Abstract 3936: Altered endosomal pH regulation: molecular mechanisms of oncogenesis and therapeutic dependencies

Abstract ATP6AP1 and ATP6AP2, which we previously identified to be recurrently altered in granular cell tumors (GCTs), encode for accessory proteins of the vacuolar (V) -ATPase, which controls endosomal acidification. The mechanistic basis of how ATP6AP1/AP2 loss of function (LOF) leads to oncogenesis remains to be elucidated. Here, we sought to unravel the molecular mechanisms downstream of ATP6AP1/AP2 inactivation resulting in oncogenesis and identify associated therapeutic dependencies. Using CRISPR/Cas9 technologies, we generated ATP6AP1-knock out (KO), ATP6AP2-KO and non-target (NT) control immortalized Schwann cells, the likeliest cell of origin of GCTs. Compared to NT controls, ATP6AP1/AP2-KOs displayed increased cellular proliferation, migration, and anchorage independent growth. ATP6AP1/AP2-KOs, but not NT controls, demonstrated in vivo tumorigenesis upon subcutaneous implantation in NSG mice. In addition, ATP6AP1/AP2-KOs, compared to NT controls, displayed abnormal bulk endocytic function, evidenced by reduced endocytic cargo delivery to lysosomes in a DQ-Red BSA assay, and increased LC3BII/LC3BI ratio and expression of the autophagy marker GABARAP, indicating altered autophagic flux. ATAC-seq analysis of our cell modes revealed changes in chromatin accessibility upon ATP6AP1/AP2 inactivation, affecting transcription factors (TFs) associated to the Wnt pathway, including LEF/TCF (canonical Wnt) and NFAT (non-canonical Wnt), and TFs associated to autophagy pathways, among others. RNA-sequencing analysis revealed expression changes in key Wnt pathway components upon ATP6AP1/AP2 LOF, such as biphasic modulators (DKK1, DKK2 and SFRP1), secreted ligands (WNT2, WNT9a and WNT7B), transcriptional regulators (TLE1, TLE2, TLE3 and TLE4) and receptors (LGR5, FZD7 and FZD1). Consistent with these findings, ATP6AP1/AP2-KOs displayed upregulation of downstream canonical (MYC, Cyclin D1) and non-canonical (NFATC1, CALM1) Wnt targets at the mRNA and protein levels, and increased sensitivity to pharmacologic inhibition of the Wnt pathway, with significantly lower IC50s to IWR-1, compared to NT controls. Lastly, ATP6AP1/AP2-KOs displayed increased sensitivity to pharmacologic inhibition or induction of autophagy and/or endocytic processes, with lower IC50s to Chloroquine, N-Ethylmaleimide, Torin-1 and Bafilomycin-A1 compared to NT controls. Taken together, we demonstrate that ATP6AP1/AP2 inactivation leads to the acquisition of oncogenic properties in vitro and in vivo, altered endocytosis and autophagy. Furthermore, our findings suggest that ATP6AP1/AP2 LOF results in chromatin accessibility changes leading to activation of an oncogenic transcriptional program with increased signaling via the Wnt pathway. Notably, inactivation of these genes results in unique and previously unknown vulnerabilities, offering potential avenues for therapeutic exploration. Citation Format: Higinio Dopeso, Yingjie Zhu, Laxmi Gusain, Thais Basili, David Brown, Fatemeh Derakhshan, Edaise M. da Silva, Richard Koche, Pierre-Jacques Hamard, Xinyu Guo, Eneda Toska, Elisa DeStanchina, Hong Zhang, Jorge Reis-Filho, Britta Weigelt, Fresia Pareja. Altered endosomal pH regulation: molecular mechanisms of oncogenesis and therapeutic dependencies abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3936.