Abstract 1385: Evaluation of kinase inhibitor efficacy and cancer-associated fibroblast-organoid crosstalk in a CRISPR-edited mouse model of bladder cancer.

Abstract Background: The PI3K/AKT/mTOR pathway is altered in more than 40% of bladder cancers (BCa), yet it remains untargeted therapeutically. We developed an immunocompetent, CRISPR-edited BCa mouse model that recapitulates common mutations in this pathway. The tumor microenvironment, particularly cancer-associated fibroblasts (CAFs), plays a critical role in BCa progression, and the molecular mechanisms underlying tumor-CAF crosstalk are incompletely understood in BCa. We focused on identifying genotype-specific kinase inhibitor combinations and elucidating cytokine-mediated crosstalk between CRISPR-edited organoids and CAFs. Methods: Urothelial organoids from Rosa26-LSL-Cas9/GFP mice were CRISPR-engineered with Trp53 knockout plus either Rb1 knockout or deletion of the region homologous to human chromosome 9p21. A third mutation in the mTOR pathway (PIK3CA H1047R hotspot mutation) was then introduced. Gene edits were confirmed and cell lines were characterized for kinase inhibitor sensitivity. Engraftment studies were assessed in immunocompromised and immunocompetent mice to evaluate therapeutic targeting of genotype-specific kinase inhibitors via bladder engraftment or subcutaneous injection. CAFs were purified from carcinogen-induced murine bladder tumors. Organoid-CAF co-injection experiments were performed to assess CAF dependency for tumorigenesis. Cytokine profiling was performed on organoid monocultures, CAF monocultures, and organoid-CAF co-cultures. GM-CSF knockout organoids were generated and co-cultured with CAFs to evaluate cytokine-mediated crosstalk. Results: Triple-mutant organoids formed tumors in immunocompromised mice and exhibited sensitivity to PI3K inhibitor treatment (pictilisib or alpelisib) vs double-mutant controls, validating our genotype-specific targeting approach. However, the same organoids were non-tumorigenic in immunocompetent hosts. Co-engraftment with CAFs promoted tumor formation, demonstrating CAF dependency for tumorigenesis in immunocompetent hosts. Cytokine profiling revealed elevated IL-6 and GM-CSF secretion from organoid-CAF co-cultures but not monocultures, with GM-CSF by organoids and IL-6 secreted by CAFs. GM-CSF knockout in organoids reduced IL-6 secretion from CAFs, supporting cytokine-mediated CAF-organoid crosstalk. Conclusions: Our findings suggest GM-CSF and IL-6 critically mediate CAF-dependent tumor engraftment and targeting this cytokine axis may disrupt CAF-organoid crosstalk to inhibit tumor growth in immunocompetent models. Future studies will evaluate immune contributions and dual cytokine targeting. These findings support precision medicine combining genotype-specific kinase inhibition with microenvironment-directed therapies for BCa patients harboring mTOR pathway alterations. Citation Format: Tahmina Haque, Henkel Valentine, Uttam Satyal, Laura Bukavina, Rhea Arya, Philip H. Abbosh, . Evaluation of kinase inhibitor efficacy and cancer-associated fibroblast-organoid crosstalk in a CRISPR-edited mouse model of bladder cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1385.