Base Editing Reveals Context-Dependent Regulation of Adhesion, Anoikis, and Motility by BAP1 in Renal Cell Models

Base Editing Reveals Context-Dependent Regulation of Adhesion, Anoikis, and Motility by BAP1 in Renal Cell Models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Base Editing Reveals Context-Dependent Regulation of Adhesion, Anoikis, and Motility by BAP1 in Renal Cell Models Chaeyeon Koo, Daye Lee, Boram Lee, Soyi Kim, Jiyeon Lee, Jongbum Kwon This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8306241/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Background 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 precise correction of the inactivating Glu31Lys mutation in KMRC-20 ccRCC cells using CRISPR/Cas9 base editing restored BAP1 activity, reinstated anchorage-dependent growth, and re-sensitized cells to anoikis. Here, we asked the converse question: whether disrupting Glu31 is sufficient to induce anchorage-independent growth and anoikis resistance in normal kidney epithelial cells. Methods Using the same adenine base-editing strategy, we introduced an inactivating Glu31Gly mutation into HK-2 normal kidney epithelial cells, generating two independent isogenic BAP1-mutant clones. As an additional control, we created a BAP1-knockout HK-2 clone via CRISPR/Cas9. Parental, mutant, and knockout cells were assessed for BAP1 enzymatic activity, DNA repair capacity, viability, proliferation, cell cycle status, anchorage-independent growth, and anoikis resistance. Migration and invasion of HK-2 mutants and knockouts were compared with KMRC-20 revertant clones in which endogenous Glu31Lys had been corrected. Results The Glu31Gly HK-2 mutants exhibited complete loss of BAP1 deubiquitinase activity and impaired UV-induced DNA damage repair—phenotypes comparable to BAP1-knockout cells—confirming successful functional inactivation. Despite this, both mutant and knockout HK-2 cells maintained parental-like morphology, viability, and proliferation. Surprisingly, Glu31Gly did not confer anchorage-independent growth or anoikis resistance: upon detachment, both mutant and knockout cells showed increased apoptosis. In contrast, in KMRC-20 cells, restoration of BAP1 activity enhanced both migration and invasion. Conversely, BAP1 inactivation or loss in HK-2 cells increased invasion but paradoxically reduced migration. These opposite outcomes indicate that BAP1 regulates motility through distinct mechanisms in normal versus malignant renal cells, likely reflecting differences in lineage state, cytoskeletal organization, and downstream signaling. Conclusions Although BAP1 restoration suppresses anchorage-independent growth and anoikis resistance in KMRC-20 ccRCC cells, BAP1 inactivation alone is insufficient to induce these oncogenic traits in normal HK-2 epithelial cells, implying that additional oncogenic alterations are required for anchorage-independent survival during kidney tumorigenesis. The divergent effects of BAP1 gain versus loss on migration and invasion further underscore the context-dependent nature of BAP1 function. These base-editing studies demonstrate that BAP1 differentially regulates adhesion, anoikis, and motility in normal and malignant renal cells and highlight the utility of precise base editing for dissecting clinically relevant mutations. BAP1 tumor suppressor clear cell renal cell carcinoma CRISPR/Cas9-mediated adenine base editing isogenic cell lines anchorage-independent growth anoikis migration invasion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Metastasis—the primary cause of cancer-related mortality—requires tumor cells to survive and disseminate beyond their tissue of origin. A central enabling trait is anchorage-independent growth, the ability to proliferate without extracellular-matrix attachment, a condition that normally activates anoikis, a specialized apoptotic program. Cancer cells evade anoikis by rewiring adhesion and survival pathways and frequently acquire enhanced migratory and invasive properties through cytoskeletal remodeling, epithelial–mesenchymal transition (EMT), and activation of integrin, cadherin, and Wnt/β-catenin signaling. Collectively, anchorage independence, anoikis resistance, and invasive potential define the metastatic phenotype and represent key biological vulnerabilities in tumor progression [ 1 – 4 ]. BAP1 is a ubiquitin C-terminal hydrolase (UCH)–containing deubiquitinase (DUB) that functions as a tumor suppressor and regulates diverse chromatin-associated processes, including transcription, DNA replication, DNA damage repair, and maintenance of genome stability [ 5 – 14 ]. Beyond its chromatin functions, BAP1 influences proliferation, differentiation, metabolism, and cell death [ 15 – 21 ]. Its nuclear–cytoplasmic distribution is tightly regulated: monoubiquitination within its nuclear localization signal (NLS) promotes cytoplasmic retention, whereas BAP1 autodeubiquitination permits nuclear entry [ 22 ]. A major substrate of BAP1 is histone H2A monoubiquitinated at lysine 119 (H2A-Ub), an epigenetic mark essential for transcriptional regulation, DNA replication, and DNA repair [ 23 ]. Germline mutations in BAP1 confer a strong predisposition to several malignancies, including clear cell renal cell carcinoma (ccRCC), mesothelioma, and uveal melanoma, collectively termed the BAP1 tumor predisposition syndrome [ 24 – 27 ]. This hereditary syndrome is characterized by remarkably high penetrance, and tumors that arise in mutation carriers frequently acquire additional somatic BAP1 alterations. Functionally, BAP1 loss has been linked to enhanced migration, invasion, and metastatic potential, in part through dysregulated EZH2 activity in ccRCC and uveal melanoma [ 28 – 32 ]. Although most cancer-associated BAP1 variants are missense or truncating mutations, the mechanistic basis by which these alterations promote tumor initiation and progression remains only partially understood [ 33 , 34 ]. Recent studies have revealed a critical role for BAP1 in the nucleotide excision repair (NER) pathway, which resolves UV-induced DNA lesions in a catalytic activity–dependent manner [ 35 ]. Following UV exposure, BAP1 is rapidly recruited to chromatin at damage sites through interactions with PARP1 and H2A-Ub. Glutamate 31 (Glu31)—a residue frequently mutated in cancers such as ccRCC and a site of PARylation—was shown to be essential for BAP1 function by enhancing its affinity for ubiquitin via a salt bridge with Arg72 and by promoting protein stability. PARylation at Glu31 further modulates BAP1 enzymatic activity, likely through reversible modification of this residue [ 35 ]. In addition, PARP1 enhances BAP1 activity in a PARylation-independent manner by cooperating with ASXL1 via its DNA-binding domain, revealing an additional layer of PARP1-mediated regulation [ 36 ]. Building on these findings, we previously employed CRISPR–Cas9 adenine base editing to precisely correct the cancer-associated Lys31 substitution to wild-type Glu31 in KMRC-20 ccRCC cells, creating isogenic cell lines that differ only at this single nucleotide [ 37 ]. This precise approach, which avoids confounding effects of overexpression or complete gene knockout, allowed us for the first time to examine the physiological consequences of restoring BAP1 activity in its endogenous context. Restoration of wild-type Glu31 reactivated BAP1 enzymatic function, re-established anchorage-dependent growth, reinstated sensitivity to anoikis, and triggered broad transcriptional remodeling, including downregulation of N-cadherin and β-catenin under non-adherent conditions [ 37 ]. These results revealed a previously unrecognized role for BAP1 in restricting anchorage-independent survival, highlighting the power of isogenic base-edited models to dissect cancer-related functions of BAP1 with single-residue precision. In the present study, we asked whether the converse is true: whether Glu31-disrupting mutations are sufficient to induce anchorage-independence phenotypes in normal epithelial cells. We generated isogenic HK-2 kidney epithelial cells harboring a BAP1-inactivating Glu31Gly substitution using CRISPR–Cas9 adenine base editing. Although this mutation completely abolished BAP1 catalytic activity, it failed to confer anchorage-independent survival or anoikis resistance, indicating that while BAP1 restoration is sufficient to suppress anchorage independence and aniokis resistance in ccRCC cells, BAP1 inactivation alone is not sufficient to induce these phenotypes in normal epithelial cells. Notably, while restoring BAP1 activity in KMRC-20 cells increased migratory and invasive behavior, BAP1 loss in HK-2 cells increased invasion but paradoxically reduced migration, suggesting complex, context-specific responses to BAP1 status. Together, these complementary gain- and loss-of-function models demonstrate that BAP1 differentially regulates adhesion, anoikis, and motility in normal versus malignant renal cells, highlighting the highly context-dependent tumor-suppressive role of BAP1 in renal carcinogenesis. Materials and Methods Cells and antibodies HK-2 (human kidney 2) cells—a proximal tubular epithelial cell line derived from normal adult male kidney and immortalized by transduction with human papillomavirus 16 (HPV-16) E6/E7 genes—were purchased from ATCC. Parental, knockout, and base-edited HK-2 cells were maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 µg/mL streptomycin. The KMRC-20 cell line (JCRB1071) and the revertant KMRC-20 clones (1–1, 1–3, and 1–34), described previously [ 37 ], were cultured in DMEM containing 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 µg/mL streptomycin. All cell lines were maintained at 37°C in a humidified incubator with 5% CO₂. The following primary antibodies were used: anti-BAP1 (mouse IgG, sc-28383; rabbit IgG, sc-28236), anti-α-tubulin (sc-8035), and anti-β-actin (sc-8432) from Santa Cruz Biotechnology; anti-H2A (07-146) from Millipore; and anti–cyclobutane pyrimidine dimers (CPD; CAC-NM-DND-001) from Cosmo Bio. Generation of based-edited and knockout HK-2 cells To convert BAP1 Glu31 to glycine (GAG→GGG) in HK-2 cells, we used the same adenine base editor—VRQR variant of ABE7.10max—previously applied to correct the Glu31Lys mutation (AAG→GAG) in KMRC-20 cells [ 37 ]. This editor recognizes an NGAG protospacer adjacent motif (PAM) and efficiently deaminates the adenine within the GAG codon. To redirect editing specificity to the adenine in the Glu31 codon, a single–nucleotide substitution was introduced into the original guide RNA (gRNA), which had been designed to correct the Glu31Lys mutation, using site-directed mutagenesis. The oligonucleotides used for mutagenesis were: forward, 5′-gaa aca ccg gga gga gat cta cga cct tcg-3′; reverse, 5′-cga agg tcg tag atc tcc tcc cgg tgt ttc-3′. The resulting construct, ABE7.10max-VRQR-BAP1-Gly31, was verified by sequencing to confirm successful alteration of the gRNA. The plasmid was transfected into HK-2 cells using FuGENE HD (Promega), after which transfected cells were selected with puromycin (2 µg/mL) and subjected to limiting dilution in 96-well plates for clonal isolation. Genomic DNA was extracted using Exgene™ Cell SV (GeneAll), and the region surrounding the target site was PCR-amplified and sequenced to verify base editing. The two top-predicted off-target sites were amplified and sequenced in an identical manner to assess off-target activity. To generate BAP1-knockout HK-2 cells, a gRNA targeting exon 2 of BAP1 (5′-acc gaa atc ttc cac gag ca-3′) was cloned into the Esp3I (BsmBI) site of LentiCRISPRv2 (Addgene #52961) to produce LentiCRISPRv2-BAP1-KO. The gRNA oligonucleotide sequences were: forward, 5′-caccg acc gaa atc ttc cac gag ca-3′; reverse: 5′-aaac tg ctc gtg gaa gat ttc ggt c-3′. LentiCRISPRv2-BAP1-KO was transfected into HK-2 cells, followed by puromycin selection and clonal isolation using the same workflow as for the base-edited clones. Sequencing of the PCR amplicon spanning the target region confirmed the presence of indel mutations. The predicted off-target sites were not present in the genome. Ub-AMC assay using BAP1 immune complexes BAP1 DUB activity was assessed using Ub-AMC substrate as previously described [ 37 ]. Briefly, parental or base edited HK-2 cell lysates were immunoprecipitated with anti-BAP1 antibody or control IgG, and the resulting immune complexes were washed and incubated with Ub-AMC (Boston Biochem) in DUB assay buffer. Fluorescence generated by AMC release was measured in black 96-well plates using a SpectraMax i3X microplate reader (excitation 350 nm, emission 450 nm) (NFEC-2019-10-258101, Ewha Fluorescence Core Imaging Center). Cell viability and cell cycle analysis Cell viability was measured by seeding 1,000 cells per well in 96-well plates and culturing for four or seven days, followed by MTS assay (CellTiter AQueous One Solution, Promega) according to the manufacturer’s protocol; absorbance was read at 490 nm. For clonogenic assays, 1,000 cells were plated in 35-mm dishes and grown for 14 days before fixation and crystal violet staining. For cell-cycle profiling, 1 × 10⁵ cells were fixed in 70% ethanol overnight, washed with PBS, stained with propidium iodide, and analyzed by flow cytometry (FACSCalibur, BD Biosciences) (NFEC-2022-04-278217, Ewha Fluorescence Core Imaging Center). Anchorage-independent growth assays Soft agar assays were performed by plating 2 × 10⁴ cells per 35-mm dish in 0.4% low-melting-point agarose over a 0.6% agarose base layer in PBS. Cultures were maintained for five weeks at 37°C in a humidified 5% CO₂ incubator. Colonies were stained with 0.001% crystal violet in 20% ethanol and quantified using ImageJ. Apoptosis assay Apoptosis was assessed by Annexin V/PI staining using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, 556547). Briefly, 2 × 10⁵ cells were washed twice with PBS, resuspended in binding buffer, and a 100 µL aliquot was incubated with 5 µL FITC–Annexin V and 5 µL PI (Invitrogen) for 20 min at room temperature in the dark. Stained cells were analyzed by flow cytometry on an LSRFortessa (BD Biosciences) (NFEC-2019-10-258102, Ewha Fluorescence Core Imaging Center). CPD assay Cyclobutane pyrimidine dimers (CPDs) were detected as previously described [ 35 ]. In brief, 1 × 10⁵ cells were seeded onto poly-L-lysine–coated coverslips, allowed to adhere, and irradiated with 10 J/m² UV-C. Cells were fixed at the indicated recovery times with methanol for 10 min at room temperature, followed by DNA denaturation in 2 M HCl for 30 min. After washing, cells were blocked in 3% BSA/PBS for 1 h and incubated with an anti-CPD monoclonal antibody (Cosmo Bio, CAC-NM-DND-001) for 1 h at room temperature. Alexa Fluor 568–conjugated goat anti-mouse IgG (Thermo Fisher Scientific) was applied for 30 min, and coverslips were washed with PBS containing 0.1% Tween-20 before mounting with VECTASHIELD containing DAPI (Vector Laboratories). Images were acquired on a Zeiss LSM 880 confocal microscope ((NFEC-2016-05-209580, Ewha Fluorescence Core Imaging Center), and CPD signal intensity was quantified using ZEN 2.3 software. Cell migration and invasion assay For the migration assay, cells were seeded in serum-free medium into Transwell inserts containing an 8-µm pore-size membrane (Corning 3422), with complete medium added to the lower chamber. For the invasion assay, cells were seeded in serum-free medium onto the upper surface of Transwell membranes pre-coated with Matrigel (Corning 354248), and complete medium was placed in the lower chamber. HK-2 cells were incubated for 6 h (migration assay) or 24 h (invasion assay), whereas KMRC-20 cells were incubated for 16 h (migration assay) or 96 h (invasion assay). After incubation, cells remaining on the upper membrane surface were removed by gentle swabbing, and cells that had migrated or invaded to the lower surface were fixed with 4% paraformaldehyde for 10 min and stained with crystal violet. Representative images were captured using an Axiovert200 microscope. Relative migration or invasion was quantified by dissolving the crystal violet stain in 10% acetic acid and measuring absorbance at 590 nm. Immunoblot analysis Whole-cell lysates were prepared in RIPA buffer (50 mM Tris-Cl, pH 8.0; 150 mM NaCl; 0.5% sodium deoxycholate; 0.1% SDS; 1% NP-40; 0.5 mM PMSF; 10 mM NaF; protease inhibitor cocktail). Protein concentrations were measured using the BCA Protein Assay Kit (Pierce). Equal amounts of protein were mixed with SDS sample buffer, boiled for 5 min, separated by SDS-PAGE, and transferred to membranes. Immunoblotting was performed using standard protocols with appropriate primary and HRP-conjugated secondary antibodies, and signals were detected by chemiluminescence. For detection of Ub-H2A and total H2A, histones were extracted as previously described. Briefly, cells were lysed in NETN buffer (20 mM Tris-Cl, pH 8.0; 150 mM NaCl; 1 mM EDTA, pH 8.0; 0.5% NP-40; 10 mM NaF; protease inhibitor cocktail) for 10 min at 4°C. Lysates were centrifuged at 8,000 rpm for 5 min at 4°C, and supernatants were discarded. Pellets were resuspended in 0.1 M HCl and incubated for 10 min at room temperature. After centrifugation at 12,000 rpm for 5 min, the supernatants were subjected to immunoblot analysis. CAPS transfer buffer (25 mM CAPS, 20% methanol) was used for membrane transfer. Full-length blots/gels are presented in Supplementary Fig. S2. Statistical analysis Statistical significance was assessed using unpaired Student’s t-tests performed with Microsoft Excel and GraphPad Prism 5 software. Data are presented as mean ± standard deviation (SD). Differences were considered statistically significant at p < 0.05. Statistical significance is indicated as follows: p < 0.05 (*), p < 0.01 (**), p 0.05 ( ns , not significant). Results Generation of isogenic HK-2 cells harboring the BAP1 Glu31Gly mutation by base editing Given that restoring wild-type BAP1 activity by correcting the Glu31 mutation rescues anchorage-dependent growth and anoikis sensitivity in ccRCC cells [ 37 ], we next tested the converse—whether introducing a Glu31-disrupting mutation is sufficient to induce anchorage-independent survival and anoikis resistance in normal kidney epithelial cells. To this end, we generated isogenic HK-2 human kidney epithelial cell lines in which Glu31 was converted to Gly using CRISPR/Cas9-mediated adenine base editing [ 38 , 39 ]. We employed the VRQR Cas9 variant of ABE7.10max, as described previously [ 37 , 40 ], which recognizes an AGAG protospacer adjacent motif (PAM) corresponding to the degenerate NGAG PAM in the BAP1 locus, together with a gRNA targeting the GAG codon encoding Glu31 to convert it to GGG (Gly31) (Fig. 1 A). This substitution has been shown to abolish BAP1 DUB activity [ 35 ]. Two independent base-edited clones, designated Gly31-1 and Gly31-2, were established, and sequencing confirmed precise on-target editing without detectable alterations at the top two predicted off-target sites (Fig. 1 B and Fig. S1 A, B). For comparison and to distinguish the effects of a single-amino acid substitution from complete loss of function, we also generated a BAP1-knockout HK-2 clone (BAP1-KO) by CRISPR/Cas9 targeting exon 2 (Fig. 1 C, D), which resulted in complete loss of BAP1 expression (Fig. 1 E). Confirmation of BAP1 inactivation in base-edited HK-2 clones To verify that the Glu31Gly substitution abolished BAP1 catalytic activity, we performed Ub-AMC assays following BAP1 immunoprecipitation. As expected, BAP1 isolated from parental HK-2 cells displayed robust DUB activity, whereas IgG control immunoprecipitates showed none, confirming assay specificity (Fig. 2 A). In contrast, BAP1 from the Gly31-1 and Gly31-2 clones exhibited no detectable activity (Fig. 2 A), demonstrating successful inactivation of BAP1 by base editing. Consistent with this loss of function, levels of H2A-Ub—a direct BAP1 substrate—were markedly elevated in both mutant clones, comparable to those observed in BAP1-KO cells (Fig. 2 B). We previously showed that Glu31 contributes to BAP1 protein stability: BAP1 variants carrying a Glu31Ala mutation exhibit substantially reduced protein levels in ccRCC cell lines (KMRC-20 and UMRC-6) and in HEK293T cells [ 35 ], and correction of the Glu31 mutation in KMRC-20 cells by base editing restores BAP1 protein abundance [ 37 ]. In contrast, the base-edited HK-2 Gly31 clones displayed BAP1 protein levels similar to parental HK-2 cells (Fig. 2 C), indicating that loss of Glu31 does not destabilize BAP1 in this cellular context. This divergence is unlikely to reflect differences between glycine and alanine substitutions—both residues lack PARylation capacity and both abolish DUB activity. Rather, the results suggest that Glu31-dependent regulation of BAP1 stability may be cell type-specific, pointing to distinct mechanisms governing BAP1 turnover in kidney epithelial versus ccRCC cells. The Glu31 mutation has minimal impact on HK-2 cell growth and proliferation BAP1 has been reported to exert either pro-survival or pro-apoptotic effects depending on cell type, genetic context, and experimental conditions. While it functions as a classic tumor suppressor in many settings—such as uveal melanoma and mesothelioma—it can promote proliferation in specific contexts, often by stabilizing pro-growth proteins through its DUB activity, as observed in certain breast cancer models [ 5 , 8 , 20 ]. In ccRCC, we previously showed that exogenous expression of wild-type BAP1, but not the Glu31Lys mutant, reduced viability of KMRC-20 cells in which endogenous mutant BAP1 was depleted by siRNA, consistent with a tumor-suppressive, anti-proliferative role [ 35 ]. However, restoring BAP1 activity under isogenic conditions by base editing did not significantly affect short-term viability or clonogenic capacity in KMRC-20 cells [ 37 ], indicating that overexpression and physiological reactivation yield distinct outcomes. To evaluate the broader cellular consequences of BAP1 loss or Glu31 inactivation in normal kidney epithelial cells, we characterized the BAP1-knockout and base-edited HK-2 clones. Morphologically, the knockout cells and the Gly31-1 and Gly31-2 mutants were indistinguishable from parental HK-2 cells (Fig. 3 A). Short-term viability assays showed a slight increase in viability and growth rate in the Glu31 mutant clones, whereas the BAP1-KO line exhibited a modest reduction (Fig. 3 B). Consistent with these observations, cell-cycle analysis revealed that neither the mutant nor knockout clones displayed major deviations from the parental profile, with all groups showing typical G1 and G2/M DNA-content peaks (Fig. 3 C). The proportion of sub-G1 cells—reflecting apoptotic populations—was also comparable across lines, indicating that the Glu31 mutation does not alter basal apoptotic activity (Fig. 3 C). Long-term clonogenic assays showed that the base-edited HK-2 clones formed slightly more colonies than parental cells, whereas the knockout clone formed slightly fewer (Fig. 3 D). Overall, these data indicate that loss of BAP1 activity through Glu31 mutation has minimal effects on HK-2 cell survival or proliferation. Taken together with our previous findings that reactivating BAP1 in ccRCC cells also does not impair growth under physiological expression levels, these results suggest that BAP1 plays a limited role in regulating baseline survival and proliferation in kidney epithelial cells. The reduced viability observed upon overexpression of wild-type BAP1 likely reflects non-physiological effects of supraphysiological expression rather than an inherent consequence of restoring BAP1 function. The Glu31 mutation impairs BAP1-dependent DNA repair in HK-2 cells To determine whether the Glu31 mutation compromises BAP1 function in HK-2 cells, we assessed its role in repairing UV-induced DNA damage, a well-established BAP1-dependent process. CPD assays revealed that parental HK-2 cells displayed time-dependent recovery following UV irradiation, with approximately 40% of damage repaired by 24 h (Fig. 4 A). In contrast, the BAP1-knockout clone showed a marked defect in DNA repair at all examined time points, achieving only 16% repair at 24 h (Fig. 4 A). Notably, both Gly31-1 and Gly31-2 clones also exhibited significant impairment in resolving UV-induced DNA lesions, mirroring the BAP1-knockout phenotype (Fig. 4 B). These findings indicate that the Glu31-to-Gly substitution not only abolishes BAP1’s catalytic activity but also disrupts its functional contribution to the cellular DNA damage response. A growing body of evidence demonstrates that BAP1 participates in multiple DNA repair pathways—including homologous recombination and non-homologous end-joining for double-strand break repair, as well as nucleotide excision repair—across a wide range of cell types. These include human cancer cell lines such as U2OS osteosarcoma, MCF7 breast cancer, KMRC-20 ccRCC, and DPM malignant pleural mesothelioma cells; normal human cells such as MEMa primary melanocytes, Met-5A mesothelial cells, LF1 fetal lung fibroblasts, and HK-2 renal epithelial cells (as shown here); the non-transformed HEK293T human embryonic kidney line; and non-human systems such as murine pancreatic cancer cells and DT40 chicken B lymphocytes [ 10 , 35 , 41 , 42 ]. Together, these findings indicate that BAP1 plays a broadly conserved and fundamental role in maintaining DNA repair capacity across diverse biological contexts. The Glu31 mutation does not induce anchorage-independent growth in HK-2 cells We next assessed whether the Glu31 mutation confers anchorage-independent growth in HK-2 cells using soft agar assays. As expected, parental HK-2 cells—which depend on adhesion for normal proliferation—displayed only minimal basal colony formation (Fig. 5 A, B). Contrary to our initial hypothesis, the Gly31-1 and Gly31-2 mutant clones did not form colonies, similar to the BAP1-knockout cells (Fig. 5 A, B). These findings indicate that, although restoration of BAP1 activity in KMRC-20 cells promotes anchorage dependence, loss of BAP1 function does not induce anchorage-independent growth in non-transformed HK-2 cells. Thus, while BAP1 activity is sufficient to restore anchorage dependence in kidney cancer cells, its inactivation alone is insufficient to drive anchorage-independent growth in normal kidney epithelial cells. The Glu31 mutation does not induce anoikis resistance in HK-2 cells Because the Glu31 mutation did not promote anchorage-independent growth and instead appeared to sensitize HK-2 cells to anchorage loss, we next examined its effect on anoikis [ 4 , 43 ]. Cells were cultured in ultra-low-attachment (ULA) dishes, which mimic non-adherent conditions and induce apoptosis triggered by loss of adhesion. Under standard adherent conditions, parental, BAP1-knockout, and Glu31Gly mutant clones all exhibited similarly low levels of apoptosis and necrosis (Fig. 6 A, B). As expected, parental HK-2 cells showed a marked increase in cell death upon anchorage loss, consistent with the behavior of normal epithelial cells. Notably, the Glu31Gly mutants did not exhibit reduced cell death in ULA conditions; instead, they showed a further increase in apoptosis, similar to the BAP1-knockout clone. This enhanced sensitivity to anchorage loss was driven predominantly by apoptosis, with minimal contribution from necrosis (Fig. 6 A, B). These findings parallel the soft agar results and indicate that BAP1 loss or inactivation does not confer anchorage-independent survival in normal kidney epithelial cells. Rather, while restoration of BAP1 is sufficient to re-establish anchorage dependence and anoikis sensitivity in kidney cancer cells [ 37 ], BAP1 loss alone is insufficient to induce anoikis resistance in a non-malignant background. Together, these observations suggest that additional oncogenic alterations beyond BAP1 inactivation are required for the acquisition of anchorage-independent growth and anoikis resistance during renal tumorigenesis. The effects of BAP1 loss and gain on cell migration and invasion are context dependent In addition to anchorage independence and anoikis resistance, metastatic progression frequently requires enhanced migratory and invasive behavior [ 44 , 45 ]. We therefore examined whether the inactivating Glu31Gly mutation alters these motility programs in HK-2 cells. Transwell assays showed that both the Glu31Gly mutant and BAP1-knockout HK-2 clones exhibited reduced migratory activity compared with parental cells (Fig. 7 A), indicating that BAP1 loss impairs the directional movement of non-malignant renal epithelial cells. In contrast, these same mutant and knockout cells displayed markedly increased invasive capacity (Fig. 7 A), demonstrating that BAP1 inactivation enhances matrix-penetrating behavior despite reduced migration. Together, these results reveal that BAP1 loss differentially influences migration and invasion in HK-2 cells, uncoupling these two motility programs. To assess the consequences of restoring BAP1 function, we evaluated the three KMRC-20 revertant clones (1–1, 1–3, and 1–34) in which the endogenous Glu31Lys mutation had been corrected to wild-type glutamate [ 37 ]. All revertant clones exhibited significant increases in migration compared with the parental KMRC-20 line, and this increase was paralleled by a marked enhancement in invasive capacity (Fig. 7 B). These findings were unexpected because BAP1 restoration in the same clones reinstated anchorage dependence and anoikis sensitivity—features typically associated with non-transformed epithelial cells. Thus, although BAP1 reactivation suppresses tumorigenic phenotypes related to anchorage independence and anoikis resistance, it simultaneously boosts motility-related behaviors in the KMRC-20 background. Taken together, these complementary gain- and loss-of-function models demonstrate that BAP1 influences invasion and migration in a highly context-dependent manner: BAP1 restoration promotes motility in ccRCC cells, whereas BAP1 inactivation reduces migration but increases invasion in normal kidney epithelial cells. These contrasting outcomes highlight the complex, pathway-specific roles of BAP1 in regulating cellular adhesion, motility, and invasive behavior during renal carcinogenesis. Discussion In this study, we used precise CRISPR base editing to dissect the functional consequences of the recurrent BAP1 Glu31 mutation in renal epithelial cells and to compare its effects with those observed in ccRCC cells harboring the corresponding cancer-associated substitution. By generating isogenic HK-2 clones carrying the Glu31Gly mutation alongside a BAP1-knockout line as a control, we were able to define the specific consequences of a single amino-acid substitution under native regulatory conditions, avoiding confounding effects commonly associated with overexpression, knockdown, or complementation approaches. Our findings confirm that the Glu31 residue is critical for BAP1's enzymatic and DNA repair functions: both the base-edited mutant and the knockout clone exhibited similarly impaired DUB activity and defective UV-induced DNA damage repair. Thus, the Glu31 residue functions as a key regulatory site required for full BAP1 activity in kidney epithelial cells. Despite the clear functional inactivation of BAP1 in the HK-2 Glu31Gly and knockout clones, these cells did not acquire hallmark oncogenic traits associated with BAP1 loss in ccRCC, such as anchorage-independent growth or anoikis resistance. Instead, both mutant and knockout cells underwent enhanced apoptosis upon detachment, behaving more like the parental cells than like their BAP1-deficient cancer cell counterparts. This finding contrasts sharply with our previous work in KMRC-20 cells, where reverting the endogenous Glu31Lys mutation to wild type restored anchorage dependence and re-sensitized cells to anoikis [ 37 ]. Taken together, these results demonstrate that while correcting BAP1 function is sufficient to suppress anchorage-independent growth in a cancer context, inactivating BAP1 alone is not sufficient to initiate these phenotypes in normal epithelial cells. These observations suggest that BAP1 loss may function primarily as a permissive or priming event, rather than acting a standalone driver of tumorigenesis. Its biological impact is highly context dependent, requiring cooperating oncogenic alterations to elicit hallmark features of cellular transformation. In normal HK-2 cells, loss of BAP1 activity is insufficient to circumvent the intrinsic apoptotic programs activated upon loss of adhesion, suggesting that additional genetic or epigenetic events—common in the evolution of ccRCC—are necessary to confer anchorage independence and anoikis resistance. This supports the model that BAP1 inactivation likely operates within a pre-existing permissive landscape—such as VHL loss or dysregulated hypoxia signaling—to overcome the robust apoptotic checkpoints characteristic of normal renal epithelium [ 26 , 33 , 46 – 49 ]. Consistently, emerging evidence indicates that the phenotypic consequences of BAP1 loss vary substantially depending on cellular lineage, metabolic state, and co-occurring mutations [ 16 , 33 ]. In renal cancer, BAP1 alterations frequently co-occur with disruptions in chromatin regulation, metabolic circuits, and hypoxia-responsive pathways, which together may enable survival in detached or mechanically stressed microenvironments [ 46 , 50 – 53 ]. An unexpected but important aspect of our findings is the context-dependent impact of BAP1 status on cell motility. In KMRC-20 cells, reactivation of BAP1 increased both migration and invasion, consistent with reports that BAP1 deficiency imposes a rigid mesenchymal-to-epithelial transition (MET)-like phenotype marked by reduced cytoskeletal dynamics and impaired single-cell movement in ccRCC cells [ 54 ]. Thus, restoring BAP1 function likely re-engages cytoskeletal remodeling programs—particularly those involving actin turnover and cell spreading—that are necessary for efficient directional migration. In contrast, introducing the BAP1-inactivating Glu31 mutation or knocking out BAP1 in non-malignant HK-2 epithelial cells produced an opposite pattern of motility regulation: migration was reduced, consistent with prior observations that BAP1 can promote cell movement in certain contexts such as breast cancer [ 20 ], yet invasion capacity increased. These parallel but divergent outcomes highlight that BAP1 controls motility through mechanistically distinct pathways in normal versus malignant renal contexts. In cancer cells, BAP1 restoration may promote differentiation-associated programs that strengthen cytoskeletal architecture and foster coherent, directed movement. In normal epithelial cells, however, BAP1 loss may relax basal constraints on extracellular matrix (ECM) engagement and remodeling, thereby supporting proteolysis-driven invasion while destabilizing the cytoskeletal polarization required for productive migration [ 55 ]. Base editing proved to be a powerful strategy for dissecting mutation-specific functions of cancer-associated genes by enabling the precise modeling of endogenous point mutations without perturbing regulatory architecture or protein stoichiometry. This approach allowed us to directly contrast the biological consequences of a clinically relevant BAP1 mutation in both non-malignant and cancer-derived isogenic backgrounds, revealing that identical mutations can generate markedly different outcomes depending on cellular lineage and oncogenic context. Our findings underscore that BAP1 does not exert uniform effects on adhesion, anoikis, or motility, but instead acts through context-dependent mechanisms that become apparent only in genetically matched systems. These results highlight the importance of evaluating cancer-associated BAP1 variants within precise isogenic settings to accurately define their functional and oncogenic consequences and point toward broader applications of base-editing platforms for studying recurrent missense mutations in ccRCC and other tumor types. Conclusions Using precise CRISPR base editing to model the recurrent BAP1 Glu31 mutation, we demonstrate that a single–amino acid substitution is sufficient to abolish BAP1 DUB activity and impair DNA repair in normal renal epithelial cells, yet is not sufficient to induce anchorage-independent growth or anoikis resistance. These results contrast with our previous finding that restoring wild-type BAP1 in ccRCC cells suppresses anchorage-independent growth and reinstates anoikis sensitivity, underscoring that the phenotypic consequences of BAP1 status are strongly context dependent. Notably, BAP1 gain and loss produced divergent effects on cell motility across malignant and normal backgrounds—BAP1 restoration increased both migration and invasion in KMRC-20 cells, whereas BAP1 inactivation in HK-2 cells reduced migration but enhanced invasion—highlighting a multifaceted, environment-specific role for BAP1 in regulating movement-related behaviors. Collectively, our findings indicate that additional oncogenic alterations beyond BAP1 mutation are required for acquisition of anchorage independence, anoikis resistance, and coordinated motility changes during renal tumorigenesis and emphasize the utility of isogenic, base-edited models for dissecting mutation-specific functions of cancer-associated genes. Abbreviations ABE, adenine base editor BAP1, BRCA1 Associated Protein-1 ccRCC, clear cell renal cell carcinoma CPD, cyclobutane pyrimidine dimer DUB, deubiquitinase ECM, extracellular matrix EMT, epithelial–mesenchymal transition gRNA, guide RNA H2A-Ub, histone H2A monoubiquitinated at lysine 119 HK-2 cells, human kidney 2 cells MET, mesenchymal-to-epithelial transition NER, nucleotide excision repair NLS, nuclear localization signal PAM, protospacer adjacent motif PARylation, poly(ADP)-ribosylation UCH, ubiquitin C-terminal hydrolase ULA, ultra-low attachment UV, ultraviolet Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All data generated or analyzed during this study are included in this published article and Supplementary Material. The detailed experimental procedures and the materials will be freely available upon request. Please contact [email protected] . Competing interests The authors declare no competing interests. Funding This work was supported by grants 2023R1A2C3005307 (JK) and 2022R1I1A1A01073047 (DL) from the National Research Foundation of Korea. Authors ’ contributions C.K., D.L., B.L., S.K., and J.L. performed the experiments and generated the data. C.K. and J.K. wrote the manuscript. J.K. conceived and supervised the research project. Acknowledgements Not applicable References Gerstberger S, Jiang Q, Ganesh K. Metastasis Cell. 2023;186(8):1564–79. Ring A, Nguyen-Strauli BD, Wicki A, Aceto N. Biology, vulnerabilities and clinical applications of circulating tumour cells. Nat Rev Cancer. 2023;23(2):95–111. Shaw P, Dey Bhowmik A, Gopinatha Pillai MS, Robbins N, Dwivedi SKD, Rao G. Anoikis resistance in Cancer: Mechanisms, therapeutic strategies, potential targets, and models for enhanced understanding. Cancer Lett. 2025;624:217750. Deng Z, Wang H, Liu J, Deng Y, Zhang N. Comprehensive understanding of anchorage-independent survival and its implication in cancer metastasis. Cell Death Dis. 2021;12(7):629. Kwon J, Lee D, Lee SA. BAP1 as a guardian of genome stability: implications in human cancer. Exp Mol Med. 2023;55(4):745–54. Szczepanski AP, Wang L. Emerging multifaceted roles of BAP1 complexes in biological processes. Cell Death Discov. 2021;7(1):20. Jensen DE, Proctor M, Marquis ST, Gardner HP, Ha SI, Chodosh LA, Ishov AM, Tommerup N, Vissing H, Sekido Y, et al. BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression. Oncogene. 1998;16(9):1097–112. Masclef L, Ahmed O, Estavoyer B, Larrivee B, Labrecque N, Nijnik A, Affar EB. Roles and mechanisms of BAP1 deubiquitinase in tumor suppression. Cell Death Differ. 2021;28(2):606–25. Lee HS, Lee SA, Hur SK, Seo JW, Kwon J. Stabilization and targeting of INO80 to replication forks by BAP1 during normal DNA synthesis. Nat Commun. 2014;5:5128. Yu H, Pak H, Hammond-Martel I, Ghram M, Rodrigue A, Daou S, Barbour H, Corbeil L, Hebert J, Drobetsky E, et al. Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair. Proc Natl Acad Sci U S A. 2014;111(1):285–90. Lee HS, Seo HR, Lee SA, Choi S, Kang D, Kwon J. BAP1 promotes stalled fork restart and cell survival via INO80 in response to replication stress. Biochem J. 2019;476(20):3053–66. Seo HR, Jeong D, Lee S, Lee HS, Lee SA, Kang SW, Kwon J. CHIP and BAP1 Act in Concert to Regulate INO80 Ubiquitination and Stability for DNA Replication. Mol Cells. 2021;44(2):101–15. Misaghi S, Ottosen S, Izrael-Tomasevic A, Arnott D, Lamkanfi M, Lee J, Liu J, O'Rourke K, Dixit VM, Wilson AC. Association of C-terminal ubiquitin hydrolase BRCA1-associated protein 1 with cell cycle regulator host cell factor 1. Mol Cell Biol. 2009;29(8):2181–92. Machida YJ, Machida Y, Vashisht AA, Wohlschlegel JA, Dutta A. The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1. J Biol Chem. 2009;284(49):34179–88. Daou S, Hammond-Martel I, Mashtalir N, Barbour H, Gagnon J, Iannantuono NV, Nkwe NS, Motorina A, Pak H, Yu H, et al. The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer. J Biol Chem. 2015;290(48):28643–63. Zhang Y, Shi J, Liu X, Feng L, Gong Z, Koppula P, Sirohi K, Li X, Wei Y, Lee H, et al. BAP1 links metabolic regulation of ferroptosis to tumour suppression. Nat Cell Biol. 2018;20(10):1181–92. Kuznetsov JN, Aguero TH, Owens DA, Kurtenbach S, Field MG, Durante MA, Rodriguez DA, King ML, Harbour JW. BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers. Sci Adv. 2019;5(9):eaax1738. Dey A, Seshasayee D, Noubade R, French DM, Liu J, Chaurushiya MS, Kirkpatrick DS, Pham VC, Lill JR, Bakalarski CE, et al. Loss of the tumor suppressor BAP1 causes myeloid transformation. Science. 2012;337(6101):1541–6. Bononi A, Giorgi C, Patergnani S, Larson D, Verbruggen K, Tanji M, Pellegrini L, Signorato V, Olivetto F, Pastorino S, et al. BAP1 regulates IP3R3-mediated Ca(2+) flux to mitochondria suppressing cell transformation. Nature. 2017;546(7659):549–53. Qin J, Zhou Z, Chen W, Wang C, Zhang H, Ge G, Shao M, You D, Fan Z, Xia H, et al. BAP1 promotes breast cancer cell proliferation and metastasis by deubiquitinating KLF5. Nat Commun. 2015;6:8471. Kang M, Park SG, Lee SA, Kim S, Lee D, Shirbhate ME, Youn SY, Kim KM, Cha SS, Kwon J. Targeting BAP1 with small compound inhibitor for colon cancer treatment. Sci Rep. 2023;13(1):2264. Mashtalir N, Daou S, Barbour H, Sen NN, Gagnon J, Hammond-Martel I, Dar HH, Therrien M. Affar el B: Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O. Mol Cell. 2014;54(3):392–406. Scheuermann JC, de Ayala Alonso AG, Oktaba K, Ly-Hartig N, McGinty RK, Fraterman S, Wilm M, Muir TW, Muller J. Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB. Nature. 2010;465(7295):243–7. Harbour JW, Onken MD, Roberson ED, Duan S, Cao L, Worley LA, Council ML, Matatall KA, Helms C, Bowcock AM. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science. 2010;330(6009):1410–3. Testa JR, Cheung M, Pei J, Below JE, Tan Y, Sementino E, Cox NJ, Dogan AU, Pass HI, Trusa S, et al. Germline BAP1 mutations predispose to malignant mesothelioma. Nat Genet. 2011;43(10):1022–5. Pena-Llopis S, Vega-Rubin-de-Celis S, Liao A, Leng N, Pavia-Jimenez A, Wang S, Yamasaki T, Zhrebker L, Sivanand S, Spence P, et al. BAP1 loss defines a new class of renal cell carcinoma. Nat Genet. 2012;44(7):751–9. Elsayed AM, Kittaneh M, Cebulla CM, Abdel-Rahman MH. An overview of BAP1 biological functions and current therapeutics. Biochim Biophys Acta Rev Cancer. 2025;1880(2):189267. LaFave LM, Beguelin W, Koche R, Teater M, Spitzer B, Chramiec A, Papalexi E, Keller MD, Hricik T, Konstantinoff K, et al. Loss of BAP1 function leads to EZH2-dependent transformation. Nat Med. 2015;21(11):1344–9. Sun C, Zhao C, Li S, Wang J, Zhou Q, Sun J, Ding Q, Liu M, Ding G. EZH2 Expression is increased in BAP1-mutant renal clear cell carcinoma and is related to poor prognosis. J Cancer. 2018;9(20):3787–96. Jin B, Zhang P, Zou H, Ye H, Wang Y, Zhang J, Yang H, Pan J. Verification of EZH2 as a druggable target in metastatic uveal melanoma. Mol Cancer. 2020;19(1):52. Hou C, Xiao L, Ren X, Cheng L, Guo B, Zhang M, Yan N. EZH2-mediated H3K27me3 is a predictive biomarker and therapeutic target in uveal melanoma. Front Genet. 2022;13:1013475. Schoumacher M, Le Corre S, Houy A, Mulugeta E, Stern MH, Roman-Roman S, Margueron R. Uveal melanoma cells are resistant to EZH2 inhibition regardless of BAP1 status. Nat Med. 2016;22(6):577–8. Carbone M, Harbour JW, Brugarolas J, Bononi A, Pagano I, Dey A, Krausz T, Pass HI, Yang H, Gaudino G. Biological Mechanisms and Clinical Significance of BAP1 Mutations in Human Cancer. Cancer Discov. 2020;10(8):1103–20. Carbone M, Adusumilli PS, Alexander HR Jr., Baas P, Bardelli F, Bononi A, Bueno R, Felley-Bosco E, Galateau-Salle F, Jablons D, et al. Mesothelioma: Scientific clues for prevention, diagnosis, and therapy. CA Cancer J Clin. 2019;69(5):402–29. Lee SA, Lee D, Kang M, Kim S, Kwon SJ, Lee HS, Seo HR, Kaushal P, Lee NS, Kim H et al. BAP1 promotes the repair of UV-induced DNA damage via PARP1-mediated recruitment to damage sites and control of activity and stability. Cell Death Differ 2022. Lee B, Lee J, Kwon J. PARP1 cooperates with ASXL1 to stimulate BAP1 deubiquitinase activity through its DNA-binding domain. Biochem Biophys Res Commun. 2025;792:152982. Lee D, Lee B, Lee J, Kwon J. Restoration of BAP1 activity via base editing suppresses anchorage-independent survival in kidney cancer. Cancer Cell Int 2025. Pickar-Oliver A, Gersbach CA. The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol. 2019;20(8):490–507. Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol. 2020;38(7):824–44. Koblan LW, Erdos MR, Wilson C, Cabral WA, Levy JM, Xiong ZM, Tavarez UL, Davison LM, Gete YG, Mao X, et al. In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice. Nature. 2021;589(7843):608–14. Sato H, Ito T, Hayashi T, Kitano S, Erdjument-Bromage H, Bott MJ, Toyooka S, Zauderer M, Ladanyi M. The BAP1 nuclear deubiquitinase is involved in the nonhomologous end-joining pathway of double-strand DNA repair through interaction with DNA-PK. Oncogene. 2024;43(15):1087–97. Perkail S, Andricovich J, Kai Y, Tzatsos A. BAP1 is a haploinsufficient tumor suppressor linking chronic pancreatitis to pancreatic cancer in mice. Nat Commun. 2020;11(1):3018. Frisch SM, Hu G. Anoikis resistance and cancer. BMC Cancer. 2025;25(1):1764. Yayan J, Franke KJ, Berger M, Windisch W, Rasche K. Adhesion, metastasis, and inhibition of cancer cells: a comprehensive review. Mol Biol Rep. 2024;51(1):165. Bouchalova P, Bouchal P. Current methods for studying metastatic potential of tumor cells. Cancer Cell Int. 2022;22(1):394. Walton J, Lawson K, Prinos P, Finelli A, Arrowsmith C, Ailles L. PBRM1, SETD2 and BAP1 - the trinity of 3p in clear cell renal cell carcinoma. Nat Rev Urol. 2023;20(2):96–115. Wang SS, Gu YF, Wolff N, Stefanius K, Christie A, Dey A, Hammer RE, Xie XJ, Rakheja D, Pedrosa I, et al. Bap1 is essential for kidney function and cooperates with Vhl in renal tumorigenesis. Proc Natl Acad Sci U S A. 2014;111(46):16538–43. Farley MN, Schmidt LS, Mester JL, Pena-Llopis S, Pavia-Jimenez A, Christie A, Vocke CD, Ricketts CJ, Peterson J, Middelton L, et al. A novel germline mutation in BAP1 predisposes to familial clear-cell renal cell carcinoma. Mol Cancer Res. 2013;11(9):1061–71. Bononi A, Wang Q, Zolondick AA, Bai F, Steele-Tanji M, Suarez JS, Pastorino S, Sipes A, Signorato V, Ferro A, et al. BAP1 is a novel regulator of HIF-1alpha. Proc Natl Acad Sci U S A. 2023;120(4):e2217840120. Wang SS, Gu YF, Wolff N, Stefanius K, Christie A, Dey A, Hammer RE, Xie XJ, Rakheja D, Pedrosa I, et al. Bap1 is essential for kidney function and cooperates with Vhl in renal tumorigenesis. Proc Natl Acad Sci U S A. 2014;111(46):16538–43. Cotta BH, Choueiri TK, Cieslik M, Ghatalia P, Mehra R, Morgan TM, Palapattu GS, Shuch B, Vaishampayan U, Van Allen E, et al. Current Landscape of Genomic Biomarkers in Clear Cell Renal Cell Carcinoma. Eur Urol. 2023;84(2):166–75. Webster BR, Gopal N, Ball MW. Tumorigenesis Mechanisms Found in Hereditary Renal Cell Carcinoma: A Review. Genes (Basel) 2022, 13(11). Gu YF, Cohn S, Christie A, McKenzie T, Wolff N, Do QN, Madhuranthakam AJ, Pedrosa I, Wang T, Dey A, et al. Modeling Renal Cell Carcinoma in Mice: Bap1 and Pbrm1 Inactivation Drive Tumor Grade. Cancer Discov. 2017;7(8):900–17. Chen P, Wang H, Zhang W, Chen Y, Lv Y, Wu D, Guo M, Deng H. Loss of BAP1 Results in Growth Inhibition and Enhances Mesenchymal-Epithelial Transition in Kidney Tumor Cells. Mol Cell Proteom. 2019;18(7):1320–9. Zanotelli MR, Zhang J, Reinhart-King CA. Mechanoresponsive metabolism in cancer cell migration and metastasis. Cell Metab. 2021;33(7):1307–21. Additional Declarations No competing interests reported. Supplementary Files SupplementarymaterialR1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 23 Jan, 2026 Reviews received at journal 18 Jan, 2026 Reviews received at journal 17 Jan, 2026 Reviews received at journal 16 Jan, 2026 Reviewers agreed at journal 13 Jan, 2026 Reviewers agreed at journal 13 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers invited by journal 07 Jan, 2026 Editor assigned by journal 05 Jan, 2026 Editor invited by journal 15 Dec, 2025 Submission checks completed at journal 12 Dec, 2025 First submitted to journal 12 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8306241","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":571256925,"identity":"cc6c8e52-16fd-4d09-b17f-f88972aa69a9","order_by":0,"name":"Chaeyeon Koo","email":"","orcid":"","institution":"Ewha Womans University","correspondingAuthor":false,"prefix":"","firstName":"Chaeyeon","middleName":"","lastName":"Koo","suffix":""},{"id":571256927,"identity":"c55310d2-3436-4b1b-9837-4d5a63590ddd","order_by":1,"name":"Daye Lee","email":"","orcid":"","institution":"Ewha Womans University","correspondingAuthor":false,"prefix":"","firstName":"Daye","middleName":"","lastName":"Lee","suffix":""},{"id":571256929,"identity":"6e418ca4-13ed-4769-9e55-65db5a961410","order_by":2,"name":"Boram Lee","email":"","orcid":"","institution":"Ewha Womans University","correspondingAuthor":false,"prefix":"","firstName":"Boram","middleName":"","lastName":"Lee","suffix":""},{"id":571256931,"identity":"2d60950d-3a4d-4d25-8ae6-c571f77b93fd","order_by":3,"name":"Soyi Kim","email":"","orcid":"","institution":"Ewha Womans University","correspondingAuthor":false,"prefix":"","firstName":"Soyi","middleName":"","lastName":"Kim","suffix":""},{"id":571256932,"identity":"f6092c3d-1cb7-4755-810f-185454ef4b92","order_by":4,"name":"Jiyeon Lee","email":"","orcid":"","institution":"Ewha Womans University","correspondingAuthor":false,"prefix":"","firstName":"Jiyeon","middleName":"","lastName":"Lee","suffix":""},{"id":571256933,"identity":"4b4dfe4b-179b-40dd-8dd6-980af091da2f","order_by":5,"name":"Jongbum Kwon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACxgYIZuBnYGCDCPEQq0WygVgtMG0MBgeI1cLc3nv45cw2mzzj4z1mD34w2Mkz8Jx9gN+GnnNplhvb0orNzpwxN+xhSDZs4G03wK9lRo6Z4cNthxO33cjdJsHDwJzAwM9GwB8wLZvnv90m+Yehnigtxg83ArVskODdJs3DcDiBgbeNgJaeM2aMM/+lJc44k/9NWsbguGEbzzH8Wgzbe4w/9pyxSexvP5Ym+aaiWp6fJ42AFmAMSiC4BvA0gBvIA6PmAyFFo2AUjIJRMMIBAB7yRI99AGUEAAAAAElFTkSuQmCC","orcid":"","institution":"Ewha Womans University","correspondingAuthor":true,"prefix":"","firstName":"Jongbum","middleName":"","lastName":"Kwon","suffix":""}],"badges":[],"createdAt":"2025-12-08 10:00:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8306241/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8306241/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100357411,"identity":"fac38924-5260-4c27-b1c0-879652acbd4d","added_by":"auto","created_at":"2026-01-16 07:19:51","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":225834,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/0aacace44ee94140c1fd23b9.tif"},{"id":99860192,"identity":"9c00e27e-dcc6-4b7e-86d2-3ef8efe9cfd1","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119434,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/c2c15219dd60e5fcfbf186a5.tif"},{"id":100357695,"identity":"bd62aa42-bfde-4ea8-9efc-60bf3295f962","added_by":"auto","created_at":"2026-01-16 07:20:13","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":193270,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/46c35932e301620b63d0c52f.tif"},{"id":100406175,"identity":"a2b330f1-79f7-48c9-a8a8-6ba02264d158","added_by":"auto","created_at":"2026-01-16 12:46:15","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153887,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptR1cleanversion.docx","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/3cce1cd61efc125814f78ef8.docx"},{"id":99860202,"identity":"38d1762a-fe9a-44c8-a4d6-f3b893db5b0a","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143778,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/810fb2f2e1c2f236576cdcf1.tif"},{"id":99860206,"identity":"a63c497c-32bc-4255-a835-60c2d11668c6","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109550,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/08f4352b5625aeeb1323cd11.tif"},{"id":99860207,"identity":"b1c43a34-9e86-4dd5-a888-916f28f2957b","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113146,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.6.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/a1843e7167ecaf8563c6d1e7.tif"},{"id":99860213,"identity":"ee83ec9e-c9cd-40ab-87b1-5cf1baadbb41","added_by":"auto","created_at":"2026-01-09 06:52:25","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":345494,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.7.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/84de6078ef428ac4959f9c52.tif"},{"id":99860217,"identity":"7ae0d159-b2f3-434f-ac4a-a98540216e31","added_by":"auto","created_at":"2026-01-09 06:52:25","extension":"json","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8824,"visible":true,"origin":"","legend":"","description":"","filename":"a6fb18df57cc4aae90eb449147d64cfa.json","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/2c578ca61dc35a02eeeb3024.json"},{"id":99860223,"identity":"66a87c7a-2576-420c-a545-6ed59a3250c5","added_by":"auto","created_at":"2026-01-09 06:52:25","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":195382,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialR1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/348c0c4131e4c04a02fbd5af.pdf"},{"id":99860216,"identity":"c5ff1bc6-1bcc-4ac9-b389-35b15b622830","added_by":"auto","created_at":"2026-01-09 06:52:25","extension":"xml","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146340,"visible":true,"origin":"","legend":"","description":"","filename":"a6fb18df57cc4aae90eb449147d64cfa1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/d46e12a2cbde8804d21a0d4a.xml"},{"id":100357267,"identity":"35ac3d2c-635c-424e-a661-3f904a5a33f3","added_by":"auto","created_at":"2026-01-16 07:19:33","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":225834,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/caa9368a7d279691c4e3ccaf.tif"},{"id":100357248,"identity":"1ea9c475-10e5-4807-8319-e61e4a4e73d6","added_by":"auto","created_at":"2026-01-16 07:19:31","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119434,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/91fc67526f80a10a41f7942a.tif"},{"id":99860214,"identity":"de1d0ad0-6d66-45f2-9352-4c84d3f4c036","added_by":"auto","created_at":"2026-01-09 06:52:25","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":193270,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/54b782f4ed6fcd9ab4692020.tif"},{"id":100357445,"identity":"d4256d70-4876-4d3e-91d5-5c45c1d69f61","added_by":"auto","created_at":"2026-01-16 07:19:52","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143778,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/bb0c4fad53c32f8932c41609.tif"},{"id":99860200,"identity":"6f369210-1ea6-45d4-a0e0-c7b316f1e09e","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109550,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/b7c38f3ca1e15f2c9f4c3df5.tif"},{"id":99860203,"identity":"5096d988-5154-4294-b840-c0567f75bf0d","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113146,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.6.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/87cb00655a6b21ffeb11389f.tif"},{"id":100357772,"identity":"74b3cd35-57eb-4e0d-9b64-3255ed018d98","added_by":"auto","created_at":"2026-01-16 07:20:18","extension":"tif","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":345494,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.7.tif","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/a75cbb6d6bebb16331f5292c.tif"},{"id":99860212,"identity":"ea0e1860-002e-4951-8490-ff7dd685687c","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":32057,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/9bfde6b888f98851ea101ff7.png"},{"id":100358187,"identity":"ed72d0ef-6f90-43ca-b42b-b59f443937a0","added_by":"auto","created_at":"2026-01-16 07:20:43","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20522,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/4657a2e55b27c7f11a1e9b79.png"},{"id":99860221,"identity":"12d3a421-60e6-4cb9-b3ad-d0492de9b2e5","added_by":"auto","created_at":"2026-01-09 06:52:25","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40057,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/71c2154fc5b6c69d3eba33d8.png"},{"id":99860215,"identity":"b1a6badd-2bdd-421b-bce0-dc47383da2af","added_by":"auto","created_at":"2026-01-09 06:52:25","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":34827,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/02f5c388e839d0052bcf0ce2.png"},{"id":99860222,"identity":"d477e1f6-8818-47ba-8d32-246e0101a4ee","added_by":"auto","created_at":"2026-01-09 06:52:25","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15223,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/3d18d2a53fc33eae3eef1d8e.png"},{"id":100357288,"identity":"8bfdc8d8-e972-4bf9-bf58-2a6700aecb07","added_by":"auto","created_at":"2026-01-16 07:19:36","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24681,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/08f79a53c87807b0ed99f3a6.png"},{"id":99860220,"identity":"7614e640-0b39-44b1-b0df-d7ff0f465802","added_by":"auto","created_at":"2026-01-09 06:52:25","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":75406,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/a6fabdcca2b094f13da3439b.png"},{"id":99860224,"identity":"11d5f7c5-b47e-4d96-9196-274780a14c24","added_by":"auto","created_at":"2026-01-09 06:52:25","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":145449,"visible":true,"origin":"","legend":"","description":"","filename":"a6fb18df57cc4aae90eb449147d64cfa1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/385ce4c44caead3de146ad68.xml"},{"id":100357774,"identity":"4bf85c06-618e-4f08-b446-1c697bcd9c78","added_by":"auto","created_at":"2026-01-16 07:20:18","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154917,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/5a535025765c11e0b4e0be08.html"},{"id":99860195,"identity":"d57c5643-fc66-4f40-a831-a86187f8ebf0","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":692855,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of isogenic HK-2 cells harboring the BAP1 Glu31Gly mutation using base editing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic of the A•T → G•C base editing strategy used to convert the Glu31 codon (GAG) to glycine (GGG) in HK-2 cells. The target adenine (green) is edited to guanine (red). The gRNA and PAM sequences are shown in blue and red, respectively. The ABE7.10max-VRQR variant, which recognizes an AGAG PAM, was engineered to target the BAP1 locus and edits the adenine at the sixth position from the 5′ end with highest efficiency. \u003cstrong\u003e(B)\u003c/strong\u003e Sequencing of genomic DNA from parental HK-2 cells and two independent base-edited clones, confirming the precise Glu31Gly substitution. \u003cstrong\u003e(C)\u003c/strong\u003e Schematic of CRISPR/Cas9-mediated BAP1 knockout targeting exon 2 of the BAP1 gene. The gRNA (blue) and PAM (red) sequences are indicated, and the predicted double-strand break site is marked by an arrowhead. The antisense strand sequence is displayed. \u003cstrong\u003e(D)\u003c/strong\u003e Sequencing analysis of parental and BAP1-knockout clones showing indel mutations at the targeted locus. Sense-strand sequences are presented in the 5′→3′ orientation, with PAM regions (CCC) indicated. \u003cstrong\u003e(E)\u003c/strong\u003e Immunoblot analysis demonstrating loss of BAP1 protein expression in the knockout clone.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/fe6d5c5d92ac9ae03262d5aa.png"},{"id":100357444,"identity":"f08f164b-829f-40ea-8655-5da7134444fa","added_by":"auto","created_at":"2026-01-16 07:19:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":477092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConfirmation of BAP1 inactivation in base-edited HK-2 clones\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Ub-AMC deubiquitinase (DUB) assay performed using BAP1 immune complexes. Lysates from parental and base-edited mutant cells were immunoprecipitated with anti-BAP1 or control IgG. One fraction was assayed for DUB activity (left), while the other was analyzed for BAP1 protein levels by immunoblotting (right). Data are representative of three independent experiments. Fluorescence values, reflecting AMC release, are shown as relative fluorescence units (RFU). \u003cstrong\u003e(B)\u003c/strong\u003eImmunoblot analysis of H2A ubiquitination (H2A-Ub) in parental, BAP1-knockout, and base-edited mutant clones. Quantification (right) shows H2A-Ub levels normalized to total H2A, with parental HK-2 set to 1 (n = 3; mean ± s.d.). \u003cstrong\u003e(C)\u003c/strong\u003eSteady-state BAP1 protein levels in parental, knockout, and mutant clones, assessed by immunoblotting.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/c410f8b8968089e0f4360356.png"},{"id":100357758,"identity":"488e0562-3866-496e-9892-a40c8208126b","added_by":"auto","created_at":"2026-01-16 07:20:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":641919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of BAP1 loss and Glu31 mutation on HK-2 cell morphology and proliferative capacity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eRepresentative microscopic images of parental, BAP1-knockout, and base-edited mutant HK-2 cells showing overall similar cell morphology. \u003cstrong\u003e(B)\u003c/strong\u003eShort-term cell viability measured by MTS assay. Relative viability was normalized to day 1 values (set to 1). Experiments were performed in triplicate (n = 3; mean ± s.d.). \u003cstrong\u003e(C)\u003c/strong\u003e Cell cycle profiles analyzed by flow cytometry. Representative histograms are shown. Quantification of sub-G1 (apoptotic), G1, S, G2/M, and polyploid cell populations is presented to the right (n = 3; mean ± s.d.). \u003cstrong\u003e(D)\u003c/strong\u003e Colony formation assays assessing long-term clonogenic potential. Representative colony images are shown with quantification of colony numbers to the right. Experiments were performed in triplicate (n = 3; mean ± s.d.).\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/7d01ef6aa721df94aab30bf5.png"},{"id":99860201,"identity":"575e01c7-3d19-4d34-9c51-b91b340805da","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":649822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Glu31 mutation impairs BAP1-dependent DNA repair in HK-2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) BAP1 knockout reduces CPD repair in HK-2 cells. Cells were exposed to UV-C irradiation (10 J/m²) and fixed immediately (0 h) or after recovery for 8, 16, or 24 h. Residual CPDs were detected by immunofluorescence staining. (Top) Representative confocal images. (Bottom left) CPD staining intensities pooled from three independent experiments displayed as a scatter plot. (Bottom right) Quantification of CPD repair shown as the percentage of average CPD signal at each time point. n = 3; error bars represent mean ± s.d.\u003c/p\u003e\n\u003cp\u003e(B) CPD repair analysis of parental, BAP1-knockout, and base-edited mutant HK-2 clones at 0 and 24 h post-irradiation. Quantification was performed as in (A), and data are presented in the same format. n = 3; error bars represent mean ±s.d.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/d2ffb01759f67c35574bcdd6.png"},{"id":99860196,"identity":"bc22e321-1e57-4365-929f-72e926fcde6e","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":291378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Glu31 mutation does not induce anchorage-independent growth in HK-2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eSoft agar assay assessing anchorage-independent growth in parental, BAP1-knockout, and base-edited mutant HK-2 cells. Representative colony images are shown, with magnified views of the boxed regions on the right. \u003cstrong\u003e(B)\u003c/strong\u003eQuantification of colony numbers from (A). Experiments were performed in triplicate. n = 3; mean ± s.d.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/d4814da3987d9a59dd8928b3.png"},{"id":100357732,"identity":"2a1f9fac-9cea-4dd6-9eb4-d924d8f41a3f","added_by":"auto","created_at":"2026-01-16 07:20:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":470530,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Glu31 mutation does not induce anoikis resistance in HK-2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Annexin V/PI apoptosis assays performed under adherent and non-adherent conditions. Parental, BAP1-knockout, and base-edited mutant HK-2 cells were cultured on standard dishes or ultra-low attachment (ULA) plates for 24 h, followed by flow cytometric analysis. Representative dot plots are shown, with percentages of necrotic cells (upper left) and apoptotic cells (upper right + lower right). \u003cstrong\u003e(B)\u003c/strong\u003eQuantification of total cell death (necrosis + apoptosis) (left) and separate apoptotic and necrotic populations (right). n = 3; mean ± s.d.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/adfa79aaf3248d6d3365c03b.png"},{"id":100357696,"identity":"9606ca57-f865-44c4-9049-2ac54b352086","added_by":"auto","created_at":"2026-01-16 07:20:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":964697,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of BAP1 loss and gain on cell migration and invasion are context dependent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Transwell migration and invasion assays in HK-2 cells. BAP1 knockout or the Glu31Gly mutation reduced migratory capacity but enhanced invasive behavior. (Top) Representative images are shown. (Bottom) Quantification of migration and invasion, normalized to parental HK-2 cells. n = 3; mean ± s.d. \u003cstrong\u003e(B)\u003c/strong\u003eTranswell migration and invasion assays in KMRC-20 cells. Restoration of BAP1 activity increased both migration and invasion. Quantification was performed as in (A). n = 3; mean ± s.d.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/3e525b657b8ea6e1456c93af.png"},{"id":100413918,"identity":"e7fa2627-242f-4c37-9710-034b7858c796","added_by":"auto","created_at":"2026-01-16 13:18:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5457211,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/a4836a15-dae3-49be-9d90-e1a4b23e57ba.pdf"},{"id":99860191,"identity":"4a7e0d0a-fb82-451b-8131-34441da5ba6f","added_by":"auto","created_at":"2026-01-09 06:52:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":195382,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialR1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8306241/v1/7954557bedfa1010903fa8c2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Base Editing Reveals Context-Dependent Regulation of Adhesion, Anoikis, and Motility by BAP1 in Renal Cell Models","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMetastasis\u0026mdash;the primary cause of cancer-related mortality\u0026mdash;requires tumor cells to survive and disseminate beyond their tissue of origin. A central enabling trait is anchorage-independent growth, the ability to proliferate without extracellular-matrix attachment, a condition that normally activates anoikis, a specialized apoptotic program. Cancer cells evade anoikis by rewiring adhesion and survival pathways and frequently acquire enhanced migratory and invasive properties through cytoskeletal remodeling, epithelial\u0026ndash;mesenchymal transition (EMT), and activation of integrin, cadherin, and Wnt/β-catenin signaling. Collectively, anchorage independence, anoikis resistance, and invasive potential define the metastatic phenotype and represent key biological vulnerabilities in tumor progression [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBAP1 is a ubiquitin C-terminal hydrolase (UCH)\u0026ndash;containing deubiquitinase (DUB) that functions as a tumor suppressor and regulates diverse chromatin-associated processes, including transcription, DNA replication, DNA damage repair, and maintenance of genome stability [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Beyond its chromatin functions, BAP1 influences proliferation, differentiation, metabolism, and cell death [\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19 CR20\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Its nuclear\u0026ndash;cytoplasmic distribution is tightly regulated: monoubiquitination within its nuclear localization signal (NLS) promotes cytoplasmic retention, whereas BAP1 autodeubiquitination permits nuclear entry [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A major substrate of BAP1 is histone H2A monoubiquitinated at lysine 119 (H2A-Ub), an epigenetic mark essential for transcriptional regulation, DNA replication, and DNA repair [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGermline mutations in BAP1 confer a strong predisposition to several malignancies, including clear cell renal cell carcinoma (ccRCC), mesothelioma, and uveal melanoma, collectively termed the BAP1 tumor predisposition syndrome [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This hereditary syndrome is characterized by remarkably high penetrance, and tumors that arise in mutation carriers frequently acquire additional somatic BAP1 alterations. Functionally, BAP1 loss has been linked to enhanced migration, invasion, and metastatic potential, in part through dysregulated EZH2 activity in ccRCC and uveal melanoma [\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Although most cancer-associated BAP1 variants are missense or truncating mutations, the mechanistic basis by which these alterations promote tumor initiation and progression remains only partially understood [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have revealed a critical role for BAP1 in the nucleotide excision repair (NER) pathway, which resolves UV-induced DNA lesions in a catalytic activity\u0026ndash;dependent manner [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Following UV exposure, BAP1 is rapidly recruited to chromatin at damage sites through interactions with PARP1 and H2A-Ub. Glutamate 31 (Glu31)\u0026mdash;a residue frequently mutated in cancers such as ccRCC and a site of PARylation\u0026mdash;was shown to be essential for BAP1 function by enhancing its affinity for ubiquitin via a salt bridge with Arg72 and by promoting protein stability. PARylation at Glu31 further modulates BAP1 enzymatic activity, likely through reversible modification of this residue [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, PARP1 enhances BAP1 activity in a PARylation-independent manner by cooperating with ASXL1 via its DNA-binding domain, revealing an additional layer of PARP1-mediated regulation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBuilding on these findings, we previously employed CRISPR\u0026ndash;Cas9 adenine base editing to precisely correct the cancer-associated Lys31 substitution to wild-type Glu31 in KMRC-20 ccRCC cells, creating isogenic cell lines that differ only at this single nucleotide [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This precise approach, which avoids confounding effects of overexpression or complete gene knockout, allowed us for the first time to examine the physiological consequences of restoring BAP1 activity in its endogenous context. Restoration of wild-type Glu31 reactivated BAP1 enzymatic function, re-established anchorage-dependent growth, reinstated sensitivity to anoikis, and triggered broad transcriptional remodeling, including downregulation of N-cadherin and β-catenin under non-adherent conditions [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These results revealed a previously unrecognized role for BAP1 in restricting anchorage-independent survival, highlighting the power of isogenic base-edited models to dissect cancer-related functions of BAP1 with single-residue precision.\u003c/p\u003e \u003cp\u003eIn the present study, we asked whether the converse is true: whether Glu31-disrupting mutations are sufficient to induce anchorage-independence phenotypes in normal epithelial cells. We generated isogenic HK-2 kidney epithelial cells harboring a BAP1-inactivating Glu31Gly substitution using CRISPR\u0026ndash;Cas9 adenine base editing. Although this mutation completely abolished BAP1 catalytic activity, it failed to confer anchorage-independent survival or anoikis resistance, indicating that while BAP1 restoration is sufficient to suppress anchorage independence and aniokis resistance in ccRCC cells, BAP1 inactivation alone is not sufficient to induce these phenotypes in normal epithelial cells. Notably, while restoring BAP1 activity in KMRC-20 cells increased migratory and invasive behavior, BAP1 loss in HK-2 cells increased invasion but paradoxically reduced migration, suggesting complex, context-specific responses to BAP1 status. Together, these complementary gain- and loss-of-function models demonstrate that BAP1 differentially regulates adhesion, anoikis, and motility in normal versus malignant renal cells, highlighting the highly context-dependent tumor-suppressive role of BAP1 in renal carcinogenesis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCells and antibodies\u003c/h2\u003e \u003cp\u003eHK-2 (human kidney 2) cells\u0026mdash;a proximal tubular epithelial cell line derived from normal adult male kidney and immortalized by transduction with human papillomavirus 16 (HPV-16) E6/E7 genes\u0026mdash;were purchased from ATCC. Parental, knockout, and base-edited HK-2 cells were maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin. The KMRC-20 cell line (JCRB1071) and the revertant KMRC-20 clones (1\u0026ndash;1, 1\u0026ndash;3, and 1\u0026ndash;34), described previously [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], were cultured in DMEM containing 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin. All cell lines were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂.\u003c/p\u003e \u003cp\u003eThe following primary antibodies were used: anti-BAP1 (mouse IgG, sc-28383; rabbit IgG, sc-28236), anti-α-tubulin (sc-8035), and anti-β-actin (sc-8432) from Santa Cruz Biotechnology; anti-H2A (07-146) from Millipore; and anti\u0026ndash;cyclobutane pyrimidine dimers (CPD; CAC-NM-DND-001) from Cosmo Bio.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeneration of based-edited and knockout HK-2 cells\u003c/h3\u003e\n\u003cp\u003eTo convert BAP1 Glu31 to glycine (GAG\u0026rarr;GGG) in HK-2 cells, we used the same adenine base editor\u0026mdash;VRQR variant of ABE7.10max\u0026mdash;previously applied to correct the Glu31Lys mutation (AAG\u0026rarr;GAG) in KMRC-20 cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This editor recognizes an NGAG protospacer adjacent motif (PAM) and efficiently deaminates the adenine within the GAG codon. To redirect editing specificity to the adenine in the Glu31 codon, a single\u0026ndash;nucleotide substitution was introduced into the original guide RNA (gRNA), which had been designed to correct the Glu31Lys mutation, using site-directed mutagenesis. The oligonucleotides used for mutagenesis were: forward, 5\u0026prime;-gaa aca ccg gga gga gat cta cga cct tcg-3\u0026prime;; reverse, 5\u0026prime;-cga agg tcg tag atc tcc tcc cgg tgt ttc-3\u0026prime;. The resulting construct, ABE7.10max-VRQR-BAP1-Gly31, was verified by sequencing to confirm successful alteration of the gRNA. The plasmid was transfected into HK-2 cells using FuGENE HD (Promega), after which transfected cells were selected with puromycin (2 \u0026micro;g/mL) and subjected to limiting dilution in 96-well plates for clonal isolation. Genomic DNA was extracted using Exgene\u0026trade; Cell SV (GeneAll), and the region surrounding the target site was PCR-amplified and sequenced to verify base editing. The two top-predicted off-target sites were amplified and sequenced in an identical manner to assess off-target activity.\u003c/p\u003e \u003cp\u003eTo generate BAP1-knockout HK-2 cells, a gRNA targeting exon 2 of BAP1 (5\u0026prime;-acc gaa atc ttc cac gag ca-3\u0026prime;) was cloned into the Esp3I (BsmBI) site of LentiCRISPRv2 (Addgene #52961) to produce LentiCRISPRv2-BAP1-KO. The gRNA oligonucleotide sequences were: forward, 5\u0026prime;-caccg acc gaa atc ttc cac gag ca-3\u0026prime;; reverse: 5\u0026prime;-aaac tg ctc gtg gaa gat ttc ggt c-3\u0026prime;. LentiCRISPRv2-BAP1-KO was transfected into HK-2 cells, followed by puromycin selection and clonal isolation using the same workflow as for the base-edited clones. Sequencing of the PCR amplicon spanning the target region confirmed the presence of indel mutations. The predicted off-target sites were not present in the genome.\u003c/p\u003e\n\u003ch3\u003eUb-AMC assay using BAP1 immune complexes\u003c/h3\u003e\n\u003cp\u003eBAP1 DUB activity was assessed using Ub-AMC substrate as previously described [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Briefly, parental or base edited HK-2 cell lysates were immunoprecipitated with anti-BAP1 antibody or control IgG, and the resulting immune complexes were washed and incubated with Ub-AMC (Boston Biochem) in DUB assay buffer. Fluorescence generated by AMC release was measured in black 96-well plates using a SpectraMax i3X microplate reader (excitation 350 nm, emission 450 nm) (NFEC-2019-10-258101, Ewha Fluorescence Core Imaging Center).\u003c/p\u003e\n\u003ch3\u003eCell viability and cell cycle analysis\u003c/h3\u003e\n\u003cp\u003eCell viability was measured by seeding 1,000 cells per well in 96-well plates and culturing for four or seven days, followed by MTS assay (CellTiter AQueous One Solution, Promega) according to the manufacturer\u0026rsquo;s protocol; absorbance was read at 490 nm. For clonogenic assays, 1,000 cells were plated in 35-mm dishes and grown for 14 days before fixation and crystal violet staining. For cell-cycle profiling, 1 \u0026times; 10⁵ cells were fixed in 70% ethanol overnight, washed with PBS, stained with propidium iodide, and analyzed by flow cytometry (FACSCalibur, BD Biosciences) (NFEC-2022-04-278217, Ewha Fluorescence Core Imaging Center).\u003c/p\u003e\n\u003ch3\u003eAnchorage-independent growth assays\u003c/h3\u003e\n\u003cp\u003eSoft agar assays were performed by plating 2 \u0026times; 10⁴ cells per 35-mm dish in 0.4% low-melting-point agarose over a 0.6% agarose base layer in PBS. Cultures were maintained for five weeks at 37\u0026deg;C in a humidified 5% CO₂ incubator. Colonies were stained with 0.001% crystal violet in 20% ethanol and quantified using ImageJ.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis assay\u003c/h2\u003e \u003cp\u003eApoptosis was assessed by Annexin V/PI staining using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, 556547). Briefly, 2 \u0026times; 10⁵ cells were washed twice with PBS, resuspended in binding buffer, and a 100 \u0026micro;L aliquot was incubated with 5 \u0026micro;L FITC\u0026ndash;Annexin V and 5 \u0026micro;L PI (Invitrogen) for 20 min at room temperature in the dark. Stained cells were analyzed by flow cytometry on an LSRFortessa (BD Biosciences) (NFEC-2019-10-258102, Ewha Fluorescence Core Imaging Center).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCPD assay\u003c/h3\u003e\n\u003cp\u003eCyclobutane pyrimidine dimers (CPDs) were detected as previously described [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In brief, 1 \u0026times; 10⁵ cells were seeded onto poly-L-lysine\u0026ndash;coated coverslips, allowed to adhere, and irradiated with 10 J/m\u0026sup2; UV-C. Cells were fixed at the indicated recovery times with methanol for 10 min at room temperature, followed by DNA denaturation in 2 M HCl for 30 min. After washing, cells were blocked in 3% BSA/PBS for 1 h and incubated with an anti-CPD monoclonal antibody (Cosmo Bio, CAC-NM-DND-001) for 1 h at room temperature. Alexa Fluor 568\u0026ndash;conjugated goat anti-mouse IgG (Thermo Fisher Scientific) was applied for 30 min, and coverslips were washed with PBS containing 0.1% Tween-20 before mounting with VECTASHIELD containing DAPI (Vector Laboratories). Images were acquired on a Zeiss LSM 880 confocal microscope ((NFEC-2016-05-209580, Ewha Fluorescence Core Imaging Center), and CPD signal intensity was quantified using ZEN 2.3 software.\u003c/p\u003e\n\u003ch3\u003eCell migration and invasion assay\u003c/h3\u003e\n\u003cp\u003eFor the migration assay, cells were seeded in serum-free medium into Transwell inserts containing an 8-\u0026micro;m pore-size membrane (Corning 3422), with complete medium added to the lower chamber. For the invasion assay, cells were seeded in serum-free medium onto the upper surface of Transwell membranes pre-coated with Matrigel (Corning 354248), and complete medium was placed in the lower chamber. HK-2 cells were incubated for 6 h (migration assay) or 24 h (invasion assay), whereas KMRC-20 cells were incubated for 16 h (migration assay) or 96 h (invasion assay). After incubation, cells remaining on the upper membrane surface were removed by gentle swabbing, and cells that had migrated or invaded to the lower surface were fixed with 4% paraformaldehyde for 10 min and stained with crystal violet. Representative images were captured using an Axiovert200 microscope. Relative migration or invasion was quantified by dissolving the crystal violet stain in 10% acetic acid and measuring absorbance at 590 nm.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblot analysis\u003c/h2\u003e \u003cp\u003eWhole-cell lysates were prepared in RIPA buffer (50 mM Tris-Cl, pH 8.0; 150 mM NaCl; 0.5% sodium deoxycholate; 0.1% SDS; 1% NP-40; 0.5 mM PMSF; 10 mM NaF; protease inhibitor cocktail). Protein concentrations were measured using the BCA Protein Assay Kit (Pierce). Equal amounts of protein were mixed with SDS sample buffer, boiled for 5 min, separated by SDS-PAGE, and transferred to membranes. Immunoblotting was performed using standard protocols with appropriate primary and HRP-conjugated secondary antibodies, and signals were detected by chemiluminescence.\u003c/p\u003e \u003cp\u003eFor detection of Ub-H2A and total H2A, histones were extracted as previously described. Briefly, cells were lysed in NETN buffer (20 mM Tris-Cl, pH 8.0; 150 mM NaCl; 1 mM EDTA, pH 8.0; 0.5% NP-40; 10 mM NaF; protease inhibitor cocktail) for 10 min at 4\u0026deg;C. Lysates were centrifuged at 8,000 rpm for 5 min at 4\u0026deg;C, and supernatants were discarded. Pellets were resuspended in 0.1 M HCl and incubated for 10 min at room temperature. After centrifugation at 12,000 rpm for 5 min, the supernatants were subjected to immunoblot analysis. CAPS transfer buffer (25 mM CAPS, 20% methanol) was used for membrane transfer. Full-length blots/gels are presented in Supplementary Fig. S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical significance was assessed using unpaired Student\u0026rsquo;s t-tests performed with Microsoft Excel and GraphPad Prism 5 software. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical significance is indicated as follows: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***), and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 (\u003cem\u003ens\u003c/em\u003e, not significant).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of isogenic HK-2 cells harboring the BAP1 Glu31Gly mutation by base editing\u003c/h2\u003e \u003cp\u003eGiven that restoring wild-type BAP1 activity by correcting the Glu31 mutation rescues anchorage-dependent growth and anoikis sensitivity in ccRCC cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], we next tested the converse\u0026mdash;whether introducing a Glu31-disrupting mutation is sufficient to induce anchorage-independent survival and anoikis resistance in normal kidney epithelial cells. To this end, we generated isogenic HK-2 human kidney epithelial cell lines in which Glu31 was converted to Gly using CRISPR/Cas9-mediated adenine base editing [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. We employed the VRQR Cas9 variant of ABE7.10max, as described previously [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], which recognizes an AGAG protospacer adjacent motif (PAM) corresponding to the degenerate NGAG PAM in the BAP1 locus, together with a gRNA targeting the GAG codon encoding Glu31 to convert it to GGG (Gly31) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This substitution has been shown to abolish BAP1 DUB activity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Two independent base-edited clones, designated Gly31-1 and Gly31-2, were established, and sequencing confirmed precise on-target editing without detectable alterations at the top two predicted off-target sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, B). For comparison and to distinguish the effects of a single-amino acid substitution from complete loss of function, we also generated a BAP1-knockout HK-2 clone (BAP1-KO) by CRISPR/Cas9 targeting exon 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D), which resulted in complete loss of BAP1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConfirmation of BAP1 inactivation in base-edited HK-2 clones\u003c/h2\u003e \u003cp\u003eTo verify that the Glu31Gly substitution abolished BAP1 catalytic activity, we performed Ub-AMC assays following BAP1 immunoprecipitation. As expected, BAP1 isolated from parental HK-2 cells displayed robust DUB activity, whereas IgG control immunoprecipitates showed none, confirming assay specificity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, BAP1 from the Gly31-1 and Gly31-2 clones exhibited no detectable activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), demonstrating successful inactivation of BAP1 by base editing. Consistent with this loss of function, levels of H2A-Ub\u0026mdash;a direct BAP1 substrate\u0026mdash;were markedly elevated in both mutant clones, comparable to those observed in BAP1-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe previously showed that Glu31 contributes to BAP1 protein stability: BAP1 variants carrying a Glu31Ala mutation exhibit substantially reduced protein levels in ccRCC cell lines (KMRC-20 and UMRC-6) and in HEK293T cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and correction of the Glu31 mutation in KMRC-20 cells by base editing restores BAP1 protein abundance [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In contrast, the base-edited HK-2 Gly31 clones displayed BAP1 protein levels similar to parental HK-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating that loss of Glu31 does not destabilize BAP1 in this cellular context. This divergence is unlikely to reflect differences between glycine and alanine substitutions\u0026mdash;both residues lack PARylation capacity and both abolish DUB activity. Rather, the results suggest that Glu31-dependent regulation of BAP1 stability may be cell type-specific, pointing to distinct mechanisms governing BAP1 turnover in kidney epithelial versus ccRCC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThe Glu31 mutation has minimal impact on HK-2 cell growth and proliferation\u003c/h2\u003e \u003cp\u003eBAP1 has been reported to exert either pro-survival or pro-apoptotic effects depending on cell type, genetic context, and experimental conditions. While it functions as a classic tumor suppressor in many settings\u0026mdash;such as uveal melanoma and mesothelioma\u0026mdash;it can promote proliferation in specific contexts, often by stabilizing pro-growth proteins through its DUB activity, as observed in certain breast cancer models [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In ccRCC, we previously showed that exogenous expression of wild-type BAP1, but not the Glu31Lys mutant, reduced viability of KMRC-20 cells in which endogenous mutant BAP1 was depleted by siRNA, consistent with a tumor-suppressive, anti-proliferative role [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, restoring BAP1 activity under isogenic conditions by base editing did not significantly affect short-term viability or clonogenic capacity in KMRC-20 cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], indicating that overexpression and physiological reactivation yield distinct outcomes.\u003c/p\u003e \u003cp\u003eTo evaluate the broader cellular consequences of BAP1 loss or Glu31 inactivation in normal kidney epithelial cells, we characterized the BAP1-knockout and base-edited HK-2 clones. Morphologically, the knockout cells and the Gly31-1 and Gly31-2 mutants were indistinguishable from parental HK-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Short-term viability assays showed a slight increase in viability and growth rate in the Glu31 mutant clones, whereas the BAP1-KO line exhibited a modest reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Consistent with these observations, cell-cycle analysis revealed that neither the mutant nor knockout clones displayed major deviations from the parental profile, with all groups showing typical G1 and G2/M DNA-content peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The proportion of sub-G1 cells\u0026mdash;reflecting apoptotic populations\u0026mdash;was also comparable across lines, indicating that the Glu31 mutation does not alter basal apoptotic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Long-term clonogenic assays showed that the base-edited HK-2 clones formed slightly more colonies than parental cells, whereas the knockout clone formed slightly fewer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Overall, these data indicate that loss of BAP1 activity through Glu31 mutation has minimal effects on HK-2 cell survival or proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together with our previous findings that reactivating BAP1 in ccRCC cells also does not impair growth under physiological expression levels, these results suggest that BAP1 plays a limited role in regulating baseline survival and proliferation in kidney epithelial cells. The reduced viability observed upon overexpression of wild-type BAP1 likely reflects non-physiological effects of supraphysiological expression rather than an inherent consequence of restoring BAP1 function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe Glu31 mutation impairs BAP1-dependent DNA repair in HK-2 cells\u003c/h2\u003e \u003cp\u003eTo determine whether the Glu31 mutation compromises BAP1 function in HK-2 cells, we assessed its role in repairing UV-induced DNA damage, a well-established BAP1-dependent process. CPD assays revealed that parental HK-2 cells displayed time-dependent recovery following UV irradiation, with approximately 40% of damage repaired by 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, the BAP1-knockout clone showed a marked defect in DNA repair at all examined time points, achieving only 16% repair at 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Notably, both Gly31-1 and Gly31-2 clones also exhibited significant impairment in resolving UV-induced DNA lesions, mirroring the BAP1-knockout phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These findings indicate that the Glu31-to-Gly substitution not only abolishes BAP1\u0026rsquo;s catalytic activity but also disrupts its functional contribution to the cellular DNA damage response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA growing body of evidence demonstrates that BAP1 participates in multiple DNA repair pathways\u0026mdash;including homologous recombination and non-homologous end-joining for double-strand break repair, as well as nucleotide excision repair\u0026mdash;across a wide range of cell types. These include human cancer cell lines such as U2OS osteosarcoma, MCF7 breast cancer, KMRC-20 ccRCC, and DPM malignant pleural mesothelioma cells; normal human cells such as MEMa primary melanocytes, Met-5A mesothelial cells, LF1 fetal lung fibroblasts, and HK-2 renal epithelial cells (as shown here); the non-transformed HEK293T human embryonic kidney line; and non-human systems such as murine pancreatic cancer cells and DT40 chicken B lymphocytes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Together, these findings indicate that BAP1 plays a broadly conserved and fundamental role in maintaining DNA repair capacity across diverse biological contexts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe Glu31 mutation does not induce anchorage-independent growth in HK-2 cells\u003c/h2\u003e \u003cp\u003eWe next assessed whether the Glu31 mutation confers anchorage-independent growth in HK-2 cells using soft agar assays. As expected, parental HK-2 cells\u0026mdash;which depend on adhesion for normal proliferation\u0026mdash;displayed only minimal basal colony formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Contrary to our initial hypothesis, the Gly31-1 and Gly31-2 mutant clones did not form colonies, similar to the BAP1-knockout cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). These findings indicate that, although restoration of BAP1 activity in KMRC-20 cells promotes anchorage dependence, loss of BAP1 function does not induce anchorage-independent growth in non-transformed HK-2 cells. Thus, while BAP1 activity is sufficient to restore anchorage dependence in kidney cancer cells, its inactivation alone is insufficient to drive anchorage-independent growth in normal kidney epithelial cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe Glu31 mutation does not induce anoikis resistance in HK-2 cells\u003c/h2\u003e \u003cp\u003eBecause the Glu31 mutation did not promote anchorage-independent growth and instead appeared to sensitize HK-2 cells to anchorage loss, we next examined its effect on anoikis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Cells were cultured in ultra-low-attachment (ULA) dishes, which mimic non-adherent conditions and induce apoptosis triggered by loss of adhesion. Under standard adherent conditions, parental, BAP1-knockout, and Glu31Gly mutant clones all exhibited similarly low levels of apoptosis and necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). As expected, parental HK-2 cells showed a marked increase in cell death upon anchorage loss, consistent with the behavior of normal epithelial cells. Notably, the Glu31Gly mutants did not exhibit reduced cell death in ULA conditions; instead, they showed a further increase in apoptosis, similar to the BAP1-knockout clone. This enhanced sensitivity to anchorage loss was driven predominantly by apoptosis, with minimal contribution from necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings parallel the soft agar results and indicate that BAP1 loss or inactivation does not confer anchorage-independent survival in normal kidney epithelial cells. Rather, while restoration of BAP1 is sufficient to re-establish anchorage dependence and anoikis sensitivity in kidney cancer cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], BAP1 loss alone is insufficient to induce anoikis resistance in a non-malignant background. Together, these observations suggest that additional oncogenic alterations beyond BAP1 inactivation are required for the acquisition of anchorage-independent growth and anoikis resistance during renal tumorigenesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of BAP1 loss and gain on cell migration and invasion are context dependent\u003c/h2\u003e \u003cp\u003eIn addition to anchorage independence and anoikis resistance, metastatic progression frequently requires enhanced migratory and invasive behavior [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. We therefore examined whether the inactivating Glu31Gly mutation alters these motility programs in HK-2 cells. Transwell assays showed that both the Glu31Gly mutant and BAP1-knockout HK-2 clones exhibited reduced migratory activity compared with parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), indicating that BAP1 loss impairs the directional movement of non-malignant renal epithelial cells. In contrast, these same mutant and knockout cells displayed markedly increased invasive capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), demonstrating that BAP1 inactivation enhances matrix-penetrating behavior despite reduced migration. Together, these results reveal that BAP1 loss differentially influences migration and invasion in HK-2 cells, uncoupling these two motility programs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the consequences of restoring BAP1 function, we evaluated the three KMRC-20 revertant clones (1\u0026ndash;1, 1\u0026ndash;3, and 1\u0026ndash;34) in which the endogenous Glu31Lys mutation had been corrected to wild-type glutamate [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. All revertant clones exhibited significant increases in migration compared with the parental KMRC-20 line, and this increase was paralleled by a marked enhancement in invasive capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These findings were unexpected because BAP1 restoration in the same clones reinstated anchorage dependence and anoikis sensitivity\u0026mdash;features typically associated with non-transformed epithelial cells. Thus, although BAP1 reactivation suppresses tumorigenic phenotypes related to anchorage independence and anoikis resistance, it simultaneously boosts motility-related behaviors in the KMRC-20 background.\u003c/p\u003e \u003cp\u003eTaken together, these complementary gain- and loss-of-function models demonstrate that BAP1 influences invasion and migration in a highly context-dependent manner: BAP1 restoration promotes motility in ccRCC cells, whereas BAP1 inactivation reduces migration but increases invasion in normal kidney epithelial cells. These contrasting outcomes highlight the complex, pathway-specific roles of BAP1 in regulating cellular adhesion, motility, and invasive behavior during renal carcinogenesis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we used precise CRISPR base editing to dissect the functional consequences of the recurrent BAP1 Glu31 mutation in renal epithelial cells and to compare its effects with those observed in ccRCC cells harboring the corresponding cancer-associated substitution. By generating isogenic HK-2 clones carrying the Glu31Gly mutation alongside a BAP1-knockout line as a control, we were able to define the specific consequences of a single amino-acid substitution under native regulatory conditions, avoiding confounding effects commonly associated with overexpression, knockdown, or complementation approaches. Our findings confirm that the Glu31 residue is critical for BAP1's enzymatic and DNA repair functions: both the base-edited mutant and the knockout clone exhibited similarly impaired DUB activity and defective UV-induced DNA damage repair. Thus, the Glu31 residue functions as a key regulatory site required for full BAP1 activity in kidney epithelial cells.\u003c/p\u003e \u003cp\u003eDespite the clear functional inactivation of BAP1 in the HK-2 Glu31Gly and knockout clones, these cells did not acquire hallmark oncogenic traits associated with BAP1 loss in ccRCC, such as anchorage-independent growth or anoikis resistance. Instead, both mutant and knockout cells underwent enhanced apoptosis upon detachment, behaving more like the parental cells than like their BAP1-deficient cancer cell counterparts. This finding contrasts sharply with our previous work in KMRC-20 cells, where reverting the endogenous Glu31Lys mutation to wild type restored anchorage dependence and re-sensitized cells to anoikis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Taken together, these results demonstrate that while correcting BAP1 function is sufficient to suppress anchorage-independent growth in a cancer context, inactivating BAP1 alone is not sufficient to initiate these phenotypes in normal epithelial cells.\u003c/p\u003e \u003cp\u003eThese observations suggest that BAP1 loss may function primarily as a permissive or priming event, rather than acting a standalone driver of tumorigenesis. Its biological impact is highly context dependent, requiring cooperating oncogenic alterations to elicit hallmark features of cellular transformation. In normal HK-2 cells, loss of BAP1 activity is insufficient to circumvent the intrinsic apoptotic programs activated upon loss of adhesion, suggesting that additional genetic or epigenetic events\u0026mdash;common in the evolution of ccRCC\u0026mdash;are necessary to confer anchorage independence and anoikis resistance. This supports the model that BAP1 inactivation likely operates within a pre-existing permissive landscape\u0026mdash;such as VHL loss or dysregulated hypoxia signaling\u0026mdash;to overcome the robust apoptotic checkpoints characteristic of normal renal epithelium [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Consistently, emerging evidence indicates that the phenotypic consequences of BAP1 loss vary substantially depending on cellular lineage, metabolic state, and co-occurring mutations [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In renal cancer, BAP1 alterations frequently co-occur with disruptions in chromatin regulation, metabolic circuits, and hypoxia-responsive pathways, which together may enable survival in detached or mechanically stressed microenvironments [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn unexpected but important aspect of our findings is the context-dependent impact of BAP1 status on cell motility. In KMRC-20 cells, reactivation of BAP1 increased both migration and invasion, consistent with reports that BAP1 deficiency imposes a rigid mesenchymal-to-epithelial transition (MET)-like phenotype marked by reduced cytoskeletal dynamics and impaired single-cell movement in ccRCC cells [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Thus, restoring BAP1 function likely re-engages cytoskeletal remodeling programs\u0026mdash;particularly those involving actin turnover and cell spreading\u0026mdash;that are necessary for efficient directional migration. In contrast, introducing the BAP1-inactivating Glu31 mutation or knocking out BAP1 in non-malignant HK-2 epithelial cells produced an opposite pattern of motility regulation: migration was reduced, consistent with prior observations that BAP1 can promote cell movement in certain contexts such as breast cancer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], yet invasion capacity increased. These parallel but divergent outcomes highlight that BAP1 controls motility through mechanistically distinct pathways in normal versus malignant renal contexts. In cancer cells, BAP1 restoration may promote differentiation-associated programs that strengthen cytoskeletal architecture and foster coherent, directed movement. In normal epithelial cells, however, BAP1 loss may relax basal constraints on extracellular matrix (ECM) engagement and remodeling, thereby supporting proteolysis-driven invasion while destabilizing the cytoskeletal polarization required for productive migration [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBase editing proved to be a powerful strategy for dissecting mutation-specific functions of cancer-associated genes by enabling the precise modeling of endogenous point mutations without perturbing regulatory architecture or protein stoichiometry. This approach allowed us to directly contrast the biological consequences of a clinically relevant BAP1 mutation in both non-malignant and cancer-derived isogenic backgrounds, revealing that identical mutations can generate markedly different outcomes depending on cellular lineage and oncogenic context. Our findings underscore that BAP1 does not exert uniform effects on adhesion, anoikis, or motility, but instead acts through context-dependent mechanisms that become apparent only in genetically matched systems. These results highlight the importance of evaluating cancer-associated BAP1 variants within precise isogenic settings to accurately define their functional and oncogenic consequences and point toward broader applications of base-editing platforms for studying recurrent missense mutations in ccRCC and other tumor types.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eUsing precise CRISPR base editing to model the recurrent BAP1 Glu31 mutation, we demonstrate that a single\u0026ndash;amino acid substitution is sufficient to abolish BAP1 DUB activity and impair DNA repair in normal renal epithelial cells, yet is not sufficient to induce anchorage-independent growth or anoikis resistance. These results contrast with our previous finding that restoring wild-type BAP1 in ccRCC cells suppresses anchorage-independent growth and reinstates anoikis sensitivity, underscoring that the phenotypic consequences of BAP1 status are strongly context dependent. Notably, BAP1 gain and loss produced divergent effects on cell motility across malignant and normal backgrounds\u0026mdash;BAP1 restoration increased both migration and invasion in KMRC-20 cells, whereas BAP1 inactivation in HK-2 cells reduced migration but enhanced invasion\u0026mdash;highlighting a multifaceted, environment-specific role for BAP1 in regulating movement-related behaviors. Collectively, our findings indicate that additional oncogenic alterations beyond BAP1 mutation are required for acquisition of anchorage independence, anoikis resistance, and coordinated motility changes during renal tumorigenesis and emphasize the utility of isogenic, base-edited models for dissecting mutation-specific functions of cancer-associated genes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eABE, adenine base editor\u003c/p\u003e\n\u003cp\u003eBAP1, BRCA1 Associated Protein-1\u003c/p\u003e\n\u003cp\u003eccRCC, clear cell renal cell carcinoma\u003c/p\u003e\n\u003cp\u003eCPD, cyclobutane pyrimidine dimer\u003c/p\u003e\n\u003cp\u003eDUB, deubiquitinase\u003c/p\u003e\n\u003cp\u003eECM, extracellular matrix\u003c/p\u003e\n\u003cp\u003eEMT, epithelial\u0026ndash;mesenchymal transition\u003c/p\u003e\n\u003cp\u003egRNA, guide RNA\u003c/p\u003e\n\u003cp\u003eH2A-Ub, histone H2A monoubiquitinated at lysine 119\u003c/p\u003e\n\u003cp\u003eHK-2 cells, human kidney 2 cells\u003c/p\u003e\n\u003cp\u003eMET, mesenchymal-to-epithelial transition\u003c/p\u003e\n\u003cp\u003eNER, nucleotide excision repair\u003c/p\u003e\n\u003cp\u003eNLS, nuclear localization signal\u003c/p\u003e\n\u003cp\u003ePAM, protospacer adjacent motif\u003c/p\u003e\n\u003cp\u003ePARylation, poly(ADP)-ribosylation\u003c/p\u003e\n\u003cp\u003eUCH, ubiquitin C-terminal hydrolase\u003c/p\u003e\n\u003cp\u003eULA, ultra-low attachment\u003c/p\u003e\n\u003cp\u003eUV, ultraviolet\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and Supplementary Material. The detailed experimental procedures and the materials will be freely available upon request. Please contact [email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants 2023R1A2C3005307 (JK) and 2022R1I1A1A01073047 (DL) from the National Research Foundation of Korea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.K., D.L., B.L., S.K., and J.L. performed the experiments and generated the data. C.K. and J.K. wrote the manuscript. J.K. conceived and supervised the research project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGerstberger S, Jiang Q, Ganesh K. Metastasis Cell. 2023;186(8):1564\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRing A, Nguyen-Strauli BD, Wicki A, Aceto N. Biology, vulnerabilities and clinical applications of circulating tumour cells. Nat Rev Cancer. 2023;23(2):95\u0026ndash;111.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaw P, Dey Bhowmik A, Gopinatha Pillai MS, Robbins N, Dwivedi SKD, Rao G. Anoikis resistance in Cancer: Mechanisms, therapeutic strategies, potential targets, and models for enhanced understanding. Cancer Lett. 2025;624:217750.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng Z, Wang H, Liu J, Deng Y, Zhang N. Comprehensive understanding of anchorage-independent survival and its implication in cancer metastasis. Cell Death Dis. 2021;12(7):629.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwon J, Lee D, Lee SA. BAP1 as a guardian of genome stability: implications in human cancer. Exp Mol Med. 2023;55(4):745\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzczepanski AP, Wang L. Emerging multifaceted roles of BAP1 complexes in biological processes. Cell Death Discov. 2021;7(1):20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJensen DE, Proctor M, Marquis ST, Gardner HP, Ha SI, Chodosh LA, Ishov AM, Tommerup N, Vissing H, Sekido Y, et al. BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression. Oncogene. 1998;16(9):1097\u0026ndash;112.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasclef L, Ahmed O, Estavoyer B, Larrivee B, Labrecque N, Nijnik A, Affar EB. Roles and mechanisms of BAP1 deubiquitinase in tumor suppression. Cell Death Differ. 2021;28(2):606\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee HS, Lee SA, Hur SK, Seo JW, Kwon J. Stabilization and targeting of INO80 to replication forks by BAP1 during normal DNA synthesis. Nat Commun. 2014;5:5128.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu H, Pak H, Hammond-Martel I, Ghram M, Rodrigue A, Daou S, Barbour H, Corbeil L, Hebert J, Drobetsky E, et al. Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair. Proc Natl Acad Sci U S A. 2014;111(1):285\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee HS, Seo HR, Lee SA, Choi S, Kang D, Kwon J. BAP1 promotes stalled fork restart and cell survival via INO80 in response to replication stress. Biochem J. 2019;476(20):3053\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeo HR, Jeong D, Lee S, Lee HS, Lee SA, Kang SW, Kwon J. CHIP and BAP1 Act in Concert to Regulate INO80 Ubiquitination and Stability for DNA Replication. Mol Cells. 2021;44(2):101\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMisaghi S, Ottosen S, Izrael-Tomasevic A, Arnott D, Lamkanfi M, Lee J, Liu J, O'Rourke K, Dixit VM, Wilson AC. Association of C-terminal ubiquitin hydrolase BRCA1-associated protein 1 with cell cycle regulator host cell factor 1. Mol Cell Biol. 2009;29(8):2181\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachida YJ, Machida Y, Vashisht AA, Wohlschlegel JA, Dutta A. The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1. J Biol Chem. 2009;284(49):34179\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaou S, Hammond-Martel I, Mashtalir N, Barbour H, Gagnon J, Iannantuono NV, Nkwe NS, Motorina A, Pak H, Yu H, et al. The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer. J Biol Chem. 2015;290(48):28643\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Shi J, Liu X, Feng L, Gong Z, Koppula P, Sirohi K, Li X, Wei Y, Lee H, et al. BAP1 links metabolic regulation of ferroptosis to tumour suppression. Nat Cell Biol. 2018;20(10):1181\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuznetsov JN, Aguero TH, Owens DA, Kurtenbach S, Field MG, Durante MA, Rodriguez DA, King ML, Harbour JW. BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers. Sci Adv. 2019;5(9):eaax1738.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDey A, Seshasayee D, Noubade R, French DM, Liu J, Chaurushiya MS, Kirkpatrick DS, Pham VC, Lill JR, Bakalarski CE, et al. Loss of the tumor suppressor BAP1 causes myeloid transformation. Science. 2012;337(6101):1541\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBononi A, Giorgi C, Patergnani S, Larson D, Verbruggen K, Tanji M, Pellegrini L, Signorato V, Olivetto F, Pastorino S, et al. BAP1 regulates IP3R3-mediated Ca(2+) flux to mitochondria suppressing cell transformation. Nature. 2017;546(7659):549\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin J, Zhou Z, Chen W, Wang C, Zhang H, Ge G, Shao M, You D, Fan Z, Xia H, et al. BAP1 promotes breast cancer cell proliferation and metastasis by deubiquitinating KLF5. Nat Commun. 2015;6:8471.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang M, Park SG, Lee SA, Kim S, Lee D, Shirbhate ME, Youn SY, Kim KM, Cha SS, Kwon J. Targeting BAP1 with small compound inhibitor for colon cancer treatment. Sci Rep. 2023;13(1):2264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMashtalir N, Daou S, Barbour H, Sen NN, Gagnon J, Hammond-Martel I, Dar HH, Therrien M. Affar el B: Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O. Mol Cell. 2014;54(3):392\u0026ndash;406.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScheuermann JC, de Ayala Alonso AG, Oktaba K, Ly-Hartig N, McGinty RK, Fraterman S, Wilm M, Muir TW, Muller J. Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB. Nature. 2010;465(7295):243\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarbour JW, Onken MD, Roberson ED, Duan S, Cao L, Worley LA, Council ML, Matatall KA, Helms C, Bowcock AM. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science. 2010;330(6009):1410\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTesta JR, Cheung M, Pei J, Below JE, Tan Y, Sementino E, Cox NJ, Dogan AU, Pass HI, Trusa S, et al. Germline BAP1 mutations predispose to malignant mesothelioma. Nat Genet. 2011;43(10):1022\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePena-Llopis S, Vega-Rubin-de-Celis S, Liao A, Leng N, Pavia-Jimenez A, Wang S, Yamasaki T, Zhrebker L, Sivanand S, Spence P, et al. BAP1 loss defines a new class of renal cell carcinoma. Nat Genet. 2012;44(7):751\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElsayed AM, Kittaneh M, Cebulla CM, Abdel-Rahman MH. An overview of BAP1 biological functions and current therapeutics. Biochim Biophys Acta Rev Cancer. 2025;1880(2):189267.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaFave LM, Beguelin W, Koche R, Teater M, Spitzer B, Chramiec A, Papalexi E, Keller MD, Hricik T, Konstantinoff K, et al. Loss of BAP1 function leads to EZH2-dependent transformation. Nat Med. 2015;21(11):1344\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun C, Zhao C, Li S, Wang J, Zhou Q, Sun J, Ding Q, Liu M, Ding G. EZH2 Expression is increased in BAP1-mutant renal clear cell carcinoma and is related to poor prognosis. J Cancer. 2018;9(20):3787\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin B, Zhang P, Zou H, Ye H, Wang Y, Zhang J, Yang H, Pan J. Verification of EZH2 as a druggable target in metastatic uveal melanoma. Mol Cancer. 2020;19(1):52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHou C, Xiao L, Ren X, Cheng L, Guo B, Zhang M, Yan N. EZH2-mediated H3K27me3 is a predictive biomarker and therapeutic target in uveal melanoma. Front Genet. 2022;13:1013475.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchoumacher M, Le Corre S, Houy A, Mulugeta E, Stern MH, Roman-Roman S, Margueron R. Uveal melanoma cells are resistant to EZH2 inhibition regardless of BAP1 status. Nat Med. 2016;22(6):577\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarbone M, Harbour JW, Brugarolas J, Bononi A, Pagano I, Dey A, Krausz T, Pass HI, Yang H, Gaudino G. Biological Mechanisms and Clinical Significance of BAP1 Mutations in Human Cancer. Cancer Discov. 2020;10(8):1103\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarbone M, Adusumilli PS, Alexander HR Jr., Baas P, Bardelli F, Bononi A, Bueno R, Felley-Bosco E, Galateau-Salle F, Jablons D, et al. Mesothelioma: Scientific clues for prevention, diagnosis, and therapy. CA Cancer J Clin. 2019;69(5):402\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SA, Lee D, Kang M, Kim S, Kwon SJ, Lee HS, Seo HR, Kaushal P, Lee NS, Kim H et al. BAP1 promotes the repair of UV-induced DNA damage via PARP1-mediated recruitment to damage sites and control of activity and stability. Cell Death Differ 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee B, Lee J, Kwon J. PARP1 cooperates with ASXL1 to stimulate BAP1 deubiquitinase activity through its DNA-binding domain. Biochem Biophys Res Commun. 2025;792:152982.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee D, Lee B, Lee J, Kwon J. Restoration of BAP1 activity via base editing suppresses anchorage-independent survival in kidney cancer. Cancer Cell Int 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePickar-Oliver A, Gersbach CA. The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol. 2019;20(8):490\u0026ndash;507.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol. 2020;38(7):824\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoblan LW, Erdos MR, Wilson C, Cabral WA, Levy JM, Xiong ZM, Tavarez UL, Davison LM, Gete YG, Mao X, et al. In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice. Nature. 2021;589(7843):608\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato H, Ito T, Hayashi T, Kitano S, Erdjument-Bromage H, Bott MJ, Toyooka S, Zauderer M, Ladanyi M. The BAP1 nuclear deubiquitinase is involved in the nonhomologous end-joining pathway of double-strand DNA repair through interaction with DNA-PK. Oncogene. 2024;43(15):1087\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerkail S, Andricovich J, Kai Y, Tzatsos A. BAP1 is a haploinsufficient tumor suppressor linking chronic pancreatitis to pancreatic cancer in mice. Nat Commun. 2020;11(1):3018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrisch SM, Hu G. Anoikis resistance and cancer. BMC Cancer. 2025;25(1):1764.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYayan J, Franke KJ, Berger M, Windisch W, Rasche K. Adhesion, metastasis, and inhibition of cancer cells: a comprehensive review. Mol Biol Rep. 2024;51(1):165.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouchalova P, Bouchal P. Current methods for studying metastatic potential of tumor cells. Cancer Cell Int. 2022;22(1):394.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalton J, Lawson K, Prinos P, Finelli A, Arrowsmith C, Ailles L. PBRM1, SETD2 and BAP1 - the trinity of 3p in clear cell renal cell carcinoma. Nat Rev Urol. 2023;20(2):96\u0026ndash;115.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang SS, Gu YF, Wolff N, Stefanius K, Christie A, Dey A, Hammer RE, Xie XJ, Rakheja D, Pedrosa I, et al. Bap1 is essential for kidney function and cooperates with Vhl in renal tumorigenesis. Proc Natl Acad Sci U S A. 2014;111(46):16538\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarley MN, Schmidt LS, Mester JL, Pena-Llopis S, Pavia-Jimenez A, Christie A, Vocke CD, Ricketts CJ, Peterson J, Middelton L, et al. A novel germline mutation in BAP1 predisposes to familial clear-cell renal cell carcinoma. Mol Cancer Res. 2013;11(9):1061\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBononi A, Wang Q, Zolondick AA, Bai F, Steele-Tanji M, Suarez JS, Pastorino S, Sipes A, Signorato V, Ferro A, et al. BAP1 is a novel regulator of HIF-1alpha. Proc Natl Acad Sci U S A. 2023;120(4):e2217840120.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang SS, Gu YF, Wolff N, Stefanius K, Christie A, Dey A, Hammer RE, Xie XJ, Rakheja D, Pedrosa I, et al. Bap1 is essential for kidney function and cooperates with Vhl in renal tumorigenesis. Proc Natl Acad Sci U S A. 2014;111(46):16538\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCotta BH, Choueiri TK, Cieslik M, Ghatalia P, Mehra R, Morgan TM, Palapattu GS, Shuch B, Vaishampayan U, Van Allen E, et al. Current Landscape of Genomic Biomarkers in Clear Cell Renal Cell Carcinoma. Eur Urol. 2023;84(2):166\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWebster BR, Gopal N, Ball MW. Tumorigenesis Mechanisms Found in Hereditary Renal Cell Carcinoma: A Review. Genes (Basel) 2022, 13(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGu YF, Cohn S, Christie A, McKenzie T, Wolff N, Do QN, Madhuranthakam AJ, Pedrosa I, Wang T, Dey A, et al. Modeling Renal Cell Carcinoma in Mice: Bap1 and Pbrm1 Inactivation Drive Tumor Grade. Cancer Discov. 2017;7(8):900\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen P, Wang H, Zhang W, Chen Y, Lv Y, Wu D, Guo M, Deng H. Loss of BAP1 Results in Growth Inhibition and Enhances Mesenchymal-Epithelial Transition in Kidney Tumor Cells. Mol Cell Proteom. 2019;18(7):1320\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZanotelli MR, Zhang J, Reinhart-King CA. Mechanoresponsive metabolism in cancer cell migration and metastasis. Cell Metab. 2021;33(7):1307\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"BAP1 tumor suppressor, clear cell renal cell carcinoma, CRISPR/Cas9-mediated adenine base editing, isogenic cell lines, anchorage-independent growth, anoikis, migration, invasion","lastPublishedDoi":"10.21203/rs.3.rs-8306241/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8306241/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBAP1 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 precise correction of the inactivating Glu31Lys mutation in KMRC-20 ccRCC cells using CRISPR/Cas9 base editing restored BAP1 activity, reinstated anchorage-dependent growth, and re-sensitized cells to anoikis. Here, we asked the converse question: whether disrupting Glu31 is sufficient to induce anchorage-independent growth and anoikis resistance in normal kidney epithelial cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eUsing the same adenine base-editing strategy, we introduced an inactivating Glu31Gly mutation into HK-2 normal kidney epithelial cells, generating two independent isogenic BAP1-mutant clones. As an additional control, we created a BAP1-knockout HK-2 clone via CRISPR/Cas9. Parental, mutant, and knockout cells were assessed for BAP1 enzymatic activity, DNA repair capacity, viability, proliferation, cell cycle status, anchorage-independent growth, and anoikis resistance. Migration and invasion of HK-2 mutants and knockouts were compared with KMRC-20 revertant clones in which endogenous Glu31Lys had been corrected.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe Glu31Gly HK-2 mutants exhibited complete loss of BAP1 deubiquitinase activity and impaired UV-induced DNA damage repair\u0026mdash;phenotypes comparable to BAP1-knockout cells\u0026mdash;confirming successful functional inactivation. Despite this, both mutant and knockout HK-2 cells maintained parental-like morphology, viability, and proliferation. Surprisingly, Glu31Gly did not confer anchorage-independent growth or anoikis resistance: upon detachment, both mutant and knockout cells showed increased apoptosis. In contrast, in KMRC-20 cells, restoration of BAP1 activity enhanced both migration and invasion. Conversely, BAP1 inactivation or loss in HK-2 cells increased invasion but paradoxically reduced migration. These opposite outcomes indicate that BAP1 regulates motility through distinct mechanisms in normal versus malignant renal cells, likely reflecting differences in lineage state, cytoskeletal organization, and downstream signaling.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAlthough BAP1 restoration suppresses anchorage-independent growth and anoikis resistance in KMRC-20 ccRCC cells, BAP1 inactivation alone is insufficient to induce these oncogenic traits in normal HK-2 epithelial cells, implying that additional oncogenic alterations are required for anchorage-independent survival during kidney tumorigenesis. The divergent effects of BAP1 gain versus loss on migration and invasion further underscore the context-dependent nature of BAP1 function. These base-editing studies demonstrate that BAP1 differentially regulates adhesion, anoikis, and motility in normal and malignant renal cells and highlight the utility of precise base editing for dissecting clinically relevant mutations.\u003c/p\u003e","manuscriptTitle":"Base Editing Reveals Context-Dependent Regulation of Adhesion, Anoikis, and Motility by BAP1 in Renal Cell Models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-09 06:52:19","doi":"10.21203/rs.3.rs-8306241/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-01-24T01:20:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-19T02:29:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-18T01:14:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-16T20:35:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49338453880120835847545114464962207850","date":"2026-01-13T08:20:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68656181446543201587210738413510634757","date":"2026-01-13T07:57:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296837005107161958513623684642057691273","date":"2026-01-08T06:34:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123569693639869532791781047262235426869","date":"2026-01-07T16:03:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-07T11:04:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-05T07:50:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-15T14:07:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-13T04:40:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-12-13T04:34:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ebb1ba28-164c-4234-a6ee-a7792ff2b283","owner":[],"postedDate":"January 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-09T06:52:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-09 06:52:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8306241","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8306241","identity":"rs-8306241","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}