES model systems. Methods: ES cell lines and normal human skeletal muscle cells (SkMC) were treated with MKIs in logarithmic fashion and IC50s were determined by CellTiter-Glo 2.0 viability assays. Incucyte® Annexin V Dye was used to evaluate apoptosis over time by Incucyte® S3 following MKI treatment. Cell cycle analysis was performed over time using Incucyte® Cell Cycle Lentivirus Reagent and analyzed using an Incucyte® S3. Western blot and qPCR analysis was used to assess protein and RNA expression changes, respectively, of targeted pathways to confirm on-target activity of these MKIs following 3-24 hr treatment. Results: LCI133 and LCI139 were nanomolar potent in ES cell lines TC71 (392.5nM/312nM) and A673 (471.5nM/407.4nM), while only LCI139 was nanomolar potent in the RDES (262.2nM) cell line. Critically, LCI133 and LCI139 were less toxic against SkMC compared to single agent CDK9 inhibitors. In TC71 and A673 cells, both compounds induced statistically significant dose-dependent apoptosis, as well as G1 phase cell cycle arrest. Three hr LCI133 and LCI139 treatment decreased expression of CDK9-RNA polymerase II response genes MCL1 (P ≤ 0.001) and MYC (P ≤ 0.001). 24 hr treatment had differential effects on pRb and pAKT levels. pRpb1 and MCL-1 were reduced in all cell lines by LCI139 and/or LCI133. Increased cleaved PARP (cPARP) was observed in all cell lines following 24 hr treatment with LCI133 and LCI139 confirming the induction of apoptosis. Conclusion: Novel small molecule MKIs allow for the simultaneous targeting of multiple pathways while mitigating the toxicity of single agent combination therapy. This is a promising potential treatment strategy for ES patients for whom new treatment approaches are desperately needed. These studies provide preclinical evidence of the potential efficacy of small molecule MKIs in ES and support the future efforts of in vivo models evaluating efficacy and safety of these agents. Citation Format: Kaitlyn H. Smith, Cody C. McHale, Kenzie Wells, Krishnaiah Maddeboina, Kimberly Q. McKinney, Poornima Gourabathini, Jeffrey S. Huo, Donald L. Durden, Javier Oesterheld. Efficacy of novel CDK9/CDK4/6/PI3K triple inhibitors, LCI133 and LCI139, in Ewing sarcoma [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 3006.

Abstract 5484: Expanding the role of western blot imaging for oncology research: novel applications for in-cell western assays and single cell analysis using the ChemiDoc Go imaging system

Abstract Western blotting is widely used to detect cellular proteins and nucleic acids in cell lysates; however, its application for analyzing intact cells remains largely unexplored. Here, we evaluate compatibility of Bio-Rad's ChemiDocTM Go Imaging System with chemiluminescence and multi-channel fluorescence detection for in-cell western immunofluorescence assays performed on 96-well plates and microscopy slides. Emphasis was placed on use of the StarBright Blue 700-conjugated secondary antibody due to its strong, stable fluorescence, wide dynamic range, and compatibility with multiplexed assays. In 96-well assays, HEK293 cells were stained with anti-mortalin primary antibodies, followed by StarBright Blue 700-conjugated secondary antibodies. These exhibited a three-fold signal increase compared to control samples lacking the primary antibody. This scalable and high-throughput platform offers a valuable tool for cancer research, particularly for evaluating protein expression in cancer cell lines, facilitating drug discovery, and assessing therapeutic efficacy across diverse cancer models. On microscope slides, HEK293 cells were stained with anti-mortalin primary and StarBright Blue 700 secondary antibodies further demonstrating the imager’s strong compatibility with immunofluorescence on fixed, intact single cells. This capability enables simultaneous detection of multiple biomarkers in cancer-related pathways, supporting target validation and pathway analysis in cancer biology. Additionally, the imager was used to validate T cell staining with CD3-labeled fluorescent antibodies prior to multiplexed flow cytometry. This step simplifies the confirmation of T cell staining before performing more complex flow cytometry assays, with direct applications in immuno-oncology. These include monitoring immune cell activation and characterizing tumor-infiltrating lymphocytes (TILs), both critical for developing immune-based cancer therapies. Potential oncology research applications of these novel techniques may include: Quantification of protein expression changes following drug treatments or genetic modifications Evaluation of protein-protein interactions relevant to cancer signaling pathways High-throughput screening of compound libraries Provide insights into the analysis of protein localization within entire cells, aiding in biomarker identification and therapeutic targeting efforts. Together, these findings highlight novel applications of western blot imaging technology as a fast, scalable, and sensitive platform for multiplexed protein analysis, supporting cancer research and therapeutic development. Citation Format: Michael Kirmiz, Deanna Woo, Nikolas Chmiel, Kenneth Oh, Angelica P. Olcott. Expanding the role of western blot imaging for oncology research: novel applications for in-cell western assays and single cell analysis using the ChemiDoc Go imaging system [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 5484.

Abstract 3837: Defining the mechanism of semaphorin4D tumorigenesis in prostate cancer models

Background: Advanced and recurrent prostate cancer exhibits a “cold” immune phenotype characterized by an influx of immunosuppressive cells and fewer tumor-infiltrating lymphocytes (TILs), limiting the efficacy of checkpoint inhibitors. Semaphorin4D (SEMA4D/CD100), via the PlexinB receptors, regulates cell-cell interactions and migration in the nervous system, also responsible for cancer metastasis. Further, SEMA4D affects angiogenesis and immune regulation in other cancers. We hypothesize that SEMA4D promotes the recruitment of immunosuppressive cells via tumor blood vessels, further impairing TIL activity and anti-tumor immunity. Loss of SEMA4D function could disrupt vascularization, creating hypoxia that induces tumor cell apoptosis. Methods: Two mouse models—subcutaneous Myc-CaP allografts in immunocompetent mice and Hi-Myc prostate cancer GEMM were treated with a SEMA4D blocking mAb (pepinemab) to inhibit SEMA4D signaling. Tumor volume changes, vascularity, and oxygen saturation were assessed during treatment using high-frequency ultrasound, Power doppler, and photoacoustic imaging followed by FACS and IHC analyses on tumor tissues to evaluate immune cell phenotypes, hypoxia, apoptosis, and microvascular damage. To further elucidate genotypic changes in the tumor immune microenvironment, RNA sequencing gene expression changes associated with SEMA4D inhibition, including signaling pathways and Immune cell constituents. Results: Blocking SEMA4D significantly arrested tumor growth and reduced vasculature in Hi-Myc mice, with no prominent changes in immune phenotype by FACS after 4 weeks pepinemab treatment. IHC will test for changes in hypoxia, apoptosis, and microvascular damage. Allografts of prostate tumor cells similarly demonstrated tumor growth arrest. Effects on vasculature and microvascular integrity, leading to hypoxia and apoptosis, in this more tractable imaging environment, are anticipated. Transcriptomically, gene ontology and enrichment analyses may reveal pathways related to vessel formation, immune regulation, and metastasis. Conclusion: Our findings suggest that SEMA4D inhibition effectively arrests tumor growth by disrupting angiogenesis and potentially regulating immune activity, alongside inducing hypoxia and apoptosis. Ongoing transcriptomic analyses will further explore molecular mechanisms, including pathways involved in angiogenesis and immune regulation. Citation Format: Kavin Velur Ganesan, Maolake Aerken, Renyuan Zhang, Kent L. Nastiuk. Defining the mechanism of semaphorin4D tumorigenesis in prostate cancer models [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 3837.

Abstract 1739: Identification of a potent and selective KAT6A/6B inhibitor: HW321005 exhibits strong antitumor activity in KAT6A-high ER+/HER2− breast cancer

KAT6A and its paralog KAT6B are histone lysine acetyltransferases (HATs) that regulate gene transcription via the acetylation of histones. Dysregulation of KAT6 leads to lineage-specific gene expression changes in cancer. Overexpression of KAT6 is associated with poorer clinical outcomes in ER+/HER2− breast cancers. Dysregulation of KAT6 activity, including gene amplification, overexpression, fusion, and mutation, is observed in various cancers, including breast cancer. We have developed potent and selective small molecule inhibitors targeting KAT6A/6B. Our proprietary compound, HW321005, inhibits KAT6A/6B with single-digit nanomolar IC50 values, demonstrating high selectivity over other KAT family members. The efficacy of HW321005 was evaluated in multiple cancer cell lines, including ZR-751 (IC50 = 1.7 nM). Treatment with HW321005 led to a marked downregulation of KAT6A-mediated histone acetylation and estrogen receptor signaling in KAT6A-high ER+ breast cancer models. Consistent with in vitro findings, HW321005 showed significant anti-tumor efficacy and a robust pharmacodynamic response (H3K23Ac, acetylation marker; ERα, estrogen receptor signaling marker) in ZR-751 xenograft model in a dose-dependent manner. HW321005 demonstrates substantial biological potency and high selectivity, with favorable drug-like properties, including desirable in vitro ADME characteristics, excellent in vivo exposure, clearance, and good oral bioavailability across multiple preclinical species. These findings highlight the potential of HW321005 as a novel and highly effective KAT6A/6B inhibitor for the treatment of KAT6A-high ER+ breast cancer. IND-enabling studies for HW321005 are planned for early 2025. Citation Format: Xin Zhao, Qun Li, Qian Wu, Zexin Tong, Yang Zang, Tiejun Bing, Lifei Liu, Xuejun Zhang. Identification of a potent and selective KAT6A/6B inhibitor: HW321005 exhibits strong antitumor activity in KAT6A-high ER+/HER2− breast 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 1739.

Abstract 7066: RING1 inhibits smoking-induced lung cancer tumorigenesis by ubiquitination and degradation of CIP2A

Abstract Lung cancer is the second incidence rate of all new cancer diagnoses and the leading cause of cancer-related deaths globally. To develop novel diagnostic and therapeutic targets for lung cancer, there are emerging needs for molecular biological research on the occurrence and progression. Among the targets, CIP2A has been studied for promoting various cancer types, but detailed underlying mechanisms are needed to be further investigated. In this study, it was verified that CIP2A is overexpressed in lung cancer tissues, particularly amongst those with a smoking history, compared to adjacent normal tissues. CIP2A knockdown in lung cancer cells efficiently reduced cell proliferation and migration capacity. Through the mass spectrometry analysis on A549 cells overexpressing CIP2A, RING1 was identified as a candidate for E3 ligase of CIP2A. The interaction of the two proteins and ubiquitin chain regulation by RING1 was verified by immunoprecipitation. RING1 knockdown enhanced proliferation and migration of lung cancer cells, and these effects result from the upregulation of CIP2A and its downstream molecules, c-Myc and cyclin B1. To further study a trigger for RING1 expression changes in lung tumorigenesis, cigarette smoke extract (CSE) was applied for the following experiments. CSE treatment depleted RING1 mRNA, including protein expression. Decreased RING1 level led to upregulation of CIP2A and c-MYC. Collectively, these results reveal novel roles of the RING1 and CIP2A in lung cancer progression caused by smoking and propose a potential biomarker candidate for the therapeutic and diagnostic targets. Citation Format: In-ho Jeong, Peter Chang Whan Lee. RING1 inhibits smoking-induced lung cancer tumorigenesis by ubiquitination and degradation of CIP2A [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 7066.

Abstract 1260: Establishment of intratumor heterogeneity models using lung cancer cells

Abstract Intratumor heterogeneity has been shown to be associated with treatment resistance in multiple cancers, including lung cancer. Elucidating intratumor heterogeneity is an issue that must be resolved to improve the prognosis of patients. Previous studies of intratumor heterogeneity have mainly involved collecting and analyzing cancer cells from patients, classifying subpopulation with different properties, and conducting comparative analysis. However, there are few studies focusing on the early phase of intratumor heterogeneity. We exposed alectinib to a commercially available ALK-positive lung cancer cell line (H2228) and established alectinib resistant cells (H2228-AR1S). H2228-AR1S was observed two cells with different appearances. In this intratumor heterogeneity model, flow cytometry (FACS) showed that the cell surface CD47 of the parent line was unimodal, whereas that of H2228-AR1S was bimodal, indicating that there were differences in the properties of migration and tumor formation between subpopulation having the CD47 high expression and subpopulation having CD47 low expression. Next, We exposed molecular targeted drugs to patient-derived lung cancer cell lines and evaluated the expression of cell surface markers using FACS. KTOR83-Dab, a cancer cell line derived from a BRAF-positive lung cancer patient that was exposed to dabrafenib for one month, showed bimodal EpCAM expression. The subpopulation of EpCAM low expression had high migration ability and showed reduced epithelial gene expression and increased mesenchymal gene expression, suggesting EMT (epithelial-mesenchymal transition). It was suggested that KTOR83-Dab is a patient-derived intratumor heterogeneity model. Furthermore, experiments are being conducted using these intratumor heterogeneity models to elucidate the mechanism of intratumor heterogeneity. H2228-AR1S is a intratumor heterogeneity model established from commercially available monoclonal cells, and single-cell analysis is performed to extract genes whose expression changes during the process of homogeneous cells becoming heterogeneous. KTOR83-Dab is a patient-derived lung cancer cell line and is therefore polyclonal. We will clone KTOR83-Dab and examine whether there are any cells that become heterogeneous when administered dabrafenib. In addition, based on the results of single-cell analysis of H2228-AR1s, we will also analyze genes involved in heterogeneity in KTOR83-Dab. Citation Format: Tomoko Funazo, Hiroaki Ozasa, Takahiro Tsuji, Yusuke Shima, Keiichiro Suminaga, Kentaro Hashimoto, Hiroshi Yoshida, Kazutaka Hosoya, Hitomi Ajimizu, Takashi Nomizo, Hironori Yoshida, Toyohiro Hirai. Establishment of intratumor heterogeneity models using lung cancer cells [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 1260.

Abstract 1469: Deciphering mechanisms of intrinsic radioresistance in breast cancer: Insights from transcriptomics and CRISPRi screens

Abstract Background: Breast cancer (BC), the most common cancer globally and a leading cause of death in women, often utilizes radiation therapy (RT) as part of treatment, with over 85% of patients receiving RT after breast-conserving surgery. Still, over 15% of women experience local recurrence despite RT, underscoring the need to better understand the molecular mechanisms driving RT response and resistance. We hypothesized that transcriptomic changes that occur after ionizing radiation in intrinsically radiosensitive and resistant BC models would offer mechanistic insight into mediators of this differential response. To test this, we conducted in vitro and in vivo RNA-seq experiments to characterize transcriptomic changes following RT across a panel of BC cell lines of varying levels of intrinsic radiation sensitivity. Furthermore, we conducted a genome-wide CRISPRi screen to identify genes which are critical to radiation survival and drive radioresistance. Methods: Eight BC cell lines representing all intrinsic subtypes were treated with 4 Gy RT and RNA was collected 24 hours after treatment for RNA-seq. For 2 cell lines, we also conducted in vivo mouse xenograft experiments in which mice received fractionated daily RT (6 fractions of 2 Gy each). Differential gene expression analysis with DeSeq2 was performed, followed by pathway analysis with Advaita Bioinformatics’ iPathwayGuide. For the CRISPRi experiments, we used the Dolcetto library in two TNBC cell lines: BT-549 and HCC-1937. Cells were transfected with the library and then treated with 3 fractions of 3 Gy RT. DNA was isolated for sequencing after 7 days and analyzed with MaGeCK to determine sgRNA abundance. Results: An average of 935 differentially expressed genes were noted in the in vitro models (S.D. 906); 142 and 324 were found in the in vivo models. Among the most highly enriched pathways from the transcriptomic data in both resistant and sensitive cell lines after RT, we found processes including cell cycle, cellular senescence, and estrogen signaling. Pathways uniquely differentially expressed in the in vivo samples for both cell lines include IL-17 signaling and transcriptional misregulation in cancer. From the CRISPRi screen, we identified genes involved in similar pathways, as well as canonical regulators of DNA damage response. Conclusions: Ionizing radiation induces transcriptomic expression changes that differ between intrinsically radiosensitive and resistant BC models. These data, coupled with results from the CRISPRi radiosensitivity screen, identify pathways that offer potential insight into the mechanisms underlying intrinsic radioresistance and suggest biologic vulnerabilities that may be targeted to more effectively treat women at a high risk of local BC recurrence. Ongoing studies are exploring the potential of therapeutic targeting of these pathways when combined with RT in breast cancer. Citation Format: Breanna McBean Priyanka S. Rana Benjamin Hauk Anna R. Michmerhuizen Samuel Lichtman-Mikol Reine Abou Zeidane Vesna Markovic Kari Wilder-Romans Mingfang Tao Alan P. Boyle Corey Speers. Deciphering mechanisms of intrinsic radioresistance in breast cancer: Insights from transcriptomics and CRISPRi screens [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 1469.

Abstract 758: Spatial profiling of matched primary and recurrent glioblastoma samples reveals changes in cellular states and the immune microenvironment

Abstract Background: Glioblastoma (GBM), the most aggressive brain tumor, is treated with surgery, chemotherapy, and radiation, yet 90% recur within two years. PD-1 blockade improves outcomes in other cancers, but shows limited effect in GBM, likely due to tumor heterogeneity and an immunosuppressive microenvironment. This study aims to investigate post-treatment transcriptional changes in tumor cells and tumor-associated macrophages (TAMs) in matched primary and recurrent GBM, focusing on neoadjuvant PD-1 blockade. Methods: We used the GeoMx Digital Spatial Profiler to spatially select and transcriptomically profile tumor cells (SOX2+) and TAMs (IBA1+). Gene expression changes between primary and recurrent GBMs were analyzed in 30 patients (26 IDHwt, 4 IDHm) with 20 paired primary samples. All patients received standard treatment, and 20 also received second-line nivolumab one week before recurrence surgery. Tumor purity was confirmed using InferCNV and cell signatures. Subsequently, differential expression and pathway enrichment analyses were performed to compare recurrent tumors treated with nivolumab to those untreated, as well as primary vs. recurrent tumors. We also evaluated Neftel signatures in matched tumors to assess treatment- and recurrence-associated malignant state changes. Results: Principal component analysis of all samples revealed distinct transcriptional profiles between tumor cells and TAMs. Separate analyses showed clear differences in tumor cell and TAM segments based on tumor status (primary/recurrent), though no separation related to nivolumab treatment. Similarly, comparing nivolumab-treated recurrent tumors with untreated controls showed no significant changes in malignant states or gene expression in tumor cells or TAMs, consistent with the lack of clinical survival benefit.To investigate overall transcriptional changes induced by standard treatment, differential expression analysis comparing tumor cell segments in paired primary and recurrent tumors revealed few significant genes, likely reflecting high inter-patient variability driven by tumor heterogeneity. Unsupervised clustering of tumor cell segments identified a distinct cluster specific to recurrent tumors, with DEGs highlighting pathways linked to this particular tumor cell phenotype. In TAM segments, increased expression of checkpoint-related genes in recurrent tumors suggests TAM phenotype changes that may influence immune dynamics in the microenvironment. Conclusion: Spatial profiling of matched primary and recurrent GBM samples reveals distinct transcriptional changes in both tumor cells and TAMs, reflecting the complex microenvironment of recurrent GBM. However, our findings point to a limited impact of neoadjuvant PD-1 blockade on transcriptional activity, highlighting ongoing challenges in targeting GBM with immunotherapy. Citation Format: Sara Artzi, Marc N. Klausen, Dylan S. Harwood, Signe R. Michaelsen, Simone B. Maarup, Alessio Locallo, Nicolai S. Bager, Nadine M. Hammouda, Benedikte Hasselbalch, Ulrik Lassen, Joachim Weischenfeldt, Bjarne W. Kristensen. Spatial profiling of matched primary and recurrent glioblastoma samples reveals changes in cellular states and the 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 758.

Abstract 1531: The role of HDAC1 in pancreatic cancer metabolism

Abstract Pancreatic cancer is a malignant disease often driven by mutations in the KRAS gene. It is associated with significant metabolic reprogramming driving tumor growth, drug resistance, and adaptation in its dense, nutrient-limited tumor microenvironments. To date, the mediators of PDAC metabolism are not fully understood. Analyzing gene expression changes in PDAC tumors compared to normal tissues identified histone deacetylase 1 (HDAC1) as a consistently upregulated gene. We found that HDAC1-high tumors display strong metabolic alterations, including the upregulation of genes in glycolysis and other metabolic processes, and that treatment with Class I selective HDAC inhibitors (HDACi) selectively blunted growth. Initial experiments altering HDAC1 expression transiently in multiple pancreatic cancer cell lines demonstrate alternate metabolic responses. Consistent with existing literature, we observe that HDACi effectively suppressed pancreatic cancer cell growth, primarily class I selective inhibitors. Furthermore, we recently developed HDAC1 knockout cell lines that demonstrate shifts in metabolic profiles of pancreatic cancer and slowed growth rates in HDACi sensitive cell lines through immunoblotting, metabolomics and Seahorse assay. We expect that our findings will show the significance and prospects of targeting HDAC1 in pancreatic cancer therapy and its potential to overcome resistance with current therapeutic. Citation Format: Don-Gerard Cavakiah Blanchard Conde, Evan J. Zhou, Maximillian Farma, Chiamaka J. Ezeh, Laiba Shiekh, Matthew Cheung, Zeribe C. Nwosu, Nwosu Lab, Cornell University. The role of HDAC1 in pancreatic cancer metabolism [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 1531.

Abstract 4339: Glucosinolates selectively target cancer cell types and exhibit antineoplastic effects

Abstract Introduction: Glucosinolates are organic compounds found in high concentrations within cruciferous vegetables. Plant tissue damage initiates the release of myrosinase, which breaks down glucosinolates into the biologically active compounds indole-3-carbinol (I3C) and sulforaphane. In vitro studies have suggested that I3C and sulforaphane can mitigate cancer progression via hormone regulation and antioxidant mechanisms, respectively. However, there has yet to be a systematic analysis of these two glucosinolates against a wide range of human cancer cell lines. We hypothesized that I3C would differentially affect cancer cell growth depending on cell line of origin, and that sulforaphane would induce a genetic signature similar to compounds and genetic perturbations with anticancer properties. Methods: Cell survival data was obtained from the NCI-60 human tumor cell lines screen. 59 tumor cell lines were seeded into 96-well microtiter plates. 100 µL of 5 different I3C 10-fold dilutions (0.01 - 100 µM) were added to the wells and incubated for 48 hours. After staining with sulforhodamine, absorbance was measured, and percent growth was calculated relative to the no-drug control and number of cells at baseline. Genetic profiling data was obtained from the Broad Institute’s L1000 assay. Sulforaphane was added to 9 cell lines in 384-well plates. mRNA was extracted and expression levels of 978 landmark genes were measured. This provided a transcriptomic signature that was compared to signatures from other compounds and genetic perturbations. A Connectivity Score was then developed to measure the similarity of each signature to the one induced by sulforaphane. Results: I3C was most potent in the KM12 cell line (IC50 = 14.9 µM; colon cancer) where it inhibited ∼90% of growth at a 100 µM concentration. Cancers of the colon and skin were most sensitive to I3C (median IC50 values of 28.3 µM and 36.1 µM, respectively), while breast and myeloid tissue cancers were least sensitive. Gene expression induced by sulforaphane was most similar to that induced by isoliquiritigenin, a flavonoid compound that exhibits antiproliferative and anti-inflammatory effects. Transcriptomic changes caused by sulforaphane were highly similar to NFE2L2 overexpression (tau score = +97.87), and SLC7A5 knockdown (tau score = +97.16). The NFE2L2 gene encodes a transcription factor (NRF2) that regulates antioxidant enzymes, and the SLC7A5 gene encodes an amino acid transporter that is often overexpressed in many cancer types. Conclusion: Glucosinolates, specifically I3C and sulforaphane, demonstrate anticancer properties by inducing cell survival and gene expression changes in several cancer cell lines. In the future, identifying the genes and proteins targeted by I3C, as well as the pathway through which sulforaphane acts, will be crucial to further elucidate the effects of glucosinolates on cancer. Citation Format: Katerina Carrozzi, Adin Aggarwal, Kenneth W. Yip. Glucosinolates selectively target cancer cell types and exhibit antineoplastic effects [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 4339.