Abstract 4309: Loss of the tumor suppressor NUMB is a hallmark of biologically and clinically aggressive bladder cancer and leads to hyperactivation of an actionable RHOA-ROCK-YAP circuitry

Abstract Advances in the personalized management of bladder cancer (BCa) have been hampered by the lack of predictive biomarkers and targeted therapies. In this study, we highlight a previously uncharacterized role for the loss of the tumor suppressor, NUMB, as a hallmark of biologically and clinically aggressive BCa. Retrospective analyses of longitudinal patient cohorts show that low NUMB expression in the primary tumor associates with increased risk of muscle invasion progression in non-muscle invasive BCa (NMIBC), and correlates with advanced stage at diagnosis, vascular invasion, and worse overall survival in post-cystectomy muscle-invasive BCa (MIBC). The causal role of NUMB loss to bladder tumorigenesis is highlighted in a transgenic mouse model where targeted Numb gene deletion in the basal layer of the bladder mucosa is per se sufficient to drive malignant transformation of the normal urothelium and sensitizes it to carcinogenic insults, accelerating tumor onset and progression. Mechanistically, through integrative transcriptomic and functional analyses in mouse and human BCa models, we reveal that the molecular hallmark associated with NUMB loss is the inhibition of the Hippo cascade with ensuing activation of the YAP oncogene via a RHOA/ROCK-dependent pathway. This ultimately results in a YAP-dependent transcriptional activation of an EMT/plasticity program and acquisition of uniquely aggressive proliferative and invasive phenotypes in BCa cells. Through pharmacological or genetic inhibition studies, we provide formal proof-of-evidence of the actionability of the RHOA/ROCK/YAP-TEAD axis to selectively inhibit proliferation and invasion of NUMB-deficient primary mouse and established human BCa cells in 3D-Matrigel organoid models, and to curb their in vivo tumorigenic potential in xenograft studies. We thereby highlight RHOA, ROCK and YAP-TEAD as selective vulnerable targets for therapeutic intervention in highly aggressive NUMB-deficient human BCa. We also describe a minimal signature composed of 27 up/down-regulated genes, characteristic of the NUMB-defective condition (the “NUMBLESS signature) that behaves as an independent prognostic predictor of risk of MIBC transition in a validation cohort study including 535 NMIBC patients enrolled in the UROMOL trial, where the NUMBLESS signature also associates with favorable response to BCG treatment. In summary, our study identifies NUMB loss as a key determinant of an underlying aggressive biology in BCa, and highlights its potential as a clinically actionable biomarker for prediction of NMIBC progression to life-threatening MIBC and response to standard therapies (i.e. BCG) or, even more importantly, to innovative anti-RHOA/ROCK/YAP-TEAD targeted treatments, thereby advancing a framework for precision oncology for high-risk NMIBC patients. Citation Format: Francesco A. Tucci, Maria Grazia Filippone, Roberta Bonfanti, Elisa Guerrera, Luca Acerbi, Giuseppe Renne, Gianluca Vago, Daniela Tosoni, Salvatore Pece. Loss of the tumor suppressor NUMB is a hallmark of biologically and clinically aggressive bladder cancer and leads to hyperactivation of an actionable RHOA-ROCK-YAP circuitry [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 4309.