Dissecting mechanism of EPHX2 deficient Bladder Cancer and targeting metabolic and immunologic vulnerabilities

/Abstract. Despite the marked genomic heterogeneity of bladder cancer (BLCA), current clinical practice treats it largely as a uniform disease, resulting in suboptimal outcomes for a substantial subset of patients who do not respond to standard therapies. Thus, there is a critical need to define molecular subtypes and identify unique genomic and metabolic alterations in BLCA that can guide the development of precision medicine approaches. To address this unmet need, we conducted an integrative analysis of transcriptomic, proteomic, and metabolomic datasets derived from BLCA patient tumors. This comprehensive approach identified xenobiotic metabolism, apical junction remodeling, and immune regulatory pathways as key contributors to BLCA progression. Our first-in-field findings demonstrate that >50% of TCGA-BLCA tumors harbor shallow or deep deletions of the gene encoding epoxide hydrolase 2 (EPHX2), a critical enzyme involved in the detoxification of endogenous epoxides. Importantly, low EPHX2 expression was associated with poor clinical outcomes, suggesting a tumor-suppressive role for EPHX2. Metabolomic profiling revealed that EPHX2 deficiency (heterozygous/deep deletions or low mRNA) leads to accumulation of epoxyeicosatrienoic acids (EETs), which are lipid signaling molecules and direct substrates of EPHX2. EETs have been shown to promote proliferation, metastasis, and immune evasion in several cancers, and our results suggest that their accumulation due to EPHX2 deficiency may have biological significance in BLCA. Our preliminary data demonstrate that EPHX2 suppressed tumor progression in vivo, while its loss activated the AKT-PKN3 axis, which is linked to aggressive phenotypes. NECTIN4, a tumor-associated antigen and therapeutic target, was also upregulated in EPHX2-deficient tumors. Pharmacological inhibition of PKN3 or targeting of NECTIN4 with enfortumab vedotin (EV) suppressed tumor growth. Interestingly, EPHX2- deficient tumors showed an “immunologically hot” profile with enhanced interferon signaling, suggesting potential for immunotherapy responsiveness. In the current proposal, we seek to further mechanistically define the role of EPHX2 loss in BLCA progression and determine whether EPHX2 loss–specific signaling pathways can be therapeutically targeted to treat BLCA. In Aim 1, we will determine the mechanisms by which EPHX2 deficiency promotes tumor progression in BLCA. In Aim 2, we will determine the mechanisms by which EPHX2 deficiency induces NECTIN4 expression and impacts the TIME. In Aim 3, we will define the impact of combination treatment with EV, anti-PD1, and PKN3 inhibitor on the TIME and anti-tumor responses in EPHX2-deficient BLCA. At the basic science level, this project seeks to define how EPHX2 deficiency drives BLCA progression through metabolic and immune remodeling, using single-cell RNA sequencing, metabolomics, ATAC-seq, and single-cell spatial transcriptomics (including Xenium 5K, COMET) to profile human tumors and relevant mouse models. Single-cell RNA sequencing will reveal cellular heterogeneity and immune signaling in EPHX2-deficient tumors, while spatial multi-omics will map tumor-immune architecture and cell interactions in situ. Translationally, these insights will support the development of a triple combination therapy with PKN3 inhibitors, NECTIN4-directed agents (e.g., EV), and immune checkpoint therapy (e.g., anti–PD-1) for EPHX2-deficient BLCA. This approach has the potential to improve upon current frontline treatments for advanced urothelial BLCA by enabling biomarker-driven combination strategies. Project Number: 1R01CA311048-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Nagireddy Putluri (+2 co-PIs) | Institution: BAYLOR COLLEGE OF MEDICINE, HOUSTON, TX | Award Amount: $681,330 | Activity Code: R01 | Study Section: Mechanisms of Cancer Therapeutics C Study Section [MCTC] View on NIH RePORTER: https://reporter.nih.gov/project-details/11336647