Base editing reveals context-dependent regulation of adhesion, anoikis, and motility by BAP1 in renal cell models

BAP1 is a tumor-suppressive deubiquitinase essential for DNA repair, and missense mutations in BAP1 are common in clear cell renal cell carcinoma (ccRCC). We previously showed that correction of the inactivating Glu31Lys mutation in KMRC-20 ccRCC cells using CRISPR/Cas9 base editing restored BAP1 function, reinstated anchorage dependence, and re-sensitized cells to anoikis. Here, we investigated whether disruption of Glu31 is sufficient to induce anchorage-independent growth and anoikis resistance in normal kidney epithelial cells. Using adenine base editing, we introduced an inactivating Glu31Gly mutation into HK-2 cells, generating two independent isogenic BAP1-mutant clones, and established a BAP1-knockout clone by CRISPR/Cas9 as an additional control. Glu31Gly mutants exhibited complete loss of BAP1 deubiquitinase activity and impaired UV-induced DNA damage repair, comparable to knockout cells. Despite the clear functional inactivation of BAP1, the Glu31Gly and knockout HK-2 cells neither acquired anchorage-independent growth nor anoikis resistance; instead, detached cells displayed increased apoptosis. In KMRC-20 cells, restoration of BAP1 enhanced both migration and invasion, whereas BAP1 inactivation or loss in HK-2 cells increased invasion but reduced migration, indicating distinct context-dependent roles for BAP1 in normal versus malignant renal cells. These findings demonstrate that BAP1 inactivation alone is insufficient to confer anchorage-independent survival in normal kidney epithelial cells and suggest that additional oncogenic alterations are required during kidney tumorigenesis. Our study further highlights the utility of precise base editing for dissecting the functional consequences of clinically relevant cancer mutations.