Potential Biomarkers Research Articles

Brain Network Functional Connectivity in Children With a Concussion.

Pediatric concussion can disrupt functional brain network connectivity, but prospective longitudinal research is needed to clarify recovery and identify moderators of change. This study investigated network functional connectivity (FC) up to 6 months after pediatric concussion. This prospective longitudinal concurrent cohort observational study consecutively recruited children (aged 8 to 17 years) at 5 Canadian pediatric hospital emergency departments within 48 hours of sustaining a concussion or mild orthopaedic injury (OI). Children completed 3T MRI scanning postacutely (2 to 33 days) and at either 3 or 6 months after injury (randomly assigned at the postacute visit). Reliable change between retrospective preinjury (based on parent report) and 1-month postinjury symptom ratings based on parent and child report was used to classify concussion with or without persisting symptoms. Within-network and between-network FC was computed for 8 brain networks from resting-state fMRI scans and analyzed using linear mixed-effects models, with multiple comparison correction. Groups (385 with concussion/198 with OI; 59% male; 69% White; age 12.42 ± 2.29 years) did not differ in within-network FC. Relative to OI, connectivity between the visual and ventral attention networks was lower after concussion, d (95% CI) = -0.46 (-0.79 to -0.14), across time. Connectivity between the visual and default mode networks was lower at 6 months after concussion, -0.60 (-0.92 to -0.27). At 3 months after concussion, connectivity between the frontoparietal and ventral attention networks was lower in younger children, -0.98 (-1.58 to -0.37), but higher in older children, 0.81 (0.20 to 1.42). For symptom groups based on parent report, connectivity between the dorsal and ventral attention networks was higher in female children at 3 months after concussion without persisting symptoms relative to concussion with persisting symptoms, 1.25 (2.05 to 0.46), and OI, 0.87 (0.25 to 1.49). Time after injury, age at injury, biological sex, and persistent symptom status are important moderators of FC after pediatric concussion for children seen in emergency department settings. Postacute FC may not enhance clinical diagnosis. Although within-network connectivity is preserved, between-network connectivity differences emerge after most children clinically recover and persist up to 6 months after pediatric concussion, providing a potential objective biomarker for lasting changes in brain function.

Abstract 5406: Evaluation of antitumor effects of plogosertib, PLK1 inhibitor in biliary tract cancer with BUBR1 as a potential biomarker

Abstract Background: Polo-like kinase 1 (PLK1) has been emerging as a promising therapeutic target in solid tumors due to its central role in cell cycle regulation and cancer cell proliferation. In biliary tract cancer (BTC), cell cycle-related factors such as PLK1, CDKs, and Aurora kinase are frequently overexpressed or aberrantly activated, driving tumor progression. However, studies targeting these proteins in BTC remain limited. BUBR1, a critical mitotic checkpoint protein, is essential for maintaining chromosomal stability by ensuring proper chromosome alignment and segregation during cell division. This study evaluated the antitumor effects of a PLK1 inhibitor plogosertib in BTC and identified BUBR1 as a potential biomarker to predict treatment response. Methods: Total 9 BTC cell lines (SNU-245, SNU-308, SNU-478, SNU-869, SNU-1196, HuCCT-1, TFK-1, SNU-2670, SNU-2773) were used. We used plogosertib (PLK1 inhibitor), ceralasertib (ATR inhibitor) and panobinostat (HDAC2/3 inhibitor). The anticancer effects of plogosertib, both alone and in combination with ceralasertib and panobinostat, were evaluated through MTT assay, Colony formation assay, Annexin-V assay, and Cell cycle analysis. The effect of plogosertib on mitotic arrest was assessed using phospho-histone H3 assay and Immunofluorescence. Results: Plogosertib monotherapy showed higher anticancer effects in BUBR1-high BTC cells (SNU2773, SNU478) compared to BUBR1-low cells (SNU869, SNU245). Plogosertib promoted mitotic checkpoint complex (MCC) formation in prometaphase, arresting mitosis and ultimately leading BTC cells to undergo apoptosis. However, plogosertib also induced ATR activation, which may limit its efficacy by initiating DNA repair pathways. ATR inhibition by ceralasertib further enhanced the anticancer effects of plogosertib in BUBR1-high cells. In BUBR1-low cells, plogosertib reduced BUBR1 protein expression, possibly through ubiquitination-proteasome-mediated degradation, limiting MCC formation and its therapeutic effects. Among several candidates regulating ubiquitination processes, HDAC2/3 inhibition by panobinostat enhanced the responsiveness of BUBR1-low cells to plogosertib. Conclusion: BTC cells with high BUBR1 expression are sensitive to the PLK1 inhibitor plogosertib, which shows synergistic effects when combined with an ATR inhibitor. In contrast, BTC cells with low BUBR1 expression which are relatively insensitive to plogosertib, HDAC2/3 inhibition increases the sensitivity to plogosertib. These findings suggest that targeting PLK1 could be an effective strategy for BTC treatment, especially with BUBR1 expression as a potential biomarker to inform optimal combination therapies. Citation Format: Yoojin Jeong, Ah-Rong Nam, Kyoung-Seok Oh, Jae-Min Kim, Ju-Hee Bang, Sea Young Choo, Hyo Jung Kim, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh. Evaluation of antitumor effects of plogosertib, PLK1 inhibitor in biliary tract cancer with BUBR1 as a potential biomarker [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 5406.

Abstract 4605: Exploring resistance mechanisms in cancer using patient-derived organoids reveals replication stress and EMT as potential biomarkers

Abstract Mutational processes such as chromosomal instability and replication stress are important molecular features of head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC), contributing to poor patient survival. Their changing influences during cancer development creates challenges in identifying response biomarkers. Patient-derived organoid 3D cancer models (PDOs) have been shown to match their primary source material and to reflect tumour biology more faithfully than cell lines. In our companion abstract, we detail how we have built the Digital Biological Twin lab to study PDOs for drug testing and biomarker discovery. In this work, we used PDOs to investigate resistance to PARP inhibitors (PARPi), which are mechanistically linked to DNA repair and replication. 10 PDOs (6 HNSCC, 4 NSCLC) generated from untreated tumor resections were bulk RNA-sequenced and treated with Niraparib. IC50, cell viability measured by flow cytometry and protein expression levels were used to classify 3 PDOs as sensitive to treatment. Expression analysis, stratifying by PARPi sensitivity, resulted in 287 differentially expressed genes (DEGs). Enrichment analyses found involvement of the endothelial-to-mesenchymal transition (EMT) and small molecule transport pathways. Knowing that EMT is linked to multi-drug resistance, we treated the PDOs with Cisplatin and found a significant correlation to PARPi IC50 values. By examining the biology of the DEGs, we categorized them using MSigDB into five gene sets, each with 1 to 14 genes. We functionally validated the gene sets by assessing PARPi susceptibility in 45 cell lines pre and post a genome-wide CRISPR knockout, finding four significantly enriched in PARPi gene dependencies. For two replication stress-derived gene sets, we demonstrated significant concordance between PARPi sensitivity status of PDOs and cell lines from DepMap, and with progression free interval of HNSCC and NSCLC patients from TCGA. Leveraging Cox analysis on these patients, one replication stress gene set stratified the patients by showing a significant decrease in relapse probability. In summary, our results indicate that PARPi resistance is associated with the deregulation of genes related to small molecule transport, multi-drug resistance and EMT. The consistent findings around replication stress across disease models strongly suggests a conserved and central role in resistance mechanisms. We also identify a replication stress gene set that significantly correlates with resistance in cancer models and relapse in patients. Further work is needed to translate our findings into a predictive model for drugs linked to replication stress. Here we showed that PDOs can be successfully used for furthering our understanding of resistance mechanisms while improving molecular stratification of patients with direct clinical implications. Citation Format: Ruben M. Drews, Finnian Firth, Paul R. Barber, Anna Pasto, Tony T. Ng. Exploring resistance mechanisms in cancer using patient-derived organoids reveals replication stress and EMT as potential biomarkers [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 4605.

Abstract 2004: Integrative bioinformatics approach reveals potential liquid biopsy-based biomarkers for breast cancer metastasis monitoring

Abstract Background: Despite recent advances in screening and therapeutic strategies, breast cancer remains the first leading cause of cancer death among females. The 5-year survival rate is high as 95% at localized stages but drops significantly to 31% once the cancer metastasizes. Therefore, developing predictive tools of metastatic recurrence is an unmet clinical need to design preventive clinical trials. Methods: This study used a public dataset (GEO ID: GSE102484), which is a microarray dataset that contains primary tumor samples (n = 683) sourced from subjects with (n = 95) and without (n = 582) distant recurrence (metastasis) of breast cancer (met and non-met, respectively). Our approach began with two sets of differentially expressed genes (DEGs). The first set of DEGs are from all the samples in the dataset regardless of breast cancer subtypes, while the second set of DEGs are from triple negative breast cancer (TNBC) samples only. To identify subjects with TNBC, basal-like breast cancer samples were selected using PAM50 algorithm with genefu R package. To select potential blood biomarker candidates, we developed an integrative bioinformatics pipeline. Briefly, 1) Differential expression analysis between met and non-met were performed using limma R package. We then selected DEGs with log2 fold change ≥ 0.58 and false discovery rate (FDR) < 0.1, 2) Genes with housekeeping/hematopoietic markers were removed, and 3) The genes that can be detectable in human bloods were selected using the human secretome database from the Human Protein Atlas (HPA). Results: The differential expression analysis of all breast cancer samples identified 134 significantly upregulated genes in met and the selection process with the pipeline resulted in 13 marker candidates including COL10A1, CD93, HPRT1, MMP1, EPHB4, HSPD1, MAP4, CCDC80, MMP11, SAFB2, CALD1, CRISP3, and GAPDH. Subsequent GO enrichment analysis identified functions associated with these markers including angiogenesis, calcium ion binding, and extracellular matrix and structure organizations. Nucleotide metabolism was observed significance in pathway. We then focused on the TNBC samples from the dataset and identified 162 significantly upregulated genes in met from TNBC. Out of the 13 marker candidates from the 134 upregulated genes, 8 marker candidates including CALD1, CD93, EPHB4, SAFB2, HSPD1, MAP4, CCDC80, and HPRT1 were also found in TNBC. Conclusion: Through an integrative computational analysis, we identified 13 markers associated with breast cancer metastasis, of which 8 were specifically linked to metastasis in TNBC. Future studies will focus on validating these biomarkers using liquid biopsy samples from breast cancer patients, potentially offering a less invasive diagnostic approach. Citation Format: Jina Kim, Cynthia Jinno, Hyoyoung Kim, Sunao Tanaka, Takeo Fujii, Sungyong You, Hideki Furuya. Integrative bioinformatics approach reveals potential liquid biopsy-based biomarkers for breast cancer metastasis monitoring [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 2004.

Abstract 4345: BH3120, a PD-L1x4-1BB bispecific antibody, exhibits favorable safety profiles in distinct in vivo and ex vivo liver toxicity evaluations

Abstract BH3120 is a novel bivalent PD-L1/4-1BB bispecific antibody targeting both inhibitory (PD-1/PD-L1) and co-stimulatory (4-1BB clustering) signaling. In multiple non-clinical safety evaluations, BH3120 consistently showed minimal modulation of T cell functions in the blood or normal tissues resulting in favorable safety profiles. Clinical evaluation of BH3120 as a monotherapy and in combination with a PD-1 inhibitor is underway (NCT06234397). While early stage clinical evaluations of 4-1BB agonists as a monotherapy or in combination with PD-1/PD-L1 inhibitors are generally associated with certain level of liver abnormalities, the potential biomarkers or patient’s backgrounds that can pre-determine the risk of liver toxicity have not been discussed in details. In the meantime, treatment discontinuation or disruption, and administration of corticosteroid due to liver toxicity would be the limitations of 4-1BB agonists in tumor management, and the mode of action and impact of 4-1BB agonists on liver inflammation need to be further investigated. To understand potential risks of liver toxicity with BH3120, sensitive in vivo and ex vivo models for liver toxicity evaluations were established: a mice model with mild liver inflammation induced by Concanavalin A, and human liver organoids co-cultured with immune cells to mimic the histological and physiological changes in patient's liver tissues. In both models, BH3120 was not associated with significant elevation of liver inflammation markers, indicating minimal risk of liver toxicity that is in line with the safety results we previously reported. The property of BH3120 to minimize systemic immune modulation enables de-coupling of anti-tumor efficacy from systemic toxicities, and would provide flexibility in combination with different anti-tumor treatments. Citation Format: Jing Wang, Jiangcheng Xu, Jun Wang, Jiangwang Liu, Kyoungwoo Lee. BH3120, a PD-L1x4-1BB bispecific antibody, exhibits favorable safety profiles in distinct in vivo and ex vivo liver toxicity evaluations [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 4345.

Abstract 3355: FTO in smoking-associated lung cancer and lung cancer tumor progression

Abstract In 2024, an estimated 234,580 patients will be diagnosed with lung cancer and 125,070 are expected to die, making lung cancer the leading cause of cancer-related deaths. Early detection of lung cancer is critical for improving prognosis, hence the need for early detection biomarkers. Fat mass and obesity-associated gene (FTO) had earlier been linked with obesity, however current studies indicate it may have a role in tumor development and could be a potential biomarker. In this study, we aimed to identify a possible link between FTO and non-small cell lung cancer (NSCLC), which accounts for 85% of lung cancer diagnosis. We hypothesize FTO is involved in increasing tumorigenicity and mortality among lung cancer patients. The role of FTO was studied in tumor progression, patient survival analysis, and smoking induced lung cancer, which is not established. Using immunohistochemistry, 115 tumor sections of NSCLC patients were studied; of these patients, 64 were early-stage NSCLC and 51 were late-stage. Patient tissues were obtained from local hospital pathology archives. Staining was completed using FTO primary antibody and a biotinylated secondary antibody. FTO brightness scores were generated using the BZX-800 analyzer software. FTO expression was also calculated using an H-score with the help of a pathologist. Both imaging studies and pathology grading indicated that late-stage lung cancer tissues had significantly (p<0.05) higher FTO expression compared to early-stage cases. When subdivided into low and high expression levels, 69% of early-stage tissue samples exhibited low expression levels and 55% of late-stage tumor tissues had high FTO expression. To assess the role of FTO expression in lung cancer survival, a Kaplan-Meier analysis was done with 47 patients. Results showed that NSCLC patients with higher expression had a shorter median survival time (14 months) compared to NSCLC patients with low expression (27 months) (p=0.03). These results suggest that patients with higher FTO expression may have poorer prognosis. Since 80-90% of lung cancer patients had a history of smoking, we studied the connection between FTO expression and smoking associated lung cancer. 50 NSCLC tumor sections were stained, 28 being smokers and 22 being non-smokers. The average FTO expression level was 24% higher in smokers compared to non-smokers (p<0.02). Using H-scores, smokers were divided into low and high expression levels. 93% of smokers had a high FTO expression compared to 7% in non-smokers indicating that smokers had higher expression of FTO compared to non-smokers (P=0.001). In conclusion, FTO may have a role in tumor progression as it is highly expressed in late-stage lung tumor tissues. High expression of FTO is also a potential indicator of patient survival, as patients who had an upregulation of FTO had shorter survival time. Finally, we found a possible link between high FTO expression and smoking-induced lung cancer. Citation Format: Rachel Campoli, Rong Qiu, Robert Hillwig, Neelu Puri. FTO in smoking-associated lung cancer and lung cancer tumor progression [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 3355.

Abstract 3309: Characterization of long non-coding RNAs in ependymomas: Uncovering potential biomarkers and therapeutic targets

Abstract Introduction: Ependymomas (EPNs), accounting for 5-6% of glial tumors, arise from radial glial cells and exhibit significant heterogeneity, leading to variable prognosis. Despite their clinical significance, the oncogenic drivers and potential therapeutic targets for EPNs remain poorly understood. Long non-coding RNAs (lncRNAs) are emerging as crucial regulators in many cancers, influencing tumorigenesis and therapeutic resistance. However, their role in EPN pathobiology has not been explored. This study aims to unravel the lncRNA landscape in EPNs to identify novel diagnostic biomarkers and therapeutic targets, improving patient stratification and treatment outcomes. Methods: 38 EPN tumor samples were classified into 21 supratentorial (ST), 13 posterior fossa (PF), and 4 spinal (SP) subtypes based on WHO 2021 criteria and subjected to RNA sequencing to map their RNA landscape, using 6 normal brain tissue samples as control. Raw data was normalized to identify significantly deregulated genes (padj < 0.05, -1 ≤ log2 fold change ≥ 1). qPCR validated the top deregulated lncRNAs (n = 60 extended cohort, p < 0.05). Pan-cancer analysis, functional annotation, target prediction, co-expression network construction, and prognostic relevance were assessed using ECORI, LncExplore, LncHub2, RNAInter, and Prognoscan respectively. Results: RNA sequencing identified 2, 271 upregulated and 1, 728 downregulated lncRNAs in EPN sub-groups, with 108 commonly upregulated and 187 downregulated across subtypes. qPCR confirmed LINC01971 overexpression in ST-EPNs (p < 0.001), FAM74A7 upregulation in PF and SP subtypes (p < 0.0001, p < 0.001), and LINC00320 downregulation in all subgroups (p < 0.001). Pan-cancer analysis revealed high LINC01971 in uterine endometrial and lung carcinoma, slightly increased FAM74A7 in kidney clear cell carcinoma, and low LINC00320 across all cancers. LINC01971, co-expressed with RELA, is involved in NF-kB-driven tumorigenesis and immune modulation. Pathway analysis associated with transcription factors (ESR1, SOX2, TP53, FOXA1) and chromatin modifiers (H3K27me3, H3K4me3), indicating a role in epigenetic reprogramming. FAM74A7, correlated with transcription factors (EOMES, SOX2, NANOG, FOXA1), is linked to tumor progression and epigenetic regulation, with disease associations (papillary, cellular, and myxopapillary EPNs) supporting its potential as a diagnostic and therapeutic target. LINC00320, reduced in malignant gliomas, regulates FOXA1 and H3K4me3, affecting metabolism and protein lipidation. Co-expression patterns in EPNs and gliomas highlight their role in tumor progression and neurodegenerative diseases Prognoscan links RELA, FOXA1, and SOX2 overexpression to poor prognosis in various cancers. This comprehensive transcriptomic analysis of EPNs unveils novel lncRNAs, as potential prognostic biomarkers and therapeutic targets. Citation Format: Rafat Malik, Dr. Amandeep Kumar, Dr. Vaishali Suri, Dr. Mehar Chand Sharma. Characterization of long non-coding RNAs in ependymomas: Uncovering potential biomarkers and therapeutic targets. [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 3309.

Abstract 5399: Placental gene PEG10 promotes onco-fetal metabolic reprogramming in hepatocellular carcinoma

Abstract Tumorigenesis is a complex biological process, accompanied by cellular dedifferentiation and metabolic reprogramming, which similarities to the metabolic characteristics of embryonic development stages. In our research, we focused particularly on the RNA-binding protein PEG10, which is highly expressed in the placenta and found to be similarly overexpressed in liver cancer cells, playing a key role in the process of aerobic glycolysis in tumor cells. This discovery suggests that there may be a phenomenon of fetal-like metabolic reprogramming in hepatocellular carcinoma (HCC), which is of significant importance for understanding the pathogenesis of HCC and for seeking new therapeutic strategies. In hepatocellular carcinoma (HCC) cells, we found that PEG10 enhances the stability of mRNA for key glycolytic genes hexokinase2 (HK2) and Glucose transporter 1 (GLUT1) by inhibiting the STAU1-mediated RNA degradation pathway, thereby promoting aerobic glycolysis in tumor cells. This process not only promotes the proliferation of tumor cells but also reduces the sensitivity of tumor cells to sorafenib. Furthermore, we observed in clinical samples that high expression of PEG10 is closely associated with poor prognosis in HCC patients, further confirming the important role of PEG10 in the development of HCC. Our research also found that retinoic acid can effectively inhibit aerobic glycolysis in tumor cells by suppressing the transcriptional expression of PEG10, thereby inhibiting the growth of tumor cells. In animal models, mice with liver-specific PEG10 gene knockout showed significant resistance to oncogene-induced liver cancer occurrence and development, providing strong evidence for PEG10 as a therapeutic target. These findings not only reveal the biological link between PEG10 and onco-fetal metabolic reprogramming at the molecular level but also indicate from a clinical perspective that PEG10 may be a potential biomarker and therapeutic target for HCC. The high expression of PEG10 is associated with poor prognosis in HCC patients, and the inhibitory effect of retinoic acid on PEG10 expression provides a new strategy for the treatment of HCC. These results provide new ideas for the future diagnosis and treatment of HCC, especially in the development of targeted therapeutic drugs against PEG10, which has important application prospects. Our research has laid a solid foundation for a deeper understanding of the metabolic reprogramming mechanisms of HCC and the development of new therapeutic methods. Citation Format: Dong Yin, Xinyi Yao, Jingyuan Zhang, Yin Zhang, Jianyou Liao, Kaishun Hu, Jiehua He, Daning Lu. Placental gene PEG10 promotes onco-fetal metabolic reprogramming in hepatocellular carcinoma [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 5399.

Abstract 1261: Assessing intratumoral genetic heterogeneity in locally advanced rectal cancer

Locally Advanced Rectal Cancer (LARC) is typically treated with neoadjuvant chemoradiotherapy (nCRT), aimed at reducing tumor size to facilitate surgical resection. Patients achieving a complete clinical response (cCR) may be eligible for organ preservation strategies, such as the Watch-and-Wait protocol. However, clinical and radiological evaluations lack precision in predicting treatment response, with tumor regrowth occurring in 30% of cases within two years. We previously demonstrated that rectal tumors present high ITGH and that this heterogeneity increases after nCRT in patients who do not achieve a complete clinical or pathological response. Therefore, ITGH has emerged as a potential biomarker for assessing nCRT response, and we herein evaluate the association between distinct ITGH estimates and nCRT response using local and public genomic datasets. We analyzed a local cohort of 71 LARC patients who underwent long-course nCRT with 5FU-based chemotherapy or Capecitabine (SP cohort). Pre-treatment tumor biopsies were subjected to whole exome sequencing (WES), and patients were classified into two groups: nCRT-R (N=16), nCRT-NR (N=55). Public WES, mRNAseq, and clinical data from 81 patients from Chatila et al. (2022, MSKCC cohort) were also used to ITGH estimates using MATH score (in WES) and DEPTH2 (in mRNAseq). The treatment response was evaluated based on cCR or pathological complete response (pCR) in patients treated with 5FU, Capecitabine or Cisplatin, receiving nCRT alone or combined with induction/consolidation. Low ITGH values correlated with nCRT response in the SP cohort (median MATH score of 28.89 versus 50.97 in nCRT-R and NR, respectively; p≤0.05, Wilcoxon test). We used ROC curve analysis to determine the best MATH score threshold (26.34, sensitivity=90%, specificity=50%, AUC=0.67). Multivariate analysis with the clinical-radiological features showed a marginal association between MATH-low with nCRT-R (OR:16.05; p=0.003). We observed opposite tendencies for the MSKCC cohort (higher ITGH in nCRT-R): median MATH score of 38.65 (nCRT-R, N=18) versus 33.51 (nCRT-NR, N=63), but the difference was not significant (p=0.2536, Wilcoxon test). Considering MATH score limitations (e.g., number of somatic mutations, WES coverage) we also inferred ITGH from expression data for a subset of these patients. Still, we observed similar, non-significant results: median ITGH of 0.667 versus 0.652 for nCRT-R (N=18) and NR (N=54), respectively (p=0.503, Wilcoxon test). The results from our cohort suggest that ITGH remains a potential biomarker of response. The conflicting data observed may simply reflect the limited statistical power of the analyses. We believe it is necessary to increase the sample size and stratify response groups based on treatment regimens. Therefore, we consider it important to disclose these findings to encourage collaboration and facilitate the expansion of our case series. Citation Format: Vandeclecio Lira da Silva, Ezequias Santos Torres, Ramon Torreglosa Do Carmo, Franciele Hinterholz Knebel, Fabiana Bettoni, Guilherme Pagin São-Julião, Bruna Vailati, Bruna Vailati, Leonardo Corbi, Natalia Mariana Felicio, Angelita Habr Gama, Anamaria Camargo, Cibele Masotti, Rodrigo Oliva Perez. Assessing intratumoral genetic heterogeneity in locally advanced rectal 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 1261.

Abstract 5207: Organoid-based drug screening identifies CDK9 as a target for stratified treatment of high-grade serous ovarian cancer

Abstract High-grade serous ovarian cancer (HGSC) is the most common and lethal ovarian cancer type. Despite favorable response to first-line therapy, most HGSC patients eventually relapse. HGSC presents few recurrent mutations that could be used as therapeutic biomarkers and hence, the progress with genomic-based precision HGSC treatment has been limited. We therefore took a functional precision medicine approach and asked whether we could identify patient-specific vulnerabilities of refractory HGSC using patient-derived organoids. We have previously established a collection of HGSC organoids1, which is currently being expanded to a publicly accessible biobank of >100 stable and expandable cultures (OvaCure Collection, ovacurecollection.com). We also demonstrated that organoid drug responses correlate with clinical treatment outcomes1. Herein, in organoids from several HGSC patients, we performed a large-scale drug-response profiling of a library of 370 compounds. Of these, we found that inhibitors of CDK9 (CDK9i), but not other CDKs, demonstrated pronounced activity in subset of patients. CDK9i have not yet been thoroughly assessed for the treatment of HGSC, and only MYC overexpression has been considered as a sensitivity biomarker2. To explore the CDK9i responses in HGSC, we profiled 18 organoid cultures against selective CDK9i (AZD4573, VIP-152, NVP-2 and KB-0742). We observed a range of CDK9i-sensitivities in the patient cohort, from resistant cultures to exceptional responders. As all organoids have been previously characterized with whole genome- and RNA sequencing, we assessed genetic and transcriptomic differences between models to identify potential biomarkers of CDK9i sensitivity. MYC expression, biomarker of sensitivity in previous CDK9i clinical trials, did not correlate with CDK9i sensitivity in organoids (Pearson r = 0.244, p=0.346), suggesting its poor predictive value. However, we identified a set of genes, expression of which closely correlated with sensitivity (36 genes with Pearson r>0.7) or resistance (84 genes with Pearson r<-0.7) to CDK9i. For instance, expression of phosphodiesterase 3B (PDE3B) was correlated with CDK9i resistance (Pearson r = -0.744, p<0.001), while expression of ERBB3 was correlated with increased CDK9i sensitivity (Pearson r = 0.841, p<0.0001). Taken together, we demonstrate the application of an HGSC organoid biobank for identifying patient-specific targets, exceptional responder groups and potential CDK9i sensitivity biomarkers in HGSC. Results of this project could serve as a basis for designing a molecularly stratified CDK9 inhibitor-focused clinical trial in refractory HGSC patients. Citation Format: Wojciech Senkowski, David Fontaneda-Arenas, Yilin Li, Kari A. Lavikka, Aura Crispino, Jakub Červinka, Francesca Gorini, Kyriaki Driva, Laura Gall-Mas, Lidia Moyano-Galceran, Jaana Oikkonen, Yan-Jun Chen, Karolin Voßgröne, Elin Pietras, Jutta Huvila, Kaisa Huhtinen, Johanna Hynninen, Sampsa Hautaniemi, Krister Wennerberg. Organoid-based drug screening identifies CDK9 as a target for stratified treatment of high-grade serous ovarian 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 5207.

Abstract 6678: Targeting long non-coding RNA LINC00324 using novel plant-derived exosome-like nanovesicles loaded with anticancer epigenetic modifiers effectively inhibits biomarkers of epithelial-mesenchymal transition in breast cancer

Abstract Despite recent advancements in the diagnostics and therapeutics of breast cancer, it continues to be the second leading cause of death among women worldwide. Therefore, identifying new potential biomarkers and molecular targets is essential for enhancing the diagnosis and treatment of BC patients. A wealth of evidence underscores the significant role of long non-coding RNAs in various biological processes, including tumorigenesis, which might offer new biomarkers and molecular targets for improved cancer detection and treatment. In this context, long intergenic non-coding RNA 00324 (LINC00324) has been associated with advanced cancer stage, larger tumor size, lymph node metastasis, and poorer prognosis across several cancer types. However, its biological function and molecular mechanisms in breast cancer remain largely unexplored. This study adopts an innovative approach aimed at challenging existing knowledge and research methodologies, with the goal of making significant advancements in breast cancer research. By exploring the role of LINC00324 as an oncogene in tumorigenesis and progression, the study may uncover crucial downstream oncogenic signaling pathways related to Epithelial-Mesenchymal Transition (EMT), which are linked to the hallmark features of breast cancer pathogenesis. This research could ultimately lead to the identification of a new biomarker for the diagnosis and prognosis of breast cancer, thereby transforming our understanding and treatment of the disease. In this study, we employed an innovative approach to downregulate the expression of LINC00324 across various breast cancer cell lines, ranging from well-differentiated to poorly differentiated types. This was accomplished using isolated novel plant-derived exosome-like nanovesicles (PELNVs), demonstrating specific biological effects, including anti-inflammatory and anti-tumor properties, while exhibiting minimal toxic side effects. PELNVs were a novel vehicle for delivering anticancer epigenetic modifier drugs and specific siRNA targeting LINC00324. Additionally, we investigated several downstream target biomarkers of epithelial-mesenchymal transition, including oncogenic targets such as Notch1, Notch4, Nanog, s6 kinase, β-catenin, and Stat3, both in vitro and in vivo. These findings suggest that LINC00324 could serve as a novel biomarker for diagnosis and prognosis. Outcome: Identifying the molecular classification of breast cancer subtypes based on the expression of individual biomarkers, such as LINC00324, might lead to the development of novel therapeutic biomarkers/targets for early detection, drug resistance, and personalized cancer treatments. Using PELNVs for drug delivery may inspire future research on plant-derived nanovesicles. Citation Format: Alaa Ashraf Al_Aaser, Nourhan El-Sayed El-Feel, Ahmed Samir Sultan. Targeting long non-coding RNA LINC00324 using novel plant-derived exosome-like nanovesicles loaded with anticancer epigenetic modifiers effectively inhibits biomarkers of epithelial-mesenchymal transition in 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 6678.

Abstract 4210: Replication stress detection using STRIDE technology platform

Abstract Replication stress, characterized by the slowing or stalling of replication forks, is a hallmark of many cancers and a key driver of genomic instability. Therapeutics targeting the ATR kinase, a central regulator of the replication stress response, have emerged as promising strategies for selectively exploiting the vulnerabilities of cancer cells. A reliable marker of replication stress is essential to better understand its dynamics and to optimize the development and application of DNA damage response (DDR) inhibitors. Such markers can provide critical insights into therapeutic mechanisms and enhance the precision of targeted treatments. Single-strand DNA breaks (SSBs) are both a cause and a consequence of replication stress, making them a potential biomarker. However, their occurrence during normal metabolism requires additional specificity, which can be provided by detecting replication stress-associated proteins. Here, utilizing our STRIDE technology platform, we combined direct in situ detection of SSBs with protein markers leading to the development of an assay aimed at measuring replication stress levels. The assay combined the detection of single-strand DNA breaks (SSBs) with replication stress-associated proteins: RPA, RAD17, and RAD9, evaluated separately in distinct assay variants. Each variant was assessed for steady-state readouts, negative control signal levels, and sensitivity to replication stress induced by known compounds. All variants effectively informed on replication stress induction, and baseline readouts of the assays correlated with the cellular response to replication stress-inducing agents. Importantly, the assays were also tested on FFPE tissue sections, demonstrating their feasibility for application to this type of material. The data collected thus far suggests that all assay variants hold potential as effective replication stress markers. Additional studies are required to further refine these assays, determine their optimal use in different contexts, and confirm their utility in identifying high replication stress backgrounds to predict responses to drugs targeting proteins involved in the replication stress response. Citation Format: Karolina Stępień, Kamila Schirmeisen, Jakub Lechowski, Magdalena Bartyńska, Magdalena Kordon-Kiszala, Kamil Solarczyk. Replication stress detection using STRIDE technology platform [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 4210.

Abstract 744: Exploration of potential biomarkers associated with treatment outcomes in locally advanced esophageal squamous cell carcinoma patients receiving neoadjuvant nivolumab plus paclitaxel and cisplatin chemoradiotherapy

Abstract Background: In a phase II trial (neo-NTP-CRT, NCT05130684), the efficacy of adding nivolumab to paclitaxel and cisplatin-based neoadjuvant chemoradiotherapy (CRT) was tested in patients with locally advanced esophageal squamous cell carcinoma (ESCC) (n=17). The study reported a pathological complete response (pCR) rate of 24%, a median relapse-free survival and overall survival (OS) of 8 and 16 months, respectively. We conducted this exploratory analysis to investigate the potential immune-related biomarkers. Materials and Methods: Pretreatment endoscopy-biopsied tumor tissues were analyzed. Immunohistochemistry (IHC) was used to measure PD-L1 expression by clone SP142 (Ventana, Tucson, AZ, USA) on tumor cells (PD-L1-TC), immune cells (PD-L1-IC), and combined positive score (CPS), and to define mature tertiary lymphoid structure (mTLS) according to CD20 and CD23 expression (n=16). The expression of immune-related genes in ESCC tumor tissues was quantitated by PanCancer Immune Profiling and analyzed by nCounter® Analysis System (nanoString, Seattle, WA, USA) (n=12). 12-chemokine TLS signature was also analyzed. The association of PD-L1-TC, PD-L1-IC, CPS, mTLS, and TLS signature with pCR was examined by Chi-square test; the association with OS was examined by log rank test. The differences of various immune cells and immune-related functions between patients with and without pCR was investigated by t-test. Results: pCR rate was significantly higher in the patients with positive PD-L1 TC (100% vs 18%, p=0.019), high PD-L1 IC (100% vs 18%, p=0.019), and CPS≥5 (100% vs 10%, p=0.0006), and trended to be higher in patients with mTLS (50% vs 14%, p=0.19) and higher 12-chemokine TLS signature (75% vs 17%, p=0.077). OS trended to be longer in patients with positive PD-L1 TC (median: not reached vs 15 months; HR: 0.24, p=0.05) and CPS≥5 (median: 26 vs 15 months; HR: 0.19, p=0.069), and patients with high PD-L1 IC had numerically longer OS (median: 25 vs 16 months; HR: 0.42, p=0.38). mTLS was not associated with OS (median: 25 vs 25 months; HR: 0.91, p=0.88); however, patients with higher 12-chemokine TLS signature had better OS (median: 25 vs 14 months; HR: 0.23, p=0.042). The analysis of immune-related gene expression revealed patients with pCR had significantly higher abundance of TILs, lower amounts of Treg, exhausted CD8 T cells, and macrophages. Significantly higher B cell function, cytotoxicity function, and Toll-like receptor function was found in tumors of pCR patients. Conclusion: We found that PD-L1 expression and 12-chemokine TLS signature may be a potential predictor of the response to neoadjuvant nivolumab plus paclitaxel and cisplatin-based CRT. (grants: NCTRC200909, MOST 108-2314-B-002-076-MY3, MOHW 111-TDU-B-221-114006) Citation Format: Ta-Chen Huang, Jhe-Cyuan Guo, Chia-Chi Lin, Hung-Yang Kuo, Chien-Huai Chuang, Jason Chia-Hsien Cheng, Feng-Ming Hsu, Jang-Ming Lee, Pei-Ming Huang, Chih-Hung Hsu. Exploration of potential biomarkers associated with treatment outcomes in locally advanced esophageal squamous cell carcinoma patients receiving neoadjuvant nivolumab plus paclitaxel and cisplatin chemoradiotherapy [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 744.

Abstract 1116: AI framework for imputing genome-wide somatic mutations in pan-cancer targeted sequencing data for biomarker discovery

Abstract Background: Tumor genotyping via high-throughput sequencing has revolutionized our understanding of various cancer types. The genotyping of tumor biopsies or liquid biopsies has now become an integral part of clinical routines to survey tumors and to identify potential markers for treatment management. This has led to the development of many assays that are either commercially or institutionally available for such testing, where they often target less than 0.5% of the entire genome, focusing on known and recurrent genes for cancer-specific panels, pan-cancer solid tumor panels, or heme panels targeting blood cancers. This enrichment of certain regions has allowed us to find mutations that occur at lower allele fractions. Nonetheless, this approach results in missing hundreds or even thousands of potential mutations, some of which could be potential biomarkers for prognostic or predictive models, or even potential targets for future drug developments. Here, we hypothesize that an artificial intelligence (AI) framework can aid us in unraveling the complex interactions between mutations and thereby impute the off-target space. Methods: Here, we introduce SPARK (Somatic mutation Prediction via Artificial intelligence for Rapid discovery and Knowledge translation), an algorithm for imputing missing genomic loci in existing genomic panels. SPARK relies on the assumption that there is a latent space in which mutations co-occur or are mutually exclusive across multiple malignancies. This AI-based framework utilizes other data modalities (e.g., gene expression) to train a unified model for solid tumors. During training, we use somatic mutations and RNA expression data, and then employ another network to transfer knowledge, enabling us to take only a small number of mutations from any sequencing panel and impute the rest. Results: We used genotyping and RNA expression data from TCGA across multiple carcinomas to train the model (n>2000 samples). This pre-trained model is then fine-tuned for each panel for further optimization. To evaluate model performance, we focused on the genes present on the MSK-IMPACT panels obtained from the GENIE Project v16. We found a number of genes that occur with over 1% frequency across multiple carcinomas in TCGA but were not covered by the MSK-IMPACT panels. We then aimed to impute these genes and achieved a precision ranging from 60% to 100% for a minority of genes (e.g., ETV4 and ETV5). The precision was much lower (less than 10%) for many genes, suggesting that those mutations are likely not functional and do not show any strong complex association with the genes in the panel. As the next step, we plan to apply this model to the entire set of samples from the GENIE Project and impute the genes that showed acceptable precision in our TCGA analysis. This larger imputed dataset will enable us to find potential biomarkers from a large number of samples in the GENIE Project. Citation Format: Amirsaman Ashtari, Mohammad Shahrokh Esfahani. AI framework for imputing genome-wide somatic mutations in pan-cancer targeted sequencing data for biomarker 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 1116.

Abstract 3292: Unraveling molecular mechanisms underlying the awakening of dormant melanomas

Abstract Tumor recurrence, which can occur many years after therapeutic intervention, represents the main challenge in clinical oncology. Clinically, this period between the “cure” of the primary tumor and its local or distal metastatic relapse is known as the “dormancy state.” Tumor dormancy has been linked to therapeutic resistance and a high metastatic potential of malignant neoplasms. During tumor immunoediting, the immune response shapes the tumor to adapt and thrive within an immunocompetent host. Epigenetic modulation is an important way by which cells can quickly regulate gene expression and respond to environmental pressures. Another way of rapid modulation can be through regulatory RNAs, such as lncRNAs which can interact at the level of transcription and translation of other molecules, such as protein-coding genes, and regulate gene expression. To better understand the molecular basis of this tumor dormancy and awakening, we established a murine immune-mediated model of awakening of dormant melanoma cells. In this model, a large number of apoptotic cells are used to generate an acute inflammation process in C57BL/6 mice that promotes a permissive environment for a sub-tumorigenic dose of viable metastatic melanoma cells grown into a fully formed tumor, that would not be formed otherwise. Our study shows that the sub-tumorigenic dose of viable cells only generates tumors in immunosuppressed (NSG) mice (100% of animals), indicating that in immunocompetent animals, the primary defense system - T and B lymphocytes, and NK cells - appears to contain it. Considering the role of epigenetic modulation in rapid cell response to the pressure of the environment, we also show that treatment with the global demethylating agent 5-aza-2’-deoxycytidine causes delay and reduction of tumor mainly through immune activation. This suggests the need for future investigations regarding the epigenetic machinery in our model. Thereby, genome-wide DNA methylome analysis was conducted (EPICarray), and differential gene expression (RNA-seq) analysis of tumors from animals treated (or not) with the demethylating agent. Differentially expressed genes are being selected for further in vitro and in vivo studies. To elucidate this model, we are carrying on assays to understand the role of acute inflammation in the awakening of melanoma dormancy by analyzing the inflammatory profile of the defense system using flow cytometry meanwhile we are conducting other in vivo experiments to better characterize our model. Now, we investigated lncRNAs that are differentially expressed in tumors of animals treated with that drug aiming for new potential biomarkers, or regulators of important immune-response protein-coding genes. All in vivo data were statistically validated using the Student's t-test. We aim to discover potential therapeutic targets with a functional role in the immune-mediated progression of melanoma. Supported by CNPq, CAPES, FAPESP Citation Format: Ianca R. Dias, Débora K. Alves-Fernandes, Lih Hongtao, Frank J. Slack, Gangning Liang, Miriam G. Jasiulionis. Unraveling molecular mechanisms underlying the awakening of dormant melanomas [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 3292.

Abstract 2440: Correlation analysis between AI-based H-score and clinical data in MET IHC stained WSIs

Abstract Background: MET protein expression is considered a potential biomarker in lung cancer, but there is no guideline for analysis, and its clinical significance has not been sufficiently analyzed. In order to explore its potential as a biomarker, it is necessary to analyze the relationship between the expression level of the biomarker and clinical data. In this study, we analyze the relationship between H-score, which is automatically measured based on cell detection detected by a deep learning model, and clinical data. Design: The dataset included 270 whole slide images (WSIs) of non-small cell lung cancer (NSCLC) immunohistochemically stained with a MET antibody (SP44), obtained through resection, biopsy, or endobronchial ultrasound (EBUS). For each of these WSIs, pathologists estimated the proportions for 0, 1+, 2+, and 3+ according to duty, and calculated the H-score based on these values. In addition, the analysis areas were manually specified to exclude the internal controls. We developed analysis models based on deep learning. The cancer area localization model identified the cancer areas for each patch, and the cell analysis model identified the cell membrane, cytoplasm, and nucleus of each cell. Additionally, the MET expression intensity of each cell was classified as 0, 1+, 2+, and 3+ based on the mean diaminobenzidine (DAB) optical density (OD) measured in the cell membrane. The H-score was calculated accordingly. Result: For each WSI, the correlation between the H-score estimated by AI and the pathologist's H-score was calculated as Pearson’s correlation coefficient = 0.6909 (p<0.0001). In addition, when analyzing the H-score estimated by AI by tumor subtype of clinical data, the mean H-score of adenocarcinoma samples was estimated to be 177, which was significantly higher than the mean H-score of 116 estimated for squamous cell carcinoma samples (Mann-Whitney test, p < 0.0001). Conclusion: For MET IHC stained WSIs, H-scores were estimated through cell analysis using deep learning and image analysis, and the correlation with pathologist's H-scores was confirmed, and H-score differences between tumor subtypes were confirmed. Semi-automated AI analysis can be used to analyze the correlation between clinical data and MET staining expression. Citation Format: Hyeon Seok Yang, Yunseob Hwang, DoKyung Kim, Yoon-La Choi, Kyungsoo Jung, Minjung Sung, Young Kee Shin, Ji-Hye Nam, Tae-Yeong Kwak, Sun Woo Kim, Hyeyoon Chang. Correlation analysis between AI-based H-score and clinical data in MET IHC stained WSIs [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 2440.

Abstract 2333: TRAIL-inducing imipridones ONC201, ONC206, and ONC212 demonstrate anti-neoplastic effects in colonic adenoma-derived organoids

Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Currently, screening is the most effective method to reduce mortality from CRC. Chemoprevention, which aims to delay or even prevent the onset of CRC through eradication or prevention of precursor colonic adenomas, is an alternative strategy to address this problem. The imipridone Dordaviprone/ONC201 is a potential chemopreventive agent that has been shown to induce apoptosis in cancer cells through both upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the integrated stress response. More recently, we have shown that ONC201 induces TRAIL and apoptosis in a dose-dependent manner in adenoma-derived organoids and reduces adenoma formation in the Apcmin/+ CRC mouse model. We hypothesized that ONC201’s analogues, ONC206 and ONC212, would similarly enhance human colonic adenoma cell death through both TRAIL-dependent and TRAIL-independent mechanisms. We therefore evaluated the effects of ONC206 and ONC212 in two adenoma-derived organoid lines: one developed from an adenoma from an individual with familial adenomatous polyposis (FAP) and the second from a sessile serrated adenoma (SSA). Methods: Sensitivity of the FAP and SSA organoid lines to ONC206 and ONC212 was assessed using CellTiter-Glo® luminescent cell viability assay. Organoids were plated in triplicates and treated with ONC206 and ONC212 (concentration ranging from 0 uM to 10 uM). Results were analyzed after 72 hours of incubation. Western blotting was performed on lysates generated from both FAP and SSA organoids treated with either DMSO, ONC206, or ONC212 for 24, 48, or 72 hours. Co-culture experiments were performed with the adenoma-derived organoids (dyed using blue CMAC dye) and human NK-92MI cells (dyed with green CMFDA dye) treated with either DMSO, ONC201, ONC206, or ONC212. Fluorescent images following administration of red live/dead dye were performed using DAPI, FITC, and cherry red channels on ImageXpress® at 0, 24, 48, and 72 hours. Results: ONC206 and ONC212 demonstrated antineoplastic effects on adenoma-derived FAP and SSA organoids, with half maximal inhibitory concentrations (IC50) less than that of ONC201 in FAP and SSA organoids. As was noted in ONC201 studies, the mechanism of action of ONC206 and ONC212 appears to be mediated through modulation of multiple pathways including the TRAIL pathway (TRAIL and DR5 upregulation) and the integrated stress responses (ATF4 upregulation). Co-culture with NK cells revealed a significant increase in NK-mediated organoid cell death. The ability of ONC206 and ONC212 to enhance NK-mediated killing is being evaluated. Further analysis of the effects of ONC206 and ONC212 on stemness markers are currently underway and may reveal other potential biomarkers and chemopreventive strategies for ONC206 and ONC212. Citation Format: Alexis J. Lannigan, Jasper Chan, Lanlan Zhou, Varun V. Prabhu, Michael Dame, Dean Brenner, Jason Spence, Wafik S. El-Deiry, Alexander G. Raufi. TRAIL-inducing imipridones ONC201, ONC206, and ONC212 demonstrate anti-neoplastic effects in colonic adenoma-derived organoids [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 2333.

Circulating Biomarkers to Predict Post-Operative Cognitive Decline in Patients Undergoing Coronary Artery Bypass Grafting

Post-operative cognitive decline (POCD) is characterized by impairments in cognitive functions. Coronary artery bypass grafting (CABG) is associated with a high risk of POCD due to its impact on neuroinflammation and oxidative stress. In this study, we investigated the dynamics of neurotrophic, inflammatory, and oxidative stress markers in a cohort of post-CABG patients to identify potential biomarkers for POCD. Blood samples were collected at baseline (immediately post-surgery) and at 3-month follow-up. Expression levels of NRF2 and other regulators of oxidative stress (GST, GSS, HMOX1, CAT, HSP27, and LOX-1), inflammatory mediators (IL-6, IP-10, and NFκB), and neuroprotective factor (BDNF) were analyzed. Cognitive assessments were performed using RBANS, TMT, TIB and MMSE. POCD patients exhibited an initial upregulation of NRF2-related antioxidant genes, which failed to sustain at 3-months follow-up, leading to a decline in HMOX1, IP-10 and BDNF protein levels, along with increased LOX-1 protein level and NFκB expression, indicating persistent oxidative stress and inflammation. In contrast, non-POCD patients demonstrated a sustained increase in antioxidant and neuroprotective markers, suggesting a more effective compensatory response. ROC analysis identified HMOX1 and BDNF as significant predictors of POCD, with LOX-1 and IP-10 emerging as diagnostic markers at follow-up. In conclusion, our findings highlight the dynamic regulation of oxidative stress and inflammatory pathways in POCD, emphasizing the failure of sustained neuroprotection in affected patients. Further large-scale studies are necessary to validate these findings, and biomarker-based screening could facilitate early risk stratification and targeted interventions to improve cognitive outcomes after cardiac surgery.Graphical Potential biomarkers of POCD (post-operative cognitive decline) in the circulatory system. It depicts patients without POCD, and highlights the activation of the NRF2 pathway to upregulate downstream genes, which exert antioxidant and anti-inflammatory effects. Central to the scheme is oxidative stress, which causes inflammation and subsequent damage, with protective biomarkers like HSP27, HMOX1, BDNF, and IP-10 counteracting its effects.

Abstract 2006: Exploring a novel crosstalk between PTK7 and EPHA2 tyrosine kinase receptors in metastatic colorectal cancer

Abstract Colorectal cancer (CRC) remains a significant public health challenge, with an urgent need of novel prognostic biomarkers and therapeutic targets. PTK7, a membrane tyrosine pseudokinase and Wnt receptor, is overexpressed in CRC and linked to poor prognosis. In CRC, PTK7 expression correlates with metastatic progression and decreased survival in non-metastatic patients, positioning PTK7 as both a potential biomarker and a promising drug target. Notably, a targeted antibody-drug conjugate (ADC) against PTK7 has recently shown efficacy in inducing tumor regression in patient-derived xenograft (PDX) models of solid tumors and has progressed to a Phase I clinical trial with promising results. Other PTK7-targeting ADCs and anti-PTK7 CAR-T cells recently entered into preclinical development. At the plasma membrane, PTK7 can interact with a range of membrane receptors, including VEGFR, EGFR, ROR2, and Plexins, enhancing their signaling capabilities. Using biotin proximity labeling in HCT116 CRC cells, we identified a novel network of PTK7-associated membrane proteins. Among these, we focused on EPHA2, an active tyrosine kinase receptor also implicated in cancer. Our findings indicate that PTK7 and EPHA2 interact via their extracellular domains (ECDs), and that PTK7 depletion significantly upregulates EPHA2 expression and signaling in response to its ligand EFNA-1 in HCT116 cells. Additionally, PTK7 regulates EPHA2’s endosomal sorting through a Rab11-dependent mechanism, as well as its K63-linked ubiquitination and subsequent lysosomal degradation. Mechanistically, PTK7 does not appear to participate directly in EFNA-1 binding but instead promotes multimerization of active EPHA2, a crucial step in amplifying EPHA2 signaling. This study aims to elucidate the functional role of the interaction between PTK7 and EPHA2 in colorectal cancer progression. By mapping the PTK7:EPHA2 interface using AlphaFold3 predictions and introducing targeted deletions and mutations within the ECDs of both receptors, we seek to understand how disrupting this interaction influences tumorigenic and metastatic processes. Our findings indicate that PTK7 depletion triggers cell rounding and detachment, with a marked increase in total and activated EPHA2 levels, which may drive pro-metastatic activity in vivo. Outcomes may significantly impact the use of anti-PTK7-ADCs or CAR-T cells and orient new therapeutic strategies to design agents interfering with PTK7 and EPHA2 interaction and function. Citation Format: Charlotte Dessaux, Constantin Semenchenko, Luc Camoin, Stéphane Audebert, Emilie Baudelet, Avais Daulat, Flavio Maina, Jean-Paul Borg. Exploring a novel crosstalk between PTK7 and EPHA2 tyrosine kinase receptors in metastatic colorectal 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 2006.

Drug Design in the Age of Network Medicine and Systems Biology: Transcriptomics Identifies Potential Drug Targets Shared by Sarcoidosis and Pulmonary Hypertension.

Network medicine considers the interconnectedness of human diseases and their underlying molecular substrates. In this context, sarcoidosis and pulmonary hypertension (PH) have long been thought of as distinct diseases, but there is growing evidence of shared molecular mechanisms. This study reports on common differentially expressed genes (DEGs), regulatory elements, and pathways between the two diseases. Publicly available transcriptomic datasets for sarcoidosis (GSE157671) and PH (GSE236251) were retrieved from the Gene Expression Omnibus database. DEGs were identified using GEO2R, followed by pathway enrichment and gene interaction analyses via GeneMANIA and STRING. Importantly, a total of 13 common DEGs were identified between sarcoidosis and PH, with 7 upregulated and 6 downregulated genes. The SMAD2/3 nuclear pathway was a shared enriched pathway, suggesting a role in fibrosis and immune regulation. There were also divergences between sarcoidosis and PH. For example, gene set enrichment analysis indicated significant associations of the IFN-gamma signaling pathway with sarcoidosis and the TNF-alpha signaling with PH. miRNA network analysis identified hsa-miR-34a-5p, hsa-let-7g-5p, and hsa-miR-19a-3p as key shared regulators linked to common genes in both sarcoidosis and PH. Finally, DGIdb analysis revealed potential therapeutic candidates targeting these genes in both diseases. This study contributes to the field of drug design and discovery from a network medicine standpoint. The shared molecular links uncovered between sarcoidosis and PH in this study point to several potential biomarkers and therapeutic targets. Further experimental validation and translational medical research are called for diagnostics and drugs, which can effectively and safely help the clinical management of both diseases.