Functional characterisation guides classification of novel bap1 germline variants

ABSTRACT We have identified six patients harbouring distinct germline _BAP1_ mutations. In this study, we functionally characterise known _BAP1_ pathogenic and likely benign germline

variants out of these six patients to aid in the evaluation and classification of unknown _BAP1_ germline variants. We found that pathogenic germline variants tend to encode truncated

proteins, show diminished expression of epithelial-mesenchymal transition (EMT) markers, are localised in the cytosol and have reduced deubiquitinase capabilities. We show that these

functional assays are useful for _BAP1_ variant curation and may be added in the American College of Medical Genetics and Genomics (ACMG) criteria for BAP1 variant classification. This will

allow clinicians to distinguish between _BAP1_ pathogenic and likely benign variants reliably and may aid to quickly benchmark newly identified _BAP1_ germline variants. Classification of

novel _BAP1_ germline variants allows clinicians to inform predisposed patients and relevant family members regarding potential cancer risks, with appropriate clinical interventions

implemented if required. SIMILAR CONTENT BEING VIEWED BY OTHERS FUNCTIONAL EVALUATION AND CLINICAL CLASSIFICATION OF _BRCA2_ VARIANTS Article Open access 08 January 2025 INTEGRATION OF

FUNCTIONAL ASSAY DATA RESULTS PROVIDES STRONG EVIDENCE FOR CLASSIFICATION OF HUNDREDS OF _BRCA1_ VARIANTS OF UNCERTAIN SIGNIFICANCE Article Open access 22 October 2020 AN INTEGRATIVE MODEL

FOR THE COMPREHENSIVE CLASSIFICATION OF _BRCA1_ AND _BRCA2_ VARIANTS OF UNCERTAIN CLINICAL SIGNIFICANCE Article Open access 03 June 2022 INTRODUCTION BAP1 (BRCA1-associated protein) is a

nuclear localised deubiquitinating enzyme that is made up of a ubiquitin carboxyl hydrolase (UCH) domain, a host cell factor 1 (HCF1) binding domain, a C-terminal domain with a coiled-coil

motif and a nuclear localisation signal1,2,3. BAP1 is known to be a tumour suppressor that is involved in many cellular processes including apoptosis, metabolism, DNA damage repair,

regulation of gene transcription and removal of ubiquitin from histones (H2AK119ub)4,5,6,7,8,9,10. The polycomb group repressive deubiqutinase complex (PR-DUB) containing BAP1 and ASXL1/2

acts in opposition to polycomb repressive complexes (PRC)2. Together PRCs and PR-DUBs ubiquitinate and deubiquitinate histones, respectively, to fine-tune gene transcription in the cells.

Loss-of-function mutations in _BAP1_ result in loss of deubiquitination activities, disrupting various cellular processes including cell cycle, therefore driving tumourigenesis1 in many

cancers such as renal cell carcinoma, mesothelioma, uveal melanoma, small cell and non-small-cell lung cancers and cholangiocarcinoma11,12,13,14. _BAP1_ germline variants predispose

individuals to high risks of development of aggressive cancers, such as, uveal melanoma, mesothelioma, cutaneous melanoma and renal cell carcinoma at a younger age with poor prognosis15.

Current therapeutic approaches for BAP1-deficient tumours are not particularly promising16,17,18,19,20. An increasing number of _BAP1_ variants have been reported. For example, in 2018, 181

families have been discovered to carry a _BAP1_ variant, of which 140 of the variants are unique21. Classification of missense _BAP1_ variants is more difficult as compared to frameshift,

nonsense and canonical splice variants even with in silico predictions21. As of April 2020, 466 _BAP1_ _germline_ variants of uncertain significance (VUS) have been reported on ClinVar, with

447 of them having single nucleotide variations and 404 of them resulting in missense mutations22. More of such _BAP1_ variants are expected to be discovered as clinical laboratories have

increasingly adopted next-generation sequencing to diagnose hereditary disorders due to its cost-effectiveness, high throughput and accuracy23. Given the increased risks and aggressiveness

of cancer development and progression in patients with _BAP1_ germline variants, understanding how variants in _BAP1_ affect function will aid interpretation of uncertain variants.

Classification of VUS allows us to inform predisposed patients and affected family members regarding potential cancer risks, with appropriate clinical interventions implemented if necessary.

Here, we study and characterise various _BAP1_ germline variants identified in our patients. These patients have family histories of kidney cancers and other cancers such as breast cancer.

Epidemiological, genetic and functional evidence are required to classify variants, and functional assays are ideal to elucidate pathogenicity status especially in rare variants24. To our

knowledge, there are currently no recommended assays to reliably evaluate the functional impact of the germline mutations on BAP1 cellular function in the assessment of variant

pathogenicity. Therefore, by using assays such as deubiquitination assays and immunofluorescence staining, we functionally compare _BAP1_ germline VUS from our patient cohort against known

pathogenic and likely benign variants, which may aid in classification of these variants. RESULTS CLINICOPATHOLOGICAL CHARACTERISTICS OF PATIENTS WITH GERMLINE _BAP1_ VARIANTS Through

clinical genetic testing, we identified six patients harbouring germline _BAP1_ mutations (Table 1, Supplementary Data 1). Three patients are carriers of pathogenic variants, two with

variants of uncertain significance (VUS) and one with a likely benign variant. One of our patients had an additional VUS identified in _MSH6_ (Supplementary Data 1). However, we prioritised

the investigation of _BAP1_ VUS in this study and the possible role of this _MSH6_ VUS in tumorigenesis was not evaluated. Among the pathogenic variant carriers, cancer spectrum was variable

but consistent with _BAP1_ tumor predisposition syndrome (Table 1): one presented mesothelioma, two with breast cancers, of whom one also presented multiple cancers including clear cell

renal cell carcinoma (ccRCC). The median age at diagnosis is 39 (range: 36–61 years). Both VUS carriers were diagnosed with ccRCC in their late thirties, whereas the likely benign carrier

presented thymoma and adrenal cortical cancer. All patients demonstrated strong family history of cancer (Table 1). Pedigrees for the cases identified with _BAP1_ germline VUS show strong

family history of cancers from the paternal side and early onset cancers, including renal and breast cancers (Fig. 1a, b). PATIENT-DERIVED LYMPHOBLASTOID CELL LINES WITH _BAP1_ PATHOGENIC

VARIANTS SHOW REDUCED EXPRESSION OF FULL-LENGTH BAP1 AND EMT MARKERS BAP1 is known to regulate migration, epithelial to mesenchymal transition (EMT) and therefore metastasis in various

cancers including cervical cancer, breast, osteosarcoma and kidney25,26,27,28,29,30. Using patient-derived lymphoblastoid cell lines, we found that carriers of pathogenic _BAP1_ germline

variants (c.852_del and c.1358_1359del) had reduced expression of full-length BAP1, Vimentin and Snail, as compared to controls with wild-type BAP1 (BAP1 WT) (Fig. 1c), whereas _BAP1_

germline VUS (c.299 T > C and c.551 A > G) or likely benign carriers were not significantly different from wild-type _BAP1_ WT. Vimentin is a type III intermediate filament that drives

EMT when overexpressed. Similarly, Snail also promotes EMT by repressing the expression of the adhesion protein E-cadherin. Loss of BAP1 has been reported to downregulate the expression of

Snail, promoting ccRCC cells towards mesenchymal-epithelial transition25. Our results suggest compromised BAP1 function among the pathogenic variants carriers, which results in altered

migration capabilities of the cancer cells, akin to the loss of BAP1. EFFECTS OF _BAP1_ VARIANTS ON CELLULAR LOCALISATION Known mutation positions of _BAP1_ pathogenic variants, _BAP1_

benign variants and _BAP1_ VUS are mapped as shown in Fig. 2a. _BAP1_ pathogenic variants are mostly located before the nuclear localisation signal (NLS) and are predominately frameshift and

nonsense mutations. Missense mutations observed in _BAP1_ pathogenic variants are all found in the UCH domain of BAP1 (Fig. 2b). _BAP1_ VUS appear to be distributed across the gene (Fig.

2a). While the majority of the missense mutations are not in the UCH domain of BAP1; 20–30% of such mutations are in the UCH domain of which function has not yet been established (Fig. 2b).

To investigate if the genetic alterations lead to changes in the cellular localisation of BAP1, we expressed the different _BAP1_ germline variants in HK-2 _BAP1_ KO cell line. In addition

to the variants identified in our patients, we also characterised the _BAP1_ c.1166 G > A variant (p.R389H) that is not located in any known important domains of BAP1 (Fig. 2a) and _BAP1_

c.271 T > G, c.272 G > C variant (p.C91A), a known mutation that abrogates the UCH function31. The nuclear localization of BAP1 is essential to its function, with the loss of nuclear

localization observed in many cancers including uveal melanoma, mesothelioma and ccRCC11,13,32,33,34,35. _BAP1_ variants that lead to protein truncation or frameshift before the nuclear

localisation signal will change the localisation of BAP1, promoting oncogenic pathways36,37. Indeed, we observed that all three of our _BAP1_ truncating variant carriers (c.588 G > A,

c.852_del and c.1358_1359del) displayed cytoplasmic localisation of BAP1, which was not seen for the likely benign variant (c.1735G > A) or WT _BAP1_ (Fig. 3), suggesting that the

cytoplasmic localisation of BAP1 is consistent with the pathogenic classification for these variants. Of note, nuclear localisation of both _BAP1_ VUS carriers (c.299 T > C and c.551 A 

> G) were unaffected (Fig. 3). EFFECT OF GENE MUTATIONS ON DEUBIQUITINASE FUNCTION We performed homology modelling of the UCH domain of BAP1 using UCH-L5 (3ris.pdb) as a template (Fig.

4a). _BAP1_ c.551 A > G (p.D184G) is in the catalytic triad of UCH (made up of cysteine, histidine and aspartic acid that abrogates its deubiquitinase function38. Deubiquitination by UCH

domain takes place in two steps39 (Fig. 4b). The thiol of cysteine residue will first be deprotonated by histidine, followed by a nucleophilic attack by the deprotonated thiol on the

carbonyl carbon of the substrate. The aspartic acid residue is likely important to keep the active site residues in a favourable geometry and to stabilise the ion pair formation of the

catalytic residues40. All remaining _BAP1_ variants are not located on the catalytic triad of the UCH domain, hence their effects on the deubiquitinase function of BAP1 protein are unknown

(Fig. 4a). Our homology model of UCH domain of BAP1 using UCH-L5 (3ris.pdb) as a template shows that L100 of BAP1 is located close to the catalytic triad (Fig. 4a). To investigate the

effects of the _BAP1_ VUS p.L100P on the deubiquitinase function of BAP1 protein due to the conformational change of protein 3D structure, we would like to perform molecular dynamics (MD)

simulations for BAP1 homology model. However, because of no structure of template covered, a long unstructured loop (10 amino acid) was modelled for Q156-M165. This region is close to H169

of catalytic triad and may affect the dynamic behaviour of catalytic triad. Instead, we performed MD simulations of its template/homologous protein, UCH-L5, for that the corresponding

residue of L100 and adjacent residues of BAP1 is highly conserved among different species of BAP1 and human UCH-L5 (Supplementary Fig. 1). The corresponding position of L100 of BAP1 on

UCH-L5 is L97 (Supplementary Fig. 1). We performed MD simulations for both wild-type and L97P of UCH-L5. The convergent and comparable Cα RMSDs over 500 ns simulations (Supplementary Fig. 2)

implied that the structures of mutant UCH-L5 was as intact and stable as wild-type during simulations. However, during the last 200 ns stable simulations, the substantial conformational

changes lasting ~30 ns were sampled on L97P mutant UCH-L5. The distance between the two members of catalytic triad (Nε2 of H164 and Oδ of D179) from L97P UCH-L5 was extended to >5 Å

compared to the constant and stable ~3 Å in wild-type protein (Fig. 4c). The falling apart between catalytic triad may affect the enzymatic activity and implies that the highly conserved

leucine (L100 of BAP1 and L97 of UCH-L5) may play an important structural role in supporting the spatial positions of catalytic triad of UCH domain. In addition, we performed in vitro

deubiquitination assay on H2AK119ub, a well-known substrate of BAP1, to assess the effect that the various mutations have on the deubiquitination function of BAP1 (Fig. 4d). _BAP1_ p.C91A

(c.271 T > G, c.272 G > C), a known amino acid mutation that abrogates the UCH function31 serves as our positive control. _BAP1_ variants within the UCH domain, including the _BAP1_

VUS p.L100P, displayed defective deubiquitination capabilities consistent with our positive control (Fig. 4d), suggesting defective UCH function. Interestingly, we found that the pathogenic

_BAP1_ germline variants also have impaired UCH function, even though not positioned within the UCH domain (Fig. 4d). By calculating the ratio of H2AK119ub band intensity compared to H2A

band intensity, normalised to FLAG expressing sample, the pathogenic germline variants showed the greatest disruption to the deubiquitination activity of BAP1, while the likely benign

variant showed the least disruption (Fig. 4d). Our results suggest that pathogenicity of the _BAP1_ germline variant may be related to the integrity of the catalytic activities. Our _BAP1_

germline VUS also showed impaired deubiquitinase function, albeit moderate compared to pathogenic _BAP1_ germline variants (Fig. 4d). FUNCTIONAL CHARACTERISATION OF _BAP1_ MISSENSE GERMLINE

VARIANTS WITH CONFLICTING INTERPRETATIONS Overall, we have functionally characterised _BAP1_ germline VUS and compared them with known _BAP1_ pathogenic and likely benign variants. In 2015,

the American College of Medical Genetics and Genomics (ACMG) has published a standard framework containing variant curation criteria to guide assessment of variant pathogenicity24. The

variants are assessed based on the strength of evidence of pathogenicity in these categories: population data, computational and predictive data, functional data, segregation data, allelic

data, de novo data, other database and other data24 Subsequently, the Clinical Genome Resource (ClinGen) Sequence Variant Interpretation Working Group (SVI) proposed to refine criteria for

variant interpretation from functional assays, recommending different levels of strength of pathogenicity to be assigned based on factors such as the type and number of controls used41.

Based on results of the functional assays and number of controls used in the study, we have summarised our findings in a flowchart to aid in the determination of the functionality of a novel

_BAP1_ germline variant (Fig. 5a). These functional assays may be useful as supporting evidence for pathogenicity in the computational and predictive data and functional data categories of

the ACMG criteria and supporting level evidence for pathogenicity according to the criteria proposed by ClinGen. Any functional abnormality detected using the functional assays described in

this study will be considered evidence for pathogenicity as per ACMG guidelines; if in agreement with other evidence, variants with abnormality detected in one assay are possibly pathogenic

and in two or more assays are likely pathogenic (Fig. 5b). Our results show that pathogenic variants tend to lead to the truncation of BAP1 and associated with reduced expression of

full-length BAP1 and epithelial-mesenchymal transition (EMT) markers in patient-derived lymphoblastoid cell lines. Pathogenic _BAP1_ variants also adversely affect deubiquitination

capabilities and cellular localisation of BAP1. Combining the profiles of these assays will allow us to reliably distinguish between _BAP1_ pathogenic and likely benign variants and

therefore aid to benchmark newly identified _BAP1_ germline variants. 17 _BAP1_ missense germline variants with conflicting interpretations are listed on ClinVar as of July 2020. Five of

these variants were picked for functional characterisation (Fig. 6a), to yield additional insights for these variants based on the proposed functional assays. Two of these variants are

located in the UCH domain, two are located in the BRCA1 binding domain and one is located in region without any known domains (Fig. 6a). The nuclear localisation of these _BAP1_ variants

were not affected (Fig. 6b). In vitro deubiquitination assay showed that _BAP1_ c.121 G > A and BAP1 c.341 G > A had impaired deubiquitinase functions, even though moderate compared to

positive control (Fig. 6c). Our results provide functional evidence that _BAP1_ c.121 G > A and _BAP1_ c.341 G > A are possibly pathogenic variants and demonstrate that the proposed

functional assays can be utilised to rapidly evaluate and facilitate classification of _BAP1_ variants of uncertain significance to resolve conflicting interpretations for these variants.

DISCUSSION _BAP1_ tumour predisposition syndrome is a hereditary tumour syndrome that is associated with germline pathogenic mutations in _BAP1_21 and increased susceptibility for uveal

melanoma, mesothelioma, cutaneous melanoma and renal cell carcinoma42. An increasing number of other cancers including cholangiocarcinoma, breast cancers and neuroendocrine tumours have also

been shown to be associated with the syndrome15,43,44,45. The mode of inheritance in _BAP1_ tumour predisposition syndrome is autosomal dominant and a majority of the germline pathogenic

variants in _BAP1_ are truncating46. Cancer patients with _BAP1_ tumour predisposition syndrome are generally associated with poorer prognosis compared to patients without

predisposition15,43,45. Germline carriers of _BAP1_ pathogenic variants demonstrate earlier onset (e.g. median age of onset in uveal melanoma patients with germline _BAP1_ mutations is 51

years compared to 62 years in the general population47) and more aggressive cancers15. Several therapeutic approaches have been explored for BAP1-deficient tumours, including

chemotherapeutic drugs (e.g. gemcitabine), radiotherapy and small molecule inhibitors (e.g. EZH2 inhibitors) with limited success16,17,18,19,20. Due to the increasingly widespread use of

next-generation sequencing in clinical laboratories23, more novel _BAP1_ variants are expected to be discovered21,22. Functional studies have emerged as one of the indispensable methods to

help classify _BAP1_ VUS in addition to family cancer history and genetic evidence. Variant classification is important as it may have impact on subsequent cancer screening, follow-up and

therapeutic strategies. Unfortunately, no standard protocol of functional assays is used to aid the classification of _BAP1_ VUS. In this study, we functionally assay each _BAP1_ germline

variant found in our patient cohort, including benign, pathogenic and VUS, using in vitro deubiquitination assay, cellular localisation determination using immunofluorescence staining and

expression of EMT markers. These assays are only valid when the _BAP1_ germline variant of interest can be expressed in cell models. By analysing known BAP1 pathogenic variants listed in

ClinVar, we found that they are mostly located before the nuclear localisation signal (NLS) and are predominately frameshift and nonsense mutations (Fig. 2), which is consistent with that

from our patient cohort. These types of mutations introduce a premature stop codon and are predicted to be target of degradation by nonsense-mediated mRNA decay (NMD) to protect the cells

from the presence of abnormal peptides48,49. While we cannot exclude the possibility that our curated pathogenic frameshift and nonsense variants will undergo NMD, we have shown that

truncated proteins can be expressed from these variants (Fig. 4d). With the exception of analysing the expression of EMT markers in the LCLs, our functional assays are based on introducing

the _BAP1_ germline variant into cell models and therefore cannot be used to investigate effects of loss of _BAP1_ expression. We found that pathogenic _BAP1_ germline variants show

defective deubiquitination function, are aberrantly localised in the cytosol or display low expression of EMT markers such as Vimentin and Snail, allowing them to be clearly distinguished

from likely benign variants. Based on our deubiquitination assay, _BAP1_ VUS c.299 T > C and c.551 A > T, both of which located in the UCH domain of BAP1, were observed to have

decreased deubiquitinase activities. These patients also have strong family history of _BAP1_-related cancers. Therefore, _BAP1_ VUS c.299 T > C (Supplementary Fig. 3a) and c.551 A > T

(Supplementary Fig. 3b) are functionally abnormal. We have proposed the use of the functional assays used in the study to augment the ACMG criteria and ClinGen recommendations for assessing

_BAP1_ variant pathogenicity (Fig. 5). Given these lines of evidence and based on the level of evidence in the other categories in the ACMG framework, _BAP1_ VUS c.299 T > C and c.551 A 

> T may therefore be considered to be reclassified as likely pathogenic (Table 2). Although our validation set of _BAP1_ germline variants is limited in size, the profiles demonstrated by

our functional assays could clearly distinguish the functional impact of pathogenic from benign variants and may be used to provide a line of functional evidence in determining the

pathogenicity of newly identified _BAP1_ variants. In conclusion, our study may allow clinicians to quickly benchmark newly identified _BAP1_ variants against known pathogenic and likely

benign variants. Even though the penetrance of having a _BAP1_ germline variant has been shown incomplete and the type of _BAP1-_related cancers has been observed to vary among different

family members42, our findings contribute to providing functional support for resolution of _BAP1_ variants with uncertain significance and facilitating clinical interpretation by clinicians

so that appropriate prognosis, therapeutic intervention strategies and care may be delivered to patients with _BAP1_ tumour predisposition syndrome. METHODS VARIANT CURATION Patients

selected for this study were referred to our Cancer Genetics Service at National Cancer Centre Singapore for clinical genetic testing on multi-gene panels (including _BAP1)_ by commercial

laboratories accredited by CLIA/CAP. Classification of identified _BAP1_ variants were performed by the respective accredited laboratories in accordance with the American College of Medical

Genetics/Association for Molecular Pathology (ACMG/AMP) guidelines24. Patients included for analysis were identified to harbour germline _BAP1_ variants, regardless of classification.

Patient clinical history and family history of cancer were assessed by a clinical geneticist or genetic counsellor at our service. This study was approved by SingHealth Centralized

Institutional Review Board (CIRB 2011/826/B) with signed informed consent from all participants. COMMERCIAL CELL LINES AND CELL CULTURE All commercial cell lines were purchased from ATCC.

HEK293T is cultured in DMEM + 10% FBS. HK-2 _BAP1_ KO cell is kind gift from Prof Teh Bin Tean’s lab and cultured in RPMI 1640 + 10% FBS. Patient-derived cell lines are cultured in RPMI 1640

 + 10% FBS. All cell lines were maintained at 37 °C in a humidified chamber in the presence of 5% CO2 and routinely confirmed to be free of mycoplasma. Cells used for experiments were

between 3 and 8 passages from thawing. At least three biological replicates were performed for each experiment. IMMUNOBLOTTING Cell pellets were collected and washed twice in cold PBS,

before they are lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Roche). The lysed cells were then and sonicated for 10 cycles at 30 s on and 30 s off,

high settings using a Biorupter (Diagenode) to ensure complete lysis. The lysates were clarified by centrifugation at 16,000 × _g_ r.c.f for 20 min and the protein extracts were quantified

using BCA assay (ThermoFisher Scientific Corporation, Waltham, MA, USA). 5–10 μg of protein was loaded onto 4–12% gradient gel, electrophoresed by SDS-PAGE and transferred to 0.2 μm

nitrocellulose membrane (Biorad). The membranes were then blocked in 5% non-fat milk diluted in 1X PBST and incubated overnight with primary antibody diluted in blocking buffer at 4 °C. The

following primary antibodies were used: Monoclonal Anti-Flag® M2 antibody (Sigma–Aldrich: F1804, 1:2000), anti- ubiquityl-Histone H2A (Cell Signaling, Beverly, MA; #2577, 1:1000),

anti-Histone H2A antibody (Abcam, Cambridge, MA, USA: ab18255, 1:1000), anti-BAP1 (Santa Cruz Biotechnology: sc-28383, 1;200), anti-Vimentin (Cell Signaling, Beverly, MA: #5741, 1:1000),

anti-Snail (Cell Signaling, Beverly, MA: #3879, 1:1000) and GAPDH (Santa Cruz Biotechnology: sc-166545, 1;1000). Membranes were washed three times for 10 min each with 1× PBST then incubated

with secondary HRP-conjugated antibody anti-rabbit or anti-mouse immunoglobulin (Promega Corporation: W4011 or W4021, 1:10,000) for 1 h. After washing three times for 10 min each with 1X

PBST after incubation, the immunoreactivity was detected with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific Corporation, Waltham, MA, USA). All blots were

derived from the same experiments and processed in parallel. Un-cropped images of blots are shown in Supplementary Fig. 4. IMMUNOFLUORESCENCE STAINING Transfected HK-2 BAP1 KO cells were

plated on µ-Dish 35 mm imaging dishes (ibidi). Fixation was performed for 10 min using 4% paraformaldehyde (Sigma–Aldrich) at room temperature, followed by permeabilization using 0.3%

Triton-X 100 (Sigma–Aldrich) on ice for 10 min. The cells were then blocked in 5% BSA in PBS for 1 h and then incubated overnight with Monoclonal Anti-Flag® M2 antibody (Sigma–Aldrich:

F1804, 1:500). Slides were washed three times with 1X PBS for 10 min and then incubated AlexaFluor A488 conjugated secondary antibodies (ThermoFisher Scientific Corporation, Waltham, MA,

USA: A11008, 1:1000). Hoechst 33342 was used to stain the nucleus. After incubation for 1 h at room temperature, the slides were washed 3 times with PBS and mounted with Prolong Gold

antifade reagent (ThermoFisher Scientific Corporation, Waltham, MA, USA). Images were acquired by confocal microscope (Zeiss LSM700). HOMOLOGY MODELING Protein sequences of human BAP1

(Q92560) and human UCH-L5 (Q9Y5K5) were obtained from Uniprot50. The sequence identity of UCH domain between human BAP1 and UCH-L5 is 44%. The homology model of UCH domain of human BAP1 was

built by MODELLER51 according to the template 3D structure of human UCH-L5 (3ris.pdb52). MD SIMULATION To investigate the effect of VUS on deubiquitination function due to conformational

change of UCH domain, we implemented the computational approach, molecular dynamics (MD) simulation. The initial 3D structure of human UCH-L5 (3ris.pdb52 chainA, amino acids 7–227) was

obtained from Protein Data Bank53. The structure of six missing residues (amino acid 155–160) were modeled by MODELLER51. Explicit hydrogen atoms and protonation states of titratable

residues of UCH-L5 were determined by the web server PDB2PQR54 (http://nbcr-222.ucsd.edu/pdb2pqr_2.1.1/). The systems of MD simulations were prepared using AmberTools package55. We adopted

ff14SB force fields for the UCH-L5. The UCH-L5 was solvated in virtual cubic boxes filled with TIP3P water molecules. To maintain charge neutralization of the systems, proper numbers of

counter ions (Na+ or Cl−) were added into the simulation boxes. All energy minimizations and dynamics simulations in this study were performed using AMBER16 package55. To release steric

clashes between atoms, we energy minimized the systems by the following protocol. The first 5000 cycles of energy minimization were performed by steepest descent method while the backbone

heavy atoms of UCH-L5 were restrained by harmonic potentials (force constant was set as 10.0 kcal/mol-Å2). The second 5000 cycles of energy minimization were followed without any restraints.

Periodic boundary condition was employed during dynamics simulations while long range electrostatic interactions were treated by the particle mesh Ewald method and short range nonbonded

cutoff was set as 12.0 Å. SHAKE algorithm was used to constrain bond length so that the simulations of production run allow a larger time step of 2 fs. To heat up the system, we ran 100 ps

simulations using time step of 1 fs. During the heating process, backbone heavy atoms of UCH-L5 were restrained by a harmonic potential with force constant of 10.0 kcal/mol-Å2. The dynamics

simulations in this study were in the NPT ensemble. The pressure was coupled to 1.0 bar and temperature was coupled to 298.15 K by Berendsen’s coupling methods with both coupling constants

of 1 ps. Backbone restraints were relaxed to 5.0 kcal/mol-Å2 for additional 100 ps simulation before production run. During the simulations of production run, we performed 200 ns simulations

for both systems with larger time step of 2 fs. The temperature coupling method of production simulations was switched to Langevin thermostat with the collision frequency as 5 ps−1 whereas

Berendsen’s method was retained for pressure coupling. The simulations of production run were performed for 500 ns. All data analyses except heavy atom RMSD were based on the trajectories

belonging to the period from 300 ns to 500 ns. IN VITRO DEUBIQUITINATION ASSAY The indicated BAP1 variants were fused in frame with N-terminal FLAG in pFLAG-CMV1 expression vector

(Sigma–Aldrich). HEK293T cells were transfected with 1 µg each of the indicated plasmids using Lipofectamine 3000 (ThermoFisher Scientific Corporation, Waltham, MA, USA). After 3 days, the

cell pellets of the transfected cells were harvested, washed in PBS and lysed (50 mM Tris HCl, pH 7.4, 1% Triton X-100, with 150 mM NaCl). The FLAG tagged BAP1 variants were allowed to bind

to Dynabeads™ Protein G (ThermoFisher Scientific Corporation, Waltham, MA, USA) that had been conjugated with Monoclonal Anti-Flag® M2 antibody overnight. The FLAG tagged BAP1 variants bound

to the beads were washed five times (0.5 M Tris HCl, pH 7.4, with 1.5 M NaCl). In vitro deubiquitination assay was performed as described56 with modifications. The washed beads with bound

FLAG tagged BAP1 variants were resuspended in deubiquitination buffer (50 mM Tris-HCl, pH 7.3, 1 mM MgCl2, 50 mM NaCl, and 1 mM DTT, 1 mM EDTA and 1% Triton X-100). 200 ng of H2AK119UB

(EpiCyher) was added to the mixture and deubiquitination was carried out at 37 °C for 3 h with gentle shaking. The reaction was quenched by adding 2× Laemmli buffer and analysed by

immunoblotting. REPORTING SUMMARY Further information on research design is available in the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY All data generated or

analysed during this study are included in this published article (and its supplementary information files). CODE AVAILABILITY Algorithms used for molecular dynamics stimulation detailed

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Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We would like to thank the patients and research participants for their contribution to the study.

This work was supported by the Khoo Postdoctoral Fellowship Award (Duke-NUS-KPFA/2019/0034 to J.H.H), the National Medical Research Council Open Fund—Young Individual Research Grant

(MOH-000232 to J.H.H), the National Medical Research Council Open Fund—Individual Research Grant (MOH-000144 to B.T.T), the National Medical Research Council Singapore Translational Research

Investigator Award (MOH-000248 to B.T.T.), the NRF-NSFC Joint Research Grant (Data Science) (NRF2016NRF-NSFC001–057 to B.T.T), the LKC Startup Grant (LKC MOE to J.N.), the National Research

Foundation Singapore under its Clinical Scientist Award (NMRC/CSA-INV/0017/2017) and administered by the Singapore Ministry of Health’s National Medical Research Council (to J.N.). This

work is also partially funded by NCC Research Fund, NCC Cancer Fund, Terry Fox and Lee Foundation supporting funds to J.N. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Cancer and Stem Cell

Biology Program, Duke-NUS Medical School, Singapore, 169857, Singapore Jing Han Hong, Hong Lee Heng & Bin Tean Teh * Institute of Molecular and Cellular Biology, Agency for Science,

Technology and Research, Singapore, 138673, Singapore Jing Han Hong, Bin Tean Teh & Joanne Ngeow * Cancer Genetics Service, Division of Medical Oncology, National Cancer Center,

Singapore, 169610, Singapore Siao Ting Chong, Nur Diana Binte Ishak, Sock Hoai Chan & Joanne Ngeow * Cancer Science Institute of Singapore, National University of Singapore, Singapore,

117599, Singapore Po-Hsien Lee & Bin Tean Teh * Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, 138672, Singapore Po-Hsien Lee & Bin Tean Teh *

Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, 510060, Guangzhou, Guangdong, China Jing Tan *

Laboratory of Cancer Epigenome, Division of Medical Sciences, National Cancer Centre Singapore, Singapore, 169610, Singapore Jing Tan, Hong Lee Heng & Bin Tean Teh * SingHealth/Duke-NUS

Institute of Precision Medicine, National Heart Centre, Singapore, Singapore Bin Tean Teh * Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, 308232, Singapore

Joanne Ngeow * Oncology Academic Clinical Program, Duke-NUS Medical School, Singapore, 169857, Singapore Joanne Ngeow Authors * Jing Han Hong View author publications You can also search for

this author inPubMed Google Scholar * Siao Ting Chong View author publications You can also search for this author inPubMed Google Scholar * Po-Hsien Lee View author publications You can

also search for this author inPubMed Google Scholar * Jing Tan View author publications You can also search for this author inPubMed Google Scholar * Hong Lee Heng View author publications

You can also search for this author inPubMed Google Scholar * Nur Diana Binte Ishak View author publications You can also search for this author inPubMed Google Scholar * Sock Hoai Chan View

author publications You can also search for this author inPubMed Google Scholar * Bin Tean Teh View author publications You can also search for this author inPubMed Google Scholar * Joanne

Ngeow View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conceptualization, J.N., B.T.T., J.H.H.; Methodology, J.H.H., S.T.C., H.P.L, T.J.,

H.L.H.; Investigation, J.H.H., S.T.C., H.P.L., T.J., H.L.H., N.D.B.I.; Writing—original draft, J.H.H., J.N; Writing—-review and editing, J.H.H., J.N., S.T.C., P.H.L., S.H.C., T.J., B.T.T.;

Funding acquisition, J.N., B.T.T., J.H.H.; Resources, J.N., B.T.T., J.H.H.; Supervision, J.N. and B.T.T. CORRESPONDING AUTHORS Correspondence to Bin Tean Teh or Joanne Ngeow. ETHICS

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