Targeting the TGFβ signaling pathway to mitigate tumor metastasis in 9p21-loss urothelial bladder cancer.

4566 Background: We previously identified that 9p21-loss (containing MTAP and CDK2NA genes) occurs in 25% of metastatic urothelial cancer (mUC) and is associated with worse survival and increased visceral metastasis. Additionally, we found significantly increased expression of the TGFβ-SMAD3 pathway in 9p21-loss UC, which is well studied to induce epithelial mesenchymal transition (EMT) and lead to metastasis. We hypothesize that the TGFβ signaling pathway mediates visceral metastasis in 9p21-loss UC. Methods: We included mUC patients at MD Anderson Cancer Center (MDACC) who had MTAP testing completed (surrogate for 9p21-loss) and received chemotherapy, immune checkpoint therapy (ICT), and/or antibody drug conjugate between 2012 and 2022. Survival and metastasis were compared between MTAP deficient patients and MTAP proficient patients. The Memorial Sloan Kettering-Metastatic Events and Tropisms (MSK-MET) cohort of mUC patients and IMVigor210 dataset were assessed for genomic alterations. Mouse models bearing CDKN2A-MTAP double knock out (DKO) MB49 tumors and wild type (WT) MB49 tumors, as well as corresponding in vitro models were used for mechanistic studies. Results: 298 mUC patients at MDACC were identified with 27% (n = 81) MTAP deficient. MTAP deficient patients experienced significantly worse overall survival (16.2 vs 21.1 months; p = 0.002; HR 1.61; 95% CI 1.2-2.2), progression-free survival (3.9 vs 5.8 months; p < 0.001; HR 1.75; 95% CI 1.3-2.4) and increased visceral metastasis (62% vs 39%; p = 0.001) with lung predominance (44% vs 26%; p = 0.003) compared to MTAP proficient patients. We also found in the MSK-MET cohort that 26% (186/714) of patients with mUC to the bladder had lung metastasis and those with lung metastasis had significantly increased frequency of CDK2NA deletion compared to patients without lung metastasis (30% vs 18%; OR 2.0; 95% CI 1.3-3.0). Our preclinical data also demonstrated that CDKN2A-MTAP DKO mice resulted in significantly larger primary bladder tumors and worse survival compared to mice bearing WT MB49 tumors. Additionally, mice bearing orthotopic CDKN2A-MTAP DKO MB49 tumors readily developed lung metastasis. Analysis of the IMvigor210 dataset (N = 298) showed that 9p21-loss UC patients had significantly increased expression of TGFβ and EMT pathway genes. These data are consistent with data from our DKO MB49 tumor models. We are currently assessing the impact of a TGFβ inhibitor on mitigating CDKN2A-MTAP DKO-mediated metastasis. Conclusions: Our clinical and pre-clinical data demonstrates that 9p21-loss mUC leads to worse survival and increased visceral metastasis, especially to the lung. We found that the TGFβ signaling pathway may play a role in the development of metastasis. Moving forward, we plan to prospectively investigate the therapeutic potential of TGFβ inhibition in patients with 9p21-loss UC.