Aberrant epigenetic reprogramming together with dysregulated mTOR signaling are hallmarks of cancer, where altered chromatin methylation and nutrient-sensing pathways cooperate to drive tumor progression. S-adenosylmethionine (SAM), the universal methyl donor, is essential for these processes, yet how tumors sustain elevated SAM availability to support oncogenic transmethylation reactions remains poorly defined. Here, using prostate cancer (PCa) as a model system, we identify nicotinamide N-methyltransferase (NNMT) as a critical metabolic-epigenetic regulator and tumor suppressor. Using a prostate-specific Nnmt knockout mouse model, we demonstrate that NNMT loss accelerates PCa progression, particularly in the context of Pten deletion, resulting in infiltrating carcinoma and reduced survival. Mechanistically, NNMT functions as a "SAM-sink," and its loss increases intracellular SAM abundance, thereby activating mTORC1 signaling through SAMTOR-dependent sensing and broadly enhancing chromatin methylation. In human PCa, recurrent genomic deletions of NNMT occur in up to 7% of cases, and NNMT protein expression is largely absent in primary tumors and metastases. NNMT-deficient PCa cells exhibit elevated SAM:SAH ratios, increased histone methylation, and heightened mTORC1 activity, enabling sustained tumor growth even under dietary methionine-restriction (MR). Notably, combined MR and pharmacologic mTORC1 inhibition synergistically suppresses the growth of NNMT-deficient tumors, revealing a previously unrecognized therapeutic vulnerability. Collectively, these findings establish NNMT as a key tumor suppressor that constrains SAM-driven epigenetic and signaling programs in PCa and suggest a rational, diet-based therapeutic strategy for advanced cancers with NNMT loss.

Proteome and transcriptome data combined can help assess the relevance of non-coding germline variants. Here, we combine germline Structural Variants (SVs) with mass spectrometry-based proteomics on tumors from 1637 cancer patients spanning various tumor tissues of origin to determine the extent SV breakpoint patterns involve differential protein expression of nearby genes. Rare and singleton SVs disrupting protein expression of known cancer susceptibility genes collectively involve 6% of patients. About 24% of the hundreds of genes with SV-associated non-coding cis-regulatory alterations at the mRNA level are similarly associated at the protein level. Both rare and common SVs may associate with differential protein expression within a specific tumor type or across multiple tissue types, including SVs differentially represented by patient ancestry. SVs involving altered methylation of CpG Islands or enhancers are also implicated in differential protein expression. Our results emphasize the contribution of germline SVs to cancer heterogeneity at the proteome level.

Objectives/Goals: This study evaluates the significance of PU.1 upregulation and the PU.1-mediated regulation of the anti-inflammatory miR-146a on pro-inflammatory NF-kB signaling in RCC-derived macrophages. The downstream effects of PU.1 targeting on key phenotypes related to RCC immunity and how this impacts RCC progression will also be examined. Methods/Study Population: Bioinformatics data identifying PU.1 upregulation within RCC tumors, correlation with prognostic clear cell RCC markers, and survival analysis were obtained using The Cancar Genome Atlas (TCGA) RNA-sequencing data. Murine RCC cell line Renca possessing a Vhl deletion are used to recapitulate a common deletion in human clear cell RCC tumors. This study utilizes two methods to target PU.1 in orthotopic RCC, a conditional PU.1 deletion model and a pharmacologic PU.1 inhibitor model. Quantitative real-time PCR, western blotting, and immunofluorescent staining will evaluate NF-kB signaling in PU.1-targeted macrophages and murine RCC tumors. Flow cytometry and single-cell RNA-sequencing will reveal the effects of PU.1 targeting on key immune cell phenotypes and key metrics of RCC outcomes. Results/Anticipated Results: PU.1 mRNA and protein expression are upregulated in human clear cell RCC tumors compared to normal kidney tissue. A 10-year overall survival analysis in clear cell RCC tumors indicates that increased PU.1 mRNA expression is unfavorable (p = 0.0298; HR = 1.24; N = 133/133). PU.1 inhibition in orthotopic RCC reduces tumor volume, compared to control (p = 0.0328; N = 5/5). NF-kB signaling effectors are upregulated in PU.1-inhibited macrophages, including canonical miR-146a targets. We anticipate that PU.1-targeted macrophages and RCC tumors will yield significant increases in NF-kB signaling, pro-inflammatory macrophage phenotypes, increased antitumor immunity, and reduced tumor volume. Discussion/Significance of Impact: This is the first study to address PU.1 upregulation in RCC and use multiple methods to examine a potential function of PU.1 in immunosuppressive macrophages and RCC immunity. This work also assesses the relevance of miR-146a on canonical, pro-inflammatory NF-kB signaling in the novel context of RCC tumor immunity.

Prostate cancer (PCa), a common malignancy, is a leading cause of cancer-related deaths among men. Advances in high-throughput technologies have led to the identification of various genetic alterations, including amplifications, deletions, mutations, gene fusions, and aberrant gene expressions, associated with PCa initiation and progression. Identifying key drivers of tumor progression and their underlying signaling pathways contributes to early diagnosis and therapeutic targeting. Here, we showed that thyroid hormone receptor-interacting protein 13 (TRIP13), a member of the AAA-ATPase family is overexpressed in PCa. Additionally, we observed amplification of the TRIP13 locus in a small subset of PCa samples. Functional studies demonstrated that TRIP13 knockdown in PCa cells reduced their proliferation and invasion. Furthermore, ectopic overexpression of TRIP13 in prostate epithelial cells (RWPE-1) resulted in enhanced cell invasion. Additionally, pharmacologic inhibition of TRIP13 by the small molecule inhibitor DCZ0415 suppressed PCa cell proliferation, induced apoptosis, modulated markers of the epithelial-mesenchymal transition (EMT), and inhibited tumor growth. Overall, these findings highlight a functional role for TRIP13 in PCa progression and demonstrate its potential as a therapeutic target in TRIP13-overexpressing PCa.

PURPOSE:In the United States, African Americans (AA) have higher Pancreatic ductal adenocarcinoma (PDAC) incidence and mortality rates than Caucasian Americans (CA). This study aimed to identify distinct gene expression signatures and differentially regulated pathways in AA and CA PDACs. METHODS:Transcriptomic analyses were conducted on FFPE sections of PDACs (n = 40) from AA (9 PDACs/3 normal) and CA (31 PDACs/5 normal) tissues to evaluate the differential expression and signaling pathways within and between racial groups and to identify distinctive and common genes/pathways. RESULTS:We identified unique differentially expressed genes in both racial groups. Distinct set genes were modulated in AA and CA PDACs, compared to their respective normal tissues. Thirteen genes (seven upregulated and six downregulated) were differentially modulated in AA PDACs vs. CA PDACs. CIBERSORT analysis revealed distinct immune cell composition, with increased resting NK cells and activated mast cells, in AA PDACs, and higher CD4 memory T cells present in CA PDACs. Canonical subtype analyses indicated a more heterogenous subtype distribution in AA PDACs, whereas CA PDACs showed a predominance of classical subtypes. Using a publicly available database, we analyzed the top 25 upregulated genes (normal vs. tumor) for AA and CA racial groups and seven differentially upregulated genes in AA PDACs vs. CA PDACs comparison for associations with survival outcomes. Eight genes (CHST15, PARP15, NUDT16, SERPINB3, PADI1, H3C8, ZNF488, and LETM2) correlated with poor patient survival. CONCLUSION:These findings show distinct gene expression profiles and modulated pathways in AA and CA PDACs, supporting development of race-based therapeutic targets.

Cancer is a major cause of death worldwide. Various genomic and proteomic alterations in cells results in initiation of cancer, disease progression, and tumor metastasis. Large scale molecular data has been generated recently that can help identify these molecular changes. These genomic, transcriptomic, proteomic and epigenetic data can be utilized to evaluate and identify cancer biomarkers and sub-class specific precision targets. In order to analyze the large scale data, user friendly tools are needed. We had earlier developed UALCAN, an integrative pan-cancer cancer data analysis platform that allows users to evaluate the expression of each genes, microRNAs and long-noncoding RNAs and identify changes between normal and cancer tissues. Previously, we have described the development and release of the UALCAN Mobile application (app) that provides cancer transcriptomic data from The Cancer Genome Atlas (TCGA) to evaluate gene expression based on cancer subtypes. Here, we describe the update to the UALCAN mobile, which now provides data analysis option for epigenetic changes due to DNA promoter methylation and Clinical Proteomic Tumor Analysis Consortium (CPTAC) cancer proteomic data. This app, which is suitable for tablets and mobile devices, provides access to large cancer molecular datasets at the fingertips of the researchers. To find changes in expression of causative genes and to identify biomarkers and therapeutic targets, app will be extremely valuable. The app is free and can be downloaded from both iOS/Apple and Android play store. Citation Format: Sooryanarayana Varambally, David Rubey, Darshan Shimoga Chandrashekar, Ahmedur Rahman Shovon, Gopi Chand Puli, Santhosh Kumar Karthikeyan, Upender Manne, Chad J. Creighton, Sidarth Kumar. UALCAN Mobile app, an update to the cancer multi-omic data analysis application [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A019.

Triple-negative breast cancer (TNBC) shows racial disparities, with higher incidence in women of African ancestry (AA) compared to European ancestry (EA). Meta-transcriptomic analysis of TNBC tumor tissues from AA (n = 17) and EA (n = 19) subjects revealed distinct microbial landscapes. Hierarchical clustering based on microbial transcripts separated samples into two groups predominantly defined by racial ancestry. Bacterial genera including Hafnia and Cedecea were more abundant in AA tumors, while Erwinia was higher in EA tumors. Cellular composition analysis by xCell revealed differences in immune cell populations, with AA tumors having higher Th1 cell abundance and EA tumors containing higher macrophage M2 cell abundance. Nonetheless, AA women with high M2 abundance experienced poorer disease-free survival (DFS) than EA women. Integrative analyses revealed that high expression of human SPDYE2B gene was associated with Hafnia abundance and decreased DFS, highlighting complex host-microbe interactions in TNBC patients.

S1. BZW2 mRNA overexpression in CRC.

Cancer is a complex disease affecting various organs and is a major cause of death worldwide. During cancer initiation, disease progression, and tumor metastasis, various genomic and proteomic alterations are observed. Recent technological advances have led to the generation of large amounts of molecular data, including genomics and transcriptomics. These large-scale datasets can be utilized to analyze and identify sub-class-specific cancer biomarkers and targets. However, there is a need for the development of user-friendly tools for large-scale data analysis, disseminating the analyzed data in a visualizable format to cancer researchers with no programming skills. We developed UALCAN, a comprehensive platform that allows users to integrate disparate data to better understand the genes, proteins, and pathways perturbed in cancer and make discoveries of potential biomarkers and targets. In the current study, we describe the development of the UALCAN Mobile application (app) that will provide cancer transcriptomic data obtained from The Cancer Genome Atlas (TCGA) project to evaluate protein-coding gene expression based on various stratifications, including stage, grade, race, gender, and molecular-subtypes across over 30 types of cancers. In addition, the UALCAN mobile provides data analysis options for epigenetic changes due to DNA promoter methylation and Clinical Proteomic Tumor Analysis Consortium (CPTAC) cancer proteomic data. The app provides access to large cancer molecular datasets on the go. To find changes in the expression of causative genes and proteins and to identify biomarkers and therapeutic targets, UALCAN mobile app will be extremely valuable. The "UALCAN Mobile" app is free to use and can be downloaded from both the iOS/Apple and the Android Play Store and has been downloaded over 100 times in each of iOS and android app stores.

KDM5B, a lysine-specific histone demethylase, is widely upregulated in breast cancer. The current study investigated the role of KDM5B in breast cancer and explored the repurposing potential of the antiviral drug abacavir (ABC). The cytotoxic effects and the effect of ABC sensitization on doxorubicin (DOX) efficacy were evaluated using 2-D and 3-D cell culture models. KDM5B expression was elevated in breast cancer tissues compared to normal breast tissues. In vitro studies demonstrated that ABC treatment reduced KDM5B expression in breast cancer cells and increased their sensitivity towards DOX. ABC induced late apoptosis and S-phase arrest, while the ABC + DOX combination led to S/G2 phase arrest, late apoptosis, and cell death. Data generated from patient-derived breast tumoroids corroborated the 2-D cell culture-based findings. Additionally, molecular docking studies indicated that the active drug metabolite carbovir triphosphate (CBV-TP) could interact with the DNA polymerase β-DNA complex, suggesting its potential mechanism to be incorporated into the DNA synthesis cycle, leading to cell cycle arrest in tumor cells. Our findings highlight the repurposing potential of ABC to target KDM5B in breast cancer. This approach enhanced the efficacy of DOX, which could allow further dose reduction and reduced side effects, offering a promising therapeutic strategy.

Quadruple-negative breast cancers (also known as AR-triple negative (TN) BC) lack the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and androgen receptor (AR). AR-TNBC exhibits aggressive characteristics and a poor prognosis. Because of the lack of expression of therapeutic targets, limited therapeutic options exist for patients with AR-TNBC. Hence, new therapeutic targets and risk-predictive biomarkers are required for patients with AR-TNBC. In this study, we investigated the role of kinesin-like protein 1 (KIFC1) in AR-TNBC. We found that C/EBPβ binds to the KIFC1 promoter and induces its expression in the AR-TNBC cells. Notably, AR status was negatively correlated with KIFC1 levels. We also found that AR transcriptionally repressed the transcription factor C/EBPβ, which regulates the expression of KIFC1. The lack of AR expression in AR-TNBC led to C/EBPβ upregulation, thereby enhancing KIFC1 expression. Moreover, upregulation of KIFC1 in AR-TNBC increased cancer cell proliferation and promoted epithelial-mesenchymal transition (EMT), contributing to the aggressive characteristics of AR-TNBC. Inhibiting KIFC1 using the small molecule inhibitor CW069 significantly reduced tumor volume in mice bearing AR-TNBC xenografts, but not in those with triple-negative breast tumors. These data suggest that upregulation of C/EBPβ and KIFC1 contributes to the aggressive characteristics and poor prognosis of AR-TNBC, providing strong evidence that targeting KIFC1 using kinesin inhibitors could be a viable therapeutic approach for patients with AR-TNBC.

Supplementary figure legends 1-8, and supplementary tables 1-5

Cyclin-dependent kinase 4 and 6 inhibitor (CDK4/6i) with endocrine therapy benefits patients with hormone receptor-positive, human epidermal growth receptor 2-negative breast carcinomas. However, most tumors develop resistance to CDK4/6i during the course of therapy. Although preclinical studies have proposed molecular mechanisms for the resistance, predictive markers are yet to be discovered. We investigated the tumor molecular profiling in 42 patients with advanced-stage breast carcinoma who received CDK4/6i therapy. The tumors carrying a GATA-binding protein 3 (GATA3) gene mutation, mainly a frameshift variant, showed a better treatment response compared with other tumors. Furthermore, we explored the potential underlying mechanism of this association. To that end, nuclear expression of p18, one of the INK family proteins, was found to be positively associated with the GATA3 mutation, as well as a CDK4/6i treatment response. Therefore, our study suggests that a GATA3 gene mutation, collaborating with p18 protein expression in tumor nuclei, may have a predictive value for CDK4/6i therapy in breast carcinoma.

Proteotoxic stress progressively leads to irreversible cardiac abnormalities. Using a mouse model of reductive stress-induced proteotoxic cardiomyopathy, we identified novel microRNA signatures, termed “ProteotoxomiRs,” which reflect stage-specific and transgene-specific responses to proteotoxic stress. Seven microRNAs were uniquely linked to the human mutant R120G-αB-Crystallin transgene, indicating their direct association with the pathogenic protein. Additionally, we uncovered two distinct microRNA profiles associated with the early (pre-onset) and late (cardiomyopathy/heart failure) stages of disease progression. Early-stage signatures primarily modulate signaling pathways essential for cardiac health, including mTOR and MAPK, while late-stage signatures reveal regulatory disruptions in calcium signaling and autophagy insufficiency, driving irreversible cardiac damage caused by reductive stress (RS) and proteotoxicity in transgenic mice. These findings reveal stage-specific miRNA biomarkers with potential diagnostic and prognostic value, offering new insights into the molecular underpinnings of proteotoxic cardiac disease. Moreover, our miRNA-mRNA interaction analysis uncovered potential targets unique to the transgene-specific, early, and late stages of the disease, including several promising druggable candidates, warranting further validation for translational applications.

Breast cancer (BCa), a leading malignancy among women, is characterized by morphological and molecular heterogeneity. While early-stage, hormone receptor, and HER2-positive BCa are treatable, triple-negative BCa and metastatic BCa remains largely untreatable. Advances in sequencing and proteomic technologies have improved our understanding of the molecular alterations that occur during BCa initiation and progression and enabled identification of subclass-specific biomarkers and therapeutic targets. Despite the availability of abundant omics data in public repositories, user-friendly tools for multi-omics data analysis and integration are scarce. To address this, we developed a comprehensive BCa data analysis platform called MammOnc-DB ( http://resource.path.uab.edu/MammOnc-Home.html ), comprising data from more than 20,000 BCa samples. MammOnc-DB facilitates hypothesis generation and testing, biomarker discovery, and therapeutic targets identification. The platform also includes pre- and post-treatment data, which can help users identify treatment resistance markers and support combination therapy strategies, offering researchers and clinicians a comprehensive tool for BCa data analysis and visualization.

Solid-tubulocystic variant of intrahepatic cholangiocarcinoma (ST-iCCA) is newly described entity characterized by two distinct histologic growth patterns: (1) solid sheets of tumor cells with focal necrosis giving pseudopapillary appearance and (2) tubular or pseudoglandular structures containing pink, colloid-like material. Tumor cells are inhibin-positive and harbor NIPBL::NACC1 fusion gene. To date, only 28 cases of ST-iCCA have been documented. While prior molecular studies provided insights into ST-iCCA, genetic profiles of individual histologic components have not been explored. This study presents first transcriptomic analysis comparing the solid/pseudopapillary and pseudoglandular components of ST-iCCA. Two cases of histologically confirmed ST-iCCA were identified for RNA sequencing which was performed on solid/pseudopapillary component, pseudoglandular component, and normal tissue. Analysis revealed distinct gene expression profiles for each pattern. Solid/pseudopapillary component uniquely overexpressed DMRTA1, NEXMIF, PRDM6, SORCS3, and NALF, while pseudoglandular component exhibited unique overexpression of HRG, ITIH3, TAT, APOA2, CP, ALDOB, CPS1, F2, KHG1, SERPINC1, HPX, C9, ADGRF1, MUC21, SAA2, SPRR2A, SAA1, FGL1, CFHR1, and LBP. These findings establish unique gene signatures for these variants of ST-iCCA, providing potential biomarkers for differential diagnosis, prognosis and targeted therapy. The distinct genetic profiles may also uncover novel therapeutic targets to address the aggressive nature of ST-iCCA.