supplementary materials for · 2017-02-17 · supplementary materials for loss of tumor suppressor...

Supplementary Materials for

Loss of tumor suppressor KDM6A amplifies PRC2-regulated

transcriptional repression in bladder cancer and can be targeted

through inhibition of EZH2

Lian Dee Ler, Sujoy Ghosh, Xiaoran Chai, Aye Aye Thike, Hong Lee Heng,

Ee Yan Siew, Sucharita Dey, Liang Kai Koh, Jing Quan Lim, Weng Khong Lim,

Swe Swe Myint, Jia Liang Loh, Pauline Ong, Xin Xiu Sam, Dachuan Huang, Tony Lim,

Puay Hoon Tan, Sanjanaa Nagarajan, Christopher Wai Sam Cheng, Henry Ho,

Lay Guat Ng, John Yuen, Po-Hung Lin, Cheng-Keng Chuang, Ying-Hsu Chang,

Wen-Hui Weng, Steven G. Rozen, Patrick Tan, Caretha L. Creasy, See-Tong Pang,*

Michael T. McCabe,* Song Ling Poon,* Bin Tean Teh*

*Corresponding author. Email: [email protected] (B.T.T.); [email protected]

(S.L.P.); [email protected] (M.T.M.); [email protected] (S.-T.P.)

Published 22 February 2017, Sci. Transl. Med. 9, eaai8312 (2017)

DOI: 10.1126/scitranslmed.aai8312

This PDF file includes:

Materials and Methods

Fig. S1. Structural modeling and molecular dynamic simulation of 10 missense

mutations in primary bladder cancer patients.

Fig. S2. The expression of KDM6A in urothelial bladder carcinoma with wild-

type or mutated KDM6A.

Fig. S3. H score of EZH2 in KDM6A-mutated or KDM6A–wild-type primary

tumors.

Fig. S4. The effects of KDM6A alterations in urothelial bladder carcinoma cell

lines.

Fig. S5. The effects of EZH2 inhibitors and cytotoxic therapeutic drugs on

KDM6A-null/inactivated or KDM6A–wild-type bladder cancer cell lines.

Fig. S6. The IC50 of GSK126 and EPZ6438 in cells with and without KDM6A

expression.

Fig. S7. Transcriptional profiling of KDM6A-mutated or KDM6A–wild-type

primary tumors and GSK343-treated KDM6A-null or KDM6A–wild-type cell

lines.

Fig. S8. The effects of EZH2 inhibitors on body weight.

www.sciencetranslationalmedicine.org/cgi/content/full/9/378/eaai8312/DC1

Fig. S9. Sanger sequencing traces of KDM6A-inactivating mutation in knockout

clones of RT-4 cell line.

Table S1. Clinicopathologic characteristics of 176 urothelial bladder carcinoma

patients.

Table S2. KDM6A mutations in a cohort of 176 urothelial bladder carcinoma

patients.

Table S3. KDM6A mutations in female urothelial carcinoma patients.

Table S4. Differentially expressed gene sets comparing KDM6A-mutated versus

KDM6A–wild-type bladder tumors.

Table S5. Enrichment of PRC2 transcriptional repression in urothelial bladder

tumors with mutated KDM6A.

Table S6. Effects of loss of KDM6A on H3K27me3 levels at specific loci.

Table S7. Differentially expressed pathways in the GSK343-treated KDM6A-null

and KDM6A-reexpressing cells.

Table S8. Differentially expressed cell adhesion molecules in KDM6A-mutated

versus KDM6A–wild-type bladder tumors.

Table S9. Differentially expressed DNA replication molecules in KDM6A-

mutated versus KDM6A–wild-type bladder tumors.

Table S10. GSK343-induced PRC2-related genes in cells with KDM6A loss.

Table S11. KDM6C/UTY copy number in urothelial bladder carcinoma patients.

Table S12. Primers for PCR amplification of KDM6A full-length coding

sequence.

Table S13. Primers for Sanger sequencing of KDM6A full-length coding

sequence.

References (58–73)

Supplementary Materials and Methods

Human tissue samples and clinical information

Urothelial bladder tumors were diagnosed and classified by histopathological

examination of surgically excised tumors, according to the WHO classification system. Snap

frozen bladder tumor tissues and corresponding matched normal samples (whole blood) from 51

patients with urothelial bladder carcinoma were obtained from Singapore General Hospital in

Singapore. 125 additional bladder tumors were obtained from Chang Gung Memorial Hospital

(Taiwan). The human biological samples were sourced ethically and their research use was in

accordance with the terms outlined in the informed consent forms, under the protocols approved

by the SingHealth Institutional Review Board and Human Research Ethics Committee of the

Chang Gung Memorial Hospital. Snap frozen tumor tissues and normal samples were prioritized

for DNA extraction and Sanger sequencing, and remaining tumor tissues were used for RNA

extraction where available. Corresponding archival formalin-fixed paraffin-embedded (FFPE)

samples from 41 patients in Singapore and 57 patients in Taiwan were also used in this study.

The background and clinicopathologic data of these patients (n = 176) treated in Singapore and

Taiwan are summarized in table S1. The somatic mutation data from 342 additional urothelial

bladder carcinoma patients were previously published (9, 11, 21, 22). A total of 518 tumors were

analyzed for KDM6A mutation status.

Sanger sequencing of KDM6A

Primers for PCR and Sanger sequencing (tables S12–S13) were designed using Primer3

(58) and Vector NTI Software (Life Technologies). Purified PCR products were sequenced in

forward and reverse directions using BigDye Terminator v3.1 Cycle Sequencing Kit and ABI

PRISM 3730 Genetic Analyzer (Applied Biosystems). When the design of primers was not

possible at a few regions, purified PCR products were sequenced twice in the same direction.

Sequencing trace chromatograms were analyzed on SeqMan Pro program from DNASTAR

multiple program package (DNASTAR Inc.) and by manual review.

Antibodies

The following primary antibodies were used for Western blot analysis: rabbit anti-

KDM6A kindly provided by Dr. Eli Canaani (20) or purchased from Sigma-Aldrich

(Cat#HPA002111), mouse monoclonal anti-EZH2 (AC22 clone; Cat#3147), rabbit monoclonal

anti-H3K27me3 (C36B11 clone; Cat#9733) , rabbit monoclonal anti-Histone H3 (D1H2 clone;

Cat#4499) from Cell Signaling Technology, and mouse monoclonal anti-β-Actin (AC-15 clone;

Cat#A1978) from Sigma-Aldrich. Secondary antibodies used were sheep anti-mouse IgG and

donkey anti-rabbit IgG, conjugated with horseradish peroxidase, from GE Healthcare Life

Sciences. Primary antibodies specific for H3K27me3 (clone C36B11; Cat#9733) and EZH2

(clone D2C9 XP; Cat#3147) from Cell Signaling Technology, and IGFBP3 (H-98; Cat#sc-9028)

from Santa Cruz Biotechnology were used for IHC. Primary antibodies specific for H3K27me3

(Abcam; Cat#ab6002) and EZH2 (Diagenode; Cat#MAb-180-050; lot #001) were used for ChIP-

Seq.

Extraction and amplification of DNA from patient samples and cell lines

Total genomic DNA from snap frozen human bladder tumor tissues, whole blood, or cell

lines was extracted and purified using Blood and Cell Culture DNA Kit (Qiagen) according to

manufacturer’s instructions. Purified genomic DNA from patient samples was amplified using

illustra GenomiPhi HY DNA Amplification Kit (GE Healthcare Life Sciences) before further

sequencing.

Extraction of RNA from patient samples and cell lines

Total RNA was extracted using Trizol reagent (Life Technologies), followed by

purification using RNeasy kit (Qiagen).

Cell proliferation assay

Cell proliferation was examined by using colorimetric BrdU cell proliferation ELISA kit

(Roche Applied Science) or CellTiter 96 AQueous One Solution Cell Proliferation Assay kit

(MTS; Promega) following manufacturers’ instructions. Briefly, cells were seeded at an initial

density of 1-6x103 per well in 96-well plate format. Upon harvest, cells were subjected to the

incorporation of bromodeoxyuridine (BrdU) and subsequent incubation with specific anti-BrdU

antibody and substrate reagent. The absorbance at 370 nm (with reference at 492 nm),

representing the amount of incorporated BrdU or cell proliferation, was measured on TECAN

Safire 2 microplate reader. All experiments were performed in replicate for a minimum of three

independent experiments.

Computational Modelling of KDM6A point mutations

Starting structures

Protein coordinates were obtained from the crystal structures of the human KDM6A

protein (apo and holo form; PDB 3AVS, 3AVR) (23). The N- and C-terminal residues were

capped with acetyl (ACE) and N-methyl (NME) groups, respectively.

Modeling the mutations and generation of the mutant complexes

The ten individual KDM6A mutants (p.N1087I, p.M1129I, p.Q958K, p.P966L, p.V967F,

p.D980N, p.E1010G, p.E1102K, p.L1103P, p.Y1249C) were modeled using the apo form of

protein KDM6A (PDB 3AVS) as template, using MODELER version 9.12 (59). The mutated

protein was then docked with the histone H3 peptide using the HADDOCK server (60). The best

docked structure for subsequent MD simulations was selected using the criteria: (1) the structure

having the smallest Haddock score, and (2) the most negative Z-score.

General molecular dynamics (MD) simulations

All MD simulations were done using version 4.5 of the Groningen machine for chemical

simulations (GROMACS) (61) and/or assisted model building with energy refinement (AMBER)

v12 (62) software packages. All analyses used GROMOS96 53a6 (63) parameters for the protein.

The binding energy calculations were performed with AMBER99SB parameters (64). The total

charge was neutralized by addition of counter ions Na+ or Cl− explicitly. Each system was

solvated with SPC water in a cubic simulation box (120 Å per side).

A step-wise protocol was employed for MD equilibration beginning with heating in the

NVT ensemble to 300 K over 50 ps. Subsequently, water, hydrogen atoms, and counter ions

were equilibrated for a further 50 ps in the NPT ensemble at 300 K at a pressure of 1 bar;

temperature was held constant at 300 K by velocity rescaling with a coupling time constant of

0.1 ps. Finally, simulations were run for another 50 ns, at which point the data were analyzed.

Analysis

The conformational properties of each of the complexes were analyzed in terms of

RMSD of the peptide backbone atoms (Cα, N, C, O) using GROMACS tool g_rms. The RMSD

values for each complex were calculated relative to the first time step of the MD simulation, and

also with reference to the experimental coordinates reported recently for KDM6A (23).

Structural stability was also seen in terms of fluctuation of each atom about its average position

using the GROMACS tool g_rmsf and on overall folding by calculation of radius of gyration. A

detailed analysis of the hydrogen bond network was carried out for each complex with a distance

cut-off of 3.5 Å and an angle cut-off of 120 degrees using GROMACS tool g_bond. This

generated a hydrogen bonding map and an index, which were further used to investigate

individual bonds and their % occupancies throughout the simulation.

Binding free energies were also analyzed from water stripped AMBER trajectories based

on the MM/GBSA (Molecular Mechanics / Generalized Born Surface Area) (65) method, using

MMPBSA.py program (66), and the solvation energy was approximated through the GB implicit

solvation model (igb = 5) (67). Alanine scanning experiment was done to identify hotspot

residues that have important contributions in binding interactions.

RNA sequencing-based transcriptional profiling

Sequencing libraries were prepared using Illumina Tru-Seq RNA Sample Preparation v2

(Illumina) according to the manufacturer’s instructions. Briefly, Poly-A enrichment was

performed on 1 g of isolated RNA using poly-T oligo attached to magnetic beads. Recovered

poly-A enriched RNA was fragmented chemically and converted to cDNA using SuperScript II

and random primers. The second strand was then synthesized using the Second Strand Master

Mix provided in the kit and then purified by AMPure XP beads. After this, 3’ to 5’ exonuclease

was used to repair the ends of cDNA. A single adenosine was then added to the 3’ end. Using T4

DNA ligase, the indexing adaptors were ligated to both ends of the 3’-adenylated cDNA. The

fragments with attached adapters were then amplified by PCR to constitute the libraries.

Libraries were validated using Agilent DNA 1000 Kit and an Agilent Bioanalyzer (Agilent

Technologies). Validated libraries were diluted to 11 pM and hybridized to an Illumina flow cell

for cluster generation using Illumina Cluster Station. Subsequently, sequencing was performed

on an Illumina High Seq2000 sequencer in paired-end 76-bp read manner according to

manufacturer’s instructions.

Interrogation of PRC2-related pathway in bladder cancer

To examine the PRC2-related pathway in bladder cancer, bladder urothelial cancer cell

lines RT-112 and KU-19-19 were treated with either GSK343 (5 M or 10 M; inhibitor of

EZH2) or vehicle control for 3 days. The total RNA was then extracted by Trizol reagent (Life

Technologies), followed by purification using RNeasy kit (Qiagen). TriGene expression profiling

was performed on isolated RNA using the Affymetrix GeneChip U133 Plus 2.0 platform

(Affymetrix) according to manufacturer’s protocol. Resulting CEL files were loaded into the R

statistical environment (http://www.r-project.org) for further analysis. After importing the CEL

files using the simpleaffy package (68), the data were pre-processed using the Robust Multi-

array Average (RMA) method (69) and then quantile normalized. Probe-level gene expression

values were summarized to gene-level expression values using the BrainArray custom CDF

(Chip Definition File) (70). For each cell line, expression values for treatment conditions (for

example 5 M or 10 M GSK343) were converted to log2 fold change values relative to vehicle

control. All values used were averages of microarray experiments from two independent

biological replicates.

Gene set enrichment analysis was then performed in the following fashion. First, log2

fold change values (10 M treatment vs vehicle control) for the two bladder cancer cell lines

(KU-19-19 and RT-112) were averaged and converted to z-scores. Genes with z-score > 2 were

considered to be significantly up-regulated. These genes were then compared against curated

gene sets (C2) from the Molecular Signatures Database (MSigDB) (51). Fisher’s exact test was

used to identify gene sets that had an over-representation of genes up-regulated in these cell lines,

and a p-value threshold of 0.05 was used. PRC2-related genesets,

BENPORATH_ES_WITH_H3K27ME3, NUYTTEN_EZH2_TARGETS_UP,

BENPORATH_SUZ12_TARGETS, BENPORATH_EED_TARGETS and

BENPORATH_PRC2_TARGETS, were among the top hits. The over-represented genes from

these genesets were used to generate a 174-gene bladder cancer EZH2 inhibitor-related

expression gene signature.

Copy number analysis

Human TaqMan Copy Number Reference Assays (RNAase P, cat# 4400293) were run with

human TaqMan Copy Number Assays (UTY, cat# 4400291:Hs01079454_cn) in a duplex real-

time PCR reaction to detect and measure the relative copy number changes in 51 tumor and

matched normal urothelial bladder carcinoma samples following the manufacturer’s instructions

(ABI). The copyCaller software (ABI) was used to classify copy number gain or loss.

shRNA procedures

shRNA-mediated EZH2 knockdown was performed by infecting RT-4 and KU-19-19

cells with lentiviral transduction particles (Dharmacon) containing a non-targeting shRNA

(shCtrl) or shRNA specific for human EZH2 (shEZH2_1 and shEZH2_2). shRNA gene target

sequences were as follows: shEZH2_1: GAAAGAACGGAAATCTTAA and shEZH2_2:

TCGAGAAAGATACAGCTGA. Three days after lentiviral infection, infected cells were

selected with 2 µg/ml puromycin (Sigma-Aldrich) before being subjected to a cell proliferation

assay. EZH2 knockdown in selected cells was assessed by Western blot analysis.

Generation of isogenic KDM6A knockout cell lines

We used transcription activator-like (TAL) effector nucleases (TALENs) to generate

isogenic KDM6A knockout RT-4 cell lines, RT-4 KO1 and RT-4 KO2. The TALEN pairs

customized to target exon 2 of KDM6A (Ensembl transcript ID: ENST00000377967) were

purchased from Cellectis bioresearch SAS and used to create KDM6A gene disruption in RT-4

cell line, according to manufacturer’s instructions. Briefly, plasmids containing TALEN pairs

with DNA binding recognition sites of KDM6A, as well as pCMV-AC-GFP plasmid (Origene

Technologies Inc.), which carries a GFP transgene and puromycin selection marker, were co-

transfected into RT-4 cell line using FuGENE HD Transfection Reagent (Promega). Transfected

cells were selected by 2 g/ml puromycin 72 h after transfection. Subsequently, antibiotic-

selected cells were serially diluted, and single clones of cells were selected. Positive clones (RT4

KO1 and RT4 KO2) containing small insertions or deletions at exon 2 of KDM6A coding

sequence were verified by both Sanger sequencing and Western Blot. The DNA binding

sequences corresponding to KDM6A gene sequences were as follows: TALEN binding site left –

TGTGTCTGTCTCCACAG, TALEN binding site right – TCGTGAGATTTCATGAA. The

spacer region of TALEN pairs corresponding to KDM6A sequences was CCGCCTCTTTGGGT.

The indels generated by TALEN in the KDM6A gene were verified by Sanger sequencing (fig.

S9).

Re-introduction of KDM6A

The full length open reading frame of KDM6A cloned into pLenti-C-mGFP was

purchased from Origene Technologies Inc. The pLenti expression vector was then packaged into

lentiviral particles using Lenti-vpak Lentiviral Packaging Kit (Origene Technologies Inc.) and

HEK293T cell line, according to manufacturer’s instruction. The viral supernatant was collected,

and the viral titer was examined by Lenti-X GoStix (Clontech) before being transduced into KU-

19-19 cell line. To select for a cell clone with stable KDM6A expression, transduced cells were

serially diluted and single clones of cells were selected. Positive clones (Clone 1 and Clone 2)

expressing KDM6A were verified by Western blot. The same procedure was applied to the

generation of empty vector expressing control of KU-19-19.

Quantitative real-time PCR

Total purified RNA (1 g) was reverse transcribed to cDNA using iScript cDNA Synthesis

Kit (Bio-Rad) following manufacturer’s instructions. Expression of target genes was examined

using specific primers, SsoFast EvaGreen Supermix, and a CFX96 Real-Time PCR Detection

System (Bio-Rad) according to manufacturer’s recommendations. Primers used for the detection

were: KDM6A-F, 5’- AGCGCAAAGGAGCCGTGGAAAA-3’; KDM6A-R, 5’-

GTCGTTCACCATTAGGACCTGC-3’; IGFBP3-F, 5’-CGCTACAAAGTTGACTACGAGTC-

3’; IGFBP3-R, 5’-GTCTTCCATTTCTCTACGGCAGG-3’; GAPDH-F, 5’-

GTCTCCTCTGACTTCAACAGCG-3’; GAPDH-R, 5’- ACCACCCTGTTGCTGTAGCCAA-3’.

Relative quantification of the mRNA for KDM6A and IGFBP3 was performed by the

comparative Cq method with GAPDH as reference gene using the formula 2-Cq. Log2 fold-

change values of gene expression across the samples was derived and used for subsequent

statistical analysis.

Immunohistochemistry (IHC) analysis

Archival, de-identified FFPE samples from bladder cancer primary tissues were obtained

from Department of Pathology at both Singapore General Hospital in Singapore and Chang

Gung Memorial Hospital in Taiwan. KU-19-19-derived xenografts removed upon termination of

mice were also portioned and embedded in paraffin. Histopathological features of the FFPE

sections were reviewed for the study by pathologists blinded to the results of IHC analysis.

IHC staining was performed with Leica BOND-MAX or BOND-III IHC instruments

(Leica Biosystems), according to manufacturer’s recommendations. Briefly, sections from FFPE

samples were cut at 4-5 m, deparaffinized in Bond Dewax solution, and rehydrated through

100% ethanol to 1 X Bond Wash Solution. Antigen retrieval was performed in Bond Epitope

Retrieval Solution for 40 min at °C and followed by proteinase K treatment. After

endogenous peroxidase blocking with 3-4% H2O2 in Tris-buffered saline (TBS) for 15 min at

room temperature (RT), the sections were incubated with 10% goat serum in TBS for 1 hour as

part of non-specific background blocking. Sections were then incubated with primary antibodies

specific for IGFBP3 clone H-98, H3K27me3 clone C36B11 or EZH2 clone D2C9 XP (Cell

Signaling) for 20 min, followed by 5 min incubation with anti-rabbit labelled polymer.

Subsequently, sections were incubated with Bond Mixed DAB Refine for the detection, before

they were counterstained with hematoxylin for 5 min. Negative controls were stained in parallel

by treating sections simultaneously omitting the primary antibody. The IHC staining of FFPE

slides from KU-19-19-derived xenografts were performed by the Advanced Molecular Pathology

Laboratory, IMCB, Singapore.

Western blot analysis

After two washes with PBS, cells were harvested on ice by RIPA lysis buffer (50 mM

Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 % SDS, 1 % Igepal CA-630, and 1 % sodium

deoxycholate) in the presence of protease and phosphatase inhibitors (Roche). Whole-cell lysates

were then collected, sonicated, and centrifuged at 20,000 g for 20 min at 4 C. Protein

concentrations of the supernatant containing protein extracts were determined with a

bicinchoninic acid (BCA) protein assay (Merck Millipore) or DC Protein Assay (Bio-rad). 20 µg

total protein extracts were separated using 8-12 % SDS-polyacrylamide gels, transferred to

nitrocellulose membrane (Bio-rad), blocked with 5 % nonfat milk, and probed with primary

antibodies at 4 C overnight. After 3 washes, the membrane was incubated with appropriate

peroxidase-linked secondary antibodies for 1 h at room temperature and washed 5 times. All

washes and incubations were done in Tris-buffered saline with 0.1 % Tween 20 (TBST; 137 mM

Sodium Chloride, 20 mM Tris pH 7.6, and 0.1 % Tween-20). Proteins were detected using

Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) or

SuperSignal West Pico Chemiluminescent Substrate (Pierce) and were visualized by

autoradiography on CL-XPosure Film (Thermo Scientific). The protein bands were quantified

using the scanner GS-800 (Bio-rad) and the software Quantity One.

Supplementary Figures

Supplementary Figure 1. Structural modeling and molecular dynamic simulation of 10

missense mutations in primary bladder cancer patients.

Crystal structures for the JmjC domain and the C-terminal region of KDM6A are available. We

evaluated the impact of missense mutations (n=10) located at the above-mentioned domains on

KDM6A function. A, Alanine substitution scanning was used to examine the effect of KDM6A

interaction with histone 3 (H3) peptide. p.N1087 and p.M1129 KDM6A mutants demonstrated

changes in free energy of binding. Therefore, we performed in-depth mutation modeling and

substrate docking simulation analysis on the crystal structure of KDM6A-H3 complex with these

two mutations. B, Both mutants (p.N1087I and p.M1129I) showed substantial increased free

energy of binding relative to the wildtype KDM6A structure, suggesting that the binding

between KDM6A mutants and H3 is unstable. C, Both p.M1129I and p.N1087I demonstrated a

decrease in the number of hydrogen bonds, indicating that the binding affinity between KDM6A

mutants and H3 is reduced. D, p.M1129I and p.N1087I are located at the binding region of

KDM6A with H3 peptide. For both mutants, their respective atomic distance at the mutated

position relative to H3 peptide increased, which may affect KDM6A demethylase function. E,

The decrease in distance between H3 to the L1127 position in p.N1087I and p.M1129I mutants,

indicating a change in the orientation of H3 with the central cavity of JmjC -barrel in KDM6A.

F, Compared to human wildtype KDM6A structure, the backbone root-mean-square deviation

(RMSD) plot showed increased structural fluctuation in p.N1087I and p.M1129I, indicating

overall structural instability in both mutants. Together, the data provide a structural basis for the

negative impact of the two missense mutants on KDM6A demethylase function.

Supplementary Figure 2. The expression of KDM6A in urothelial bladder carcinoma with

wild-type or mutated KDM6A. mRNA expression of KDM6A quantified by quantitative real-

time PCR (qRT-PCR). *, p-value < 0.0001.

Supplementary Figure 3. H score of EZH2 in KDM6A-mutated or KDM6A–wild-type

primary tumors. The bladder tumors are stratified into ‘Wildtype KDM6A’ (n= 52) and ‘Mutant

KDM6A’ (n= 46) groups according to their KDM6A gene status. Scatter plot shows H3K27me3

protein expression of the FFPE sections as represented by the H-score; ns, not significant.

Supplementary Figure 4. The effects of KDM6A alterations in urothelial bladder carcinoma

cell lines. Parental RT-4 cells and KDM6A knockout isogenic cells (RT-4 KO1 and RT-4 KO2),

parental KU-19-19 cells, and KDM6A re-expressing cells (Clone 1 and Clone 2) were subjected

to BrDu assays to test their respective basal proliferation activity. Data are represented as mean ±

SD from three biological replicates. *, p-value = 0.0091 for RT-4 KO1; p-value = 0.0009 for RT-

4 KO2.

Supplementary Figure 5. The effects of EZH2 inhibitors and cytotoxic therapeutic drugs

on KDM6A-null/inactivated or KDM6A–wild-type bladder cancer cell lines.

KDM6A-null or KDM6A–wild type bladder cancer cell lines were treated with (A) EZH2

inhibitors (GSK126 and EPZ6438) or (B) gemcitabine or (C) cisplatin. The proliferation relative

to respective vehicle-treated control over a period of 6 days (GSK126 and EPZ6438) or 3 days

(gemcitabine and cisplatin) were plotted for each cell line. Data are represented as mean ± SD

from three biological replicates.

Supplementary Figure 6. The IC50 of GSK126 and EPZ6438 in cells with and without

KDM6A expression.

KDM6A-null KU-19-19 and KDM6A re-expressing cells, KDM6A–wild-type RT-4 cell line, and

KDM6A knockout clones, UM-UC-1 and EJ-138 were treated with GSK126 (0-10 M) or

EPZ6438 (0-10 mM) or vehicle control for 6 days before being subjected to BrDu assays. Dose-

response curves were fitted, and IC50 of GSK126 and EPZ6438 were determined by curve

fitting.

Supplementary Figure 7. Transcriptional profiling of KDM6A-mutated or KDM6A–wild-

type primary tumors and GSK343-treated KDM6A-null or KDM6A–wild-type cell lines.

Heatmap representation of GSK343-associated changes in pathway gene expression in either

RT-4 (KDM6A–wild-type) or KU-19-19 (KDM6A-null) cell lines over a period of 7 or 14 days

(left, three biological replicates). Heatmap representation of KDM6A mutation-associated

changes (right, 10 KDM6A-mutated tumors compared with 10 KDM6A–wild-type tumors) in

pathway gene expression. Blue indicates down-regulated pathways, whereas red indicates up-

regulated pathways. FDR < 0.10.

Supplementary Figure 8. The effects of EZH2 inhibitors on body weight. Average body weight measurements of KDM6A-null and KDM6A–wild-type patient-derived

xenograft models during treatment with either vehicle (20% Captisol) or 100 mg/kg/day

GSK503. Data are represented as percentage of body weight at the start of GSK503 dosing.

Supplementary Figure 9. Sanger sequencing traces of KDM6A-inactivating mutation in

knockout clones of RT-4 cell line.

Sequencing trace chromatograms of KDM6A indels in RT-4 KO1 and RT-4 KO2 cell lines. The

horizontal red or green arrow shows the direction of genomic DNA sequencing. The mutant

sequences show homozygous frame-shifting mutations in both clones (highlighted in red boxes).

Supplementary Table 1

Clinicopathologic characteristics of 176 urothelial bladder carcinoma patients

No Sample ID Tumor type Gender Age, y TNM Stage

1 55561884 MI UBC M 66 T4N2M0

2 85262131 NMI UBC M 76 T1N0M0

3 48647323 NMI UBC M 69 TisN0M0

4 43368963 NMI UBC F 42 TaN0M0

5 91161285 NMI UBC M 76 T1N0M0

6 39168035 NMI UBC M 62 T1N0M0

7 61487606 MI UBC M 73 T2NXM1

8 68096177 MI UBC M 61 T2N0M0

9 97917945 NMI UBC M 69 TaN0M0

10 33324197 NMI UBC M 80 TaN0M0

11 18997671 MI UBC M 82 T2N0M0

12 35177468 NMI UBC M 61 T1N0M0

13 77067111 MI UBC M 55 T2N0M0

14 92130677 MI UBC M 83 T2N0M0

15 31085175 NMI UBC M 68 T1N0M0

16 27977057 NMI UBC F 65 T1N0M0

17 12960991 NMI UBC M 69 T1N0M0

18 44442154 MI UBC M 63 T2N0M0

19 90868356 MI UBC M 53 T2N0M0

20 17475125 NMI UBC M 80 TaN0M0

21 00929697 NMI UBC M 43 T1N0M0

22 42011796 NMI UBC M 84 TaN0M0

23 01828434 MI UBC F 67 T2N0M0

24 00980134 MI UBC F 55 T2NXMX

25 Z1229 MI UBC M 74 T2N0M0

26 26836983 NMI UBC M 57 T1N0M0

27 69024895 NMI UBC M 70 T1N0M0

28 81878157 NMI UBC F 59 TaN0M0

29 84591949 MI UBC M 67 T2N1M0

30 49738784 MI UBC M 52 T3N2M0

31 96739451 NMI UBC M 64 TaNXMX

32 39565985 NMI UBC F 79 T1NXM0

33 64694706 MI UBC M 56 T2N0M0

34 88064942 NMI UBC F 76 TaN0M0

35 81819543 NMI UBC M 74 TaN0M0

36 03721368 MI UBC M 72 T2N0M0


37 30163859 NMI UBC M 46 TaN0M0

38 98249921 NMI UBC M 61 TaN0M0

39 66839848 NMI UBC M 71 T1N0M0

40 44702557 NMI UBC M 79 TisN0M0

41 23182620 MI UBC F 60 T3bN0M0

42 09640870 NMI UBC F 75 TaN0M0

43 56320093 MI UBC M 75 T2N0M0

44 Z1085 MI UBC M 68 T3NXM0

45 Z1128 MI UBC M 63 T2N0M0

46 Z1360 NMI UBC M 56 T1N0M0

47 Z1837 MI UBC M 69 T2N0M0

48 Z1750 MI UBC M 76 T2NXM0

49 Z1658 MI UBC F 67 T2N0M0

50 Z2225 MI UBC M 60 T2N0M0

51 Z2492 NMI UBC M 65 T1N0M0

52 TCC'B-5 NMI UBC F 56 Ta

53 TCC'B-6 MI UBC M 76 T2

54 TCC'B-9 NMI UBC M 62 T1

55 TCC'B-11 MI UBC F 70 T3a

56 TCC'B-15 NMI UBC F 34 T1


58 TCC'B-17 NMI UBC F 80 T1NXMX


60 TCC'B-33 NMI UBC M 50 Ta









69 TCC'B-100 MI UBC F 94 T2






75 TCC'B-106 MI UBC F 46 T2bN1

76 TCC'B-109 MI UBC M 57 T3N2MX


77 TCC'B-110 MI UBC F 77 T2a

78 TCC'B-111 MI UBC M 60 T4N0MX





83 TCC'B-116 MI UBC F 81 T2










93 TCC'B-130 NMI UBC M 76 T1N0M0



96 TCC'B-133 MI UBC M 77 T4a



99 TCC'B-136 MI UBC M 79 T4N2M0




103 TCC'B-142 MI UBC M 80 T3


105 TCC'B-144 MI UBC F 56 T2

106 TCC'B-145 MI UBC F 83 T2

107 TCC'B-146 MI UBC F 73 T2N0M0

108 TCC'B-147 NA F 76 NA

109 TCC'B-148 MI UBC M 69 T3bN1M0




113 TCC'B-153 NA M 69 NA

114 TCC'B-154 NA M 55 NA

115 TCC'B-155 MI UBC F 74 T3N0MX



117 TCC'B-157 NA M 76 NA

118 TCC'B-158 MI UBC M 77 T3N1


120 TCC'B-161 NMI UBC F 69 T1N0M0

121 TCC'B-162 MI UBC F 84 T3N2MX

122 TCC'B-163 NA M 63 NA

123 TCC'B-165 NA M 76 NA


125 TCC'B-168 MI UBC M 74 T2



128 TCC'B-173 NA F 45 NA



131 TCC'B-176 MI UBC M 65 T3bN2MX








139 TCC'B-185 NMI UBC F 79 TaN0M0


141 TCC'B-189 MI UBC M 65 T2b

142 TCC'B-190 MI UBC M 73 T2aN0M0






148 TCC'B-197 MI UBC M 60 T2


150 TCC'B-199 MI UBC F 74 T2


152 TCC'B-201 MI UBC M 64 T2














165 TCC'B-216 MI UBC F 69 T2





170 TCC'B-221 MI UBC F 65 T2




174 TCC'B-228 MI UBC F 72 T3bN0MX



MI UBC, muscle-invasive urothelial bladder carcinoma; NMI UBC, non-muscle-invasive

urothelial bladder carcinoma


KDM6A mutations in a cohort of 176 urothelial bladder carcinoma patients

Sample ID KDM6A nucleotide change

KDM6A amino

acid change

KDM6A

mutation type

85262131 c.884C>G p.S295X Nonsense

48647323 c.2509A>CC p.837fs9X Indel

43368963 c.1610C>G p.S537X Nonsense

91161285 c.1679_1689delCTGCCTGCCCT p.560fs17X Indel

61487606 c.1663C>T p.Q555X Nonsense

33324197 Exon 21 splice site -2A>T splice site Indel

92130677 c.4120delG p.1374fs2X Indel

31085175 c.3304G>A p.E1102K Missense

17475125 c.1875delC p.625fs66X Indel

42011796 c.3028G>T p.E1010X Nonsense

*Z1229 c.857_858CC>TG

c.872delG

p.S286L

p.291fs34X Missense

Indel

96739451 c.1906_1909delCTAT p.636fs54X Indel

81819543 c.1906_1909delCTAT p.636fs54X Indel

66839848 c.514C>T p.R172X Nonsense

44702557 c.2587C>T p.Q863X Nonsense

23182620 c.3746A>G p.Y1249C Missense

Z1750 c.1663C>T p.Q555X Nonsense

Z1658 c.3952_3953insA p.1318fs15X Indel

TCC'B-6 c.2414C>T p.P805L Missense

*TCC'B-11 c.446C>T

c.4127_4146delAACAGTACAAAATGGA

GGAC

p.A149V

p.1376fs6X

Missense

Indel

TCC'B-94 Exon 9 splice side -2A>G splice site Indel

*TCC'B-98 c.1193A>C

c.1682C>T

Exon 19 splice side -1G>T

Exon26 splice site -2A>T

p.Q398P

p.A561V

splice site

splice site

Missense

Missense

Indel

Indel

TCC'B-100 c.1516C>T p.Q506X Nonsense

TCC'B-101 c.3478-3479GG>TT p.G1160F Missense

TCC'B-104 c.2363C>T p.A788V Missense

TCC'B-110 c.2471C>G p.S824X Nonsense

TCC'B-113 c.4124T>C p.L1375P Missense

TCC'B-114 c.2357delG p.786fs81X Indel

*TCC'B-115 c.2897C>T

c.3110_3119delAGTACCAGGC

p.P966L

p.1037fs8X

Missense

Indel


KDM6A amino

acid change

KDM6A

mutation type

TCC'B-122 c.4072T>G p.C1358G Missense

TCC'B-123 c.2021_2025delCTGGT p.674fs12X Indel

*TCC'B-124 c.35C>T

c.3397C>T

p.A12V

p.Q1133X

Missense

Nonsense

*TCC'B-130 Exon 7 splice side -2A>T

Exon 21 splice side -1G>A

c.3145G>A

splice site

splice site

p.E1049K

Indel

Indel

Missense




*TCC'B-136 c.2126A>T

exon 27 splice side -2A>T

p.Q709L

splice site

Missense

Indel

TCC'B-137 c.2872C>A

c.3260-3261AT>TC

p.Q958K

p.N1087I

Missense

Missense

TCC'B-138 c.1654A>T p.S552C Missense

TCC'B-139 c.379_380insT p.127 f.s Indel

TCC'B-143 Exon 23 splice site -1 G>A splice site Indel

TCC'B-150 c.3029A>G p.E1010G Missense

*TCC'B-153 c.1570C>A

c.3425_exon23+19delAACACCAGGTAA

TTTGTATGAACTAAC

p.P524T

p.1143fs5X

Missense

Indel

TCC'B-154 c.1667_1668insTCCA p.556fs25X Indel

*TCC'B-155 c.807delT

c.2899G>T

c.2938G>A

c.2943delG

c.3415delC

c.4000T>A

p.270fs55X

p.V967F

p.D980N

p.982fs17X

p.1139fs19X

p.C1334S

Indel

Missense

Missense

Indel

Indel

Missense


TCC'B-161 c.608C>A p.S203Y Missense

TCC'B-162 c.2174T>A p.L725X Nonsense

TCC'B-166 c.3182C>G p.S1061X Nonsense

*TCC'B-168 c.151G>A

c.2872C>A

p.G51R

p.Q958K

Missense

Missense

*TCC'B-174 exon 14 splice side -2A>G

c.3408_3409insA

splice site

1137fs14X

Indel

Indel

TCC'B-175 exon 18 splice side -1G>T splice site Indel

*TCC'B-177 c.3793A>T

c.3008delA

p.K1265X

p.1003fs45X

Nonsense

Indel


KDM6A amino

acid change

KDM6A

mutation type

TCC'B-179 c.1689delT p.565fs28X Indel

*TCC'B-181 c.673A>T

exon 12 splice site -1G>A

p.K225X

splice site

Nonsense

Indel

TCC'B-184 c.4141G>T p.E1381X Nonsense

TCC'B-192 exon 11 splice site +1_+2 insGG splice site Indel

*TCC'B-193 c.1489C>T

c.1663C>T

c.3387G>A

p.Q497X

p.Q555X

p.M1129I

Nonsense

Nonsense

Missense

*TCC'B-197 c.1054G>A

c.1111G>A

p.A352T

p.A371T

Missense

Missense

TCC'B-202 c.3308T>C p.L1103P Missense

TCC'B-203 c.2039_2069

delACGGACATCCCACCCTGCCTAGCA

ATTCAGT

p.680fs25X Indel


TCC'B-209 exon 4 splice side -2A>T splice site Indel

TCC'B-214 c. 1067_1068insT p.356fs8X Indel

TCC'B-215 c.3409A>T p.K1137X Nonsense



*TCC'B-226 c.2047delC

c.3017delA

p.684fs7X

p.1006fs42X

Indel

Indel

*, samples with multiple KDM6A mutations


KDM6A mutations in female urothelial carcinoma patients


KDM6A

amino acid

change

KDM6A

mutation

type Zygosity

43368963 c.1610C>G p.S537X Nonsense Heterozygous

23182620 c.3746A>G p.Y1249C Missense Heterozygous

Z1658 c.3952_3953insA p.1318fs15X Indel Heterozygous

*TCC'B-11 c.446C>T

c.4127_4146delAACAGTA

CAAAATGGAGGAC

p.A149V

p.1376fs6X

Missense

Indel

Heterozygous

Heterozygous

*TCC'B-98 c.1193A>C

c.1682C>T

Exon 19 splice side -1G>T

Exon26 splice site -2A>T

p.Q398P

p.A561V

splice site

splice site

Missense

Missense

Indel

Indel

Heterozygous

Heterozygous

Heterozygous

Heterozygous

TCC'B-100 c.1516C>T p.Q506X Nonsense Heterozygous

TCC'B-110 c.2471C>G p.S824X Nonsense Heterozygous

TCC'B-143 Exon 23 splice site -1 G>A splice site Indel Heterozygous

TCC'B-150 c.3029A>G p.E1010G Missense Heterozygous

*TCC'B-155 c.807delT

c.2899G>T

c.2938G>A

c.2943delG

c.3415delC

c.4000T>A

p.270fs55X

p.V967F

p.D980N

p.982fs17X

p.1139fs19X

p.C1334S

Indel

Missense

Missense

Indel

Indel

Missense

Homozygous

Heterozygous

Heterozygous

Heterozygous

Heterozygous

Heterozygous

TCC'B-156 c.1646delG p.549fs44X Indel Heterozygous

TCC'B-161 c.608C>A p.S203Y Missense Homozygous

TCC'B-162 c.2174T>A p.L725X Nonsense Heterozygous

*TCC'B-174 exon 14 splice side -2A>G

c.3408_3409insA

splice site

1137fs14X

Indel

Indel

Heterozygous

Heterozygous

*TCC'B-177 c.3793A>T

c.3008delA

p.K1265X

p.1003fs45X

Nonsense

Indel

Heterozygous

Heterozygous

*TCC'B-181 c.673A>T

exon 12 splice site -1G>A

p.K225X

splice site

Nonsense

Indel

Heterozygous

Heterozygous

TCC'B-214 c. 1067_1068insT p.356fs8X Indel Heterozygous

TCC'B-215 c.3409A>T p.K1137X Nonsense Heterozygous

TCC'B-223 c.1663C>T p.Q555X Nonsense Heterozygous

*TCC'B-226 c.2047delC

c.3017delA

p.684fs7X

p.1006fs42X

Indel

Indel

Heterozygous

Heterozygous

*, samples with multiple KDM6A mutations


Differentially expressed gene sets comparing KDM6A-mutated versus KDM6A–wild-

type bladder tumors

NAME NES NOM

p-val

FDR

q-val

SIZE

KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION -5.152 0.000 0.000 98

KEGG_COMPLEMENT_AND_COAGULATION_CASCADES -4.224 0.000 0.000 30

KEGG_ECM_RECEPTOR_INTERACTION -4.173 0.000 0.000 53

KEGG_HEMATOPOIETIC_CELL_LINEAGE -4.011 0.000 0.000 42

EZH2 INHIBITOR_RELATED_PATHWAY -3.524 0.000 0.000 106

KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION -3.316 0.000 0.000 37

KEGG_DNA_REPLICATION -2.983 0.000 0.000 33

KEGG_CELL_CYCLE -2.839 0.000 0.000 110

KEGG_FOCAL_ADHESION -2.830 0.000 0.000 145

KEGG_CHEMOKINE_SIGNALING_PATHWAY -2.823 0.000 0.000 113

KEGG_ARRHYTHMOGENIC_RIGHT_VENTRICULAR_CARDIO

MYOPATHY_ARVC

-2.755 0.000 0.000 36

KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS -2.750 0.000 0.000 40

KEGG_CELL_ADHESION_MOLECULES_CAMS -2.672 0.000 0.000 61

KEGG_REGULATION_OF_ACTIN_CYTOSKELETON -2.567 0.000 0.001 144

KEGG_JAK_STAT_SIGNALING_PATHWAY -2.554 0.000 0.001 70

KEGG_LEISHMANIA_INFECTION -2.523 0.000 0.001 46

KEGG_PROGESTERONE_MEDIATED_OOCYTE_MATURATIO

N

-2.479 0.000 0.001 59

KEGG_DILATED_CARDIOMYOPATHY -2.452 0.000 0.001 38

KEGG_PRION_DISEASES -2.451 0.000 0.001 24

KEGG_MAPK_SIGNALING_PATHWAY -2.416 0.000 0.002 168

KEGG_VASCULAR_SMOOTH_MUSCLE_CONTRACTION -2.393 0.002 0.002 64

KEGG_HYPERTROPHIC_CARDIOMYOPATHY_HCM -2.316 0.000 0.004 36

KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICIT

Y

-1.993 0.004 0.024 72

KEGG_GLIOMA -1.985 0.004 0.024 48

KEGG_OOCYTE_MEIOSIS -1.976 0.006 0.025 81

KEGG_FC_GAMMA_R_MEDIATED_PHAGOCYTOSIS -1.962 0.010 0.025 77

KEGG_INTESTINAL_IMMUNE_NETWORK_FOR_IGA_PRODU

CTION

-1.907 0.008 0.035 18

KEGG_SMALL_CELL_LUNG_CANCER -1.896 0.008 0.035 67

KEGG_PANCREATIC_CANCER -1.866 0.014 0.042 61

KEGG_CHRONIC_MYELOID_LEUKEMIA -1.827 0.014 0.049 63

KEGG_MELANOMA -1.762 0.029 0.069 43

KEGG_RENAL_CELL_CARCINOMA -1.719 0.018 0.084 60

KEGG_LEUKOCYTE_TRANSENDOTHELIAL_MIGRATION -1.700 0.039 0.089 72

NAME NES NOM

p-val

FDR

q-val

SIZE

KEGG_GAP_JUNCTION -1.694 0.028 0.089 54

KEGG_LONG_TERM_POTENTIATION -1.692 0.039 0.088 42

KEGG_NEUROTROPHIN_SIGNALING_PATHWAY -1.691 0.027 0.085 93

KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_CHONDROI

TIN_SULFATE

-1.684 0.030 0.086 15

KEGG_GRAFT_VERSUS_HOST_DISEASE -1.680 0.022 0.085 13

KEGG_PROSTATE_CANCER -1.677 0.023 0.085 71

KEGG_NUCLEOTIDE_EXCISION_REPAIR -1.644 0.045 0.096 37

KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY -1.632 0.041 0.098 75

KEGG_RIBOSOME 5.571 0.000 0.000 77

KEGG_OXIDATIVE_PHOSPHORYLATION 3.296 0.000 0.000 97

KEGG_PARKINSONS_DISEASE 3.135 0.000 0.000 93

KEGG_HUNTINGTONS_DISEASE 2.826 0.000 0.000 138

KEGG_STEROID_HORMONE_BIOSYNTHESIS 2.201 0.002 0.018 13

KEGG_ALZHEIMERS_DISEASE 2.194 0.000 0.016 121

KEGG_DRUG_METABOLISM_CYTOCHROME_P450 2.185 0.004 0.015 22

KEGG_METABOLISM_OF_XENOBIOTICS_BY_CYTOCHROME

_P450

2.021 0.002 0.036 23

KEGG_VALINE_LEUCINE_AND_ISOLEUCINE_DEGRADATIO

N

1.990 0.002 0.040 39

KEGG_CARDIAC_MUSCLE_CONTRACTION 1.871 0.017 0.072 35

Supplementary Table 5.

Enrichment of PRC2 transcriptional repression in urothelial bladder tumors with mutated

KDM6A

Gene sets NES NOM p-val FDR q-val

MEISSNER_BRAIN_HCP_WITH_H3K4ME3_AND_H3K27ME3

(71)

-6.71 < 0.001 < 0.001

NUYTTEN_EZH2_TARGETS_UP (72) -5.35 < 0.001 < 0.001

BENPORATH_SUZ12_TARGETS (73) -4.71 < 0.001 < 0.001

BENPORATH_ES_WITH_H3K27ME3 (73) -4.45 < 0.001 < 0.001

BENPORATH_EED_TARGETS (73) -3.75 < 0.001 < 0.001

BENPORATH_PRC2_TARGETS (73) -3.27 < 0.001 < 0.001


Effects of loss of KDM6A on H3K27me3 levels at specific loci

Comparison Promoter Exon Intron CpG_island Total

KO1 vs

RT-4

H3K27me3_KO1 vs RT-4

unique to KO1 362 338 1040 606 2702


common 5256 5325 23683 4894 61523

KO2 vs

RT-4


unique to KO2 75 73 243 99 646


common 5094 5452 21469 5785 56112


Differentially expressed pathways in the GSK343-treated KDM6A-null and KDM6A-

reexpressing cells

KU-19-19: Ctrl versus

GSK343

treatment

Genesets NOM p-val FDR q-val

KEGG_GLYCOSAMINOGLYCAN_BIOSY

NTHESIS_KERATAN_SULFATE < 0.001 < 0.001

KEGG_FATTY_ACID_METABOLISM < 0.001 < 0.001

KEGG_CELL_ADHESION_MOLECULES_

CAMS < 0.001 < 0.001

Clone 1: Ctrl versus

GSK343

treatment



NTHESIS_KERATAN_SULFATE > 0.001 > 0.001

KEGG_FATTY_ACID_METABOLISM > 0.001 > 0.001


CAMS > 0.001 > 0.001

Clone 2: Ctrl versus

GSK343

treatment



NTHESIS_CHONDROITIN_SULFATE > 0.001 > 0.001

KEGG_FATTY_ACID_METABOLISM > 0.001 > 0.001


CAMS > 0.001 > 0.001


Differentially expressed cell adhesion molecules in KDM6A-mutated versus KDM6A–wild-

type bladder tumors

Gene ID

Rank

Metric

Score

GSEA Core

Enrichment

CDH2 -0.66295737 YES

NLGN2 -0.701846063 YES

CNTN1 -0.713806152 YES

NRXN2 -0.737018645 YES

ITGB7 -0.742584467 YES

CNTNAP2 -0.792684793 YES

SDC3 -0.816892862 YES

ICOSLG -0.86352849 YES

CD40LG -0.929691851 YES

CD34 -0.998430312 YES

CD226 -1.012130499 YES

NRXN1 -1.045967102 YES

CD276 -1.059631824 YES

NRCAM -1.074645877 YES

CD4 -1.100297451 YES

CD80 -1.104243517 YES

PDCD1 -1.113559008 YES

ESAM -1.209498405 YES

CDH5 -1.266846418 YES

ITGA9 -1.269753456 YES

CD274 -1.282839656 YES

CNTNAP1 -1.330945492 YES

ICAM3 -1.361379623 YES

CADM3 -1.426323771 YES

CD8B -1.429209828 YES

CLDN2 -1.548209071 YES

NFASC -1.557807684 YES

CD8A -1.626329064 YES

PDCD1LG2 -1.633134723 YES

MPZ -1.6700629 YES

MADCAM1 -1.682909012 YES

ITGB8 -1.716550231 YES

JAM3 -1.753076434 YES

JAM2 -1.753827929 YES

Gene ID Rank Metric GSEA Core

Score Enrichment

SPN -1.879507184 YES

ICOS -1.920187354 YES

SDC2 -1.929414749 YES

NEGR1 -2.037558079 YES

ITGA8 -2.269280434 YES

CLDN5 -2.274779081 YES

CD86 -2.290999651 YES

CLDN11 -2.333554268 YES

CD28 -2.38535285 YES

HLA-DRB5 -2.455762386 YES

SIGLEC1 -2.49187398 YES

CD22 -2.507254124 YES

CD6 -2.528774738 YES

ITGA4 -2.594025135 YES

ITGAM -2.629947662 YES

CD2 -2.746627808 YES

ICAM1 -2.841643095 YES

CTLA4 -2.924205065 YES

HLA-DRB1 -2.998133659 YES

ITGAL -3.063920975 YES

HLA-DMA -3.086145878 YES

SELPLG -3.111727476 YES

SELE -3.140482426 YES

VCAM1 -3.244471788 YES

SELP -3.27270174 YES

ITGB2 -3.430675745 YES

PTPRC -3.604540586 YES

VCAN -3.689659834 YES

SELL -4.167525291 YES

SDC1 -7.007856846 YES


Differentially expressed DNA replication molecules in KDM6A-mutated versus KDM6A–

wild-type bladder tumors

Gene ID

Rank Metric

Score

GSEA Core

Enrichment

RFC1 -0.183349654 YES

POLD2 -0.214434788 YES

RPA4 -0.216803744 YES

RNASEH1 -0.344447553 YES

RNASEH2B -0.386798501 YES

PRIM2 -0.398551643 YES

POLE4 -0.559066117 YES

POLA1 -0.56798166 YES

RFC3 -0.656368136 YES

DNA2 -0.684004068 YES

RFC5 -0.772996128 YES

POLD3 -0.801892161 YES

POLA2 -0.849106669 YES

MCM7 -0.891861737 YES

MCM3 -0.929967165 YES

MCM4 -1.009983301 YES

RPA3 -1.031189799 YES

PCNA -1.074128509 YES

LIG1 -1.186130881 YES

MCM5 -1.281409025 YES

RNASEH2A -1.374159694 YES

POLD1 -1.428000927 YES

POLE -1.45439291 YES

PRIM1 -1.544605851 YES

POLE2 -1.679383039 YES

MCM6 -2.043511629 YES

RFC4 -2.092530489 YES

FEN1 -2.197273731 YES

MCM2 -2.54959774 YES


GSK343-induced PRC2-related genes in cells with KDM6A loss

Gene ID Day 7

log2FC

Day 14

log2FC

Day 7 GSEA

core enrichment

Day 14 GSEA core

enrichment

IGFBP3 1.378 1.738 Yes Yes

SLIT3 1.367 1.553 Yes Yes

DHRS9 1.059 1.954 Yes Yes

PAPPA 1.001 NA Yes NA

FLRT3 0.965 0.546 Yes Yes

GDF15 0.840 -0.188 Yes No

CEACAM1 0.830 1.151 Yes Yes

SRPX2 0.704 0.654 Yes Yes

ST3GAL6 0.679 1.016 Yes Yes

TSPAN8 0.645 1.040 Yes Yes

IL8 0.641 -0.256 Yes No

PIR 0.583 0.573 Yes Yes

FOXA1 0.542 0.410 Yes Yes

ULBP2 0.496 0.206 Yes Yes

RDH10 0.493 0.858 Yes Yes

TNFAIP3 0.492 0.400 Yes Yes

SLC2A13 0.463 0.778 Yes Yes

PPIC 0.458 0.718 Yes Yes

MSX2 0.421 0.700 Yes Yes

LBH 0.392 NA Yes NA

SLC16A7 0.344 0.544 Yes Yes

GBP2 0.343 0.983 Yes Yes

SQSTM1 0.342 0.220 Yes Yes

TNFSF9 0.331 0.473 Yes Yes

MOSPD1 0.328 0.047 Yes No

INHBA 0.323 -0.009 Yes No

UST 0.322 0.338 Yes Yes

ASAH1 0.318 0.488 Yes Yes

FAM89A 0.312 0.207 Yes Yes

SERPINE2 0.306 -0.103 Yes No

NPAS2 0.304 -0.094 Yes No

CDKN1A 0.294 -0.048 Yes No

UBE2E2 0.292 0.219 Yes Yes

ENTPD3 0.288 0.213 Yes Yes

BACE2 0.287 0.575 Yes Yes

Gene ID Day 7

log2FC

Day 14

log2FC

Day 7 GSEA

core enrichment

Day 14 GSEA core

enrichment

FGFBP1 0.283 0.349 Yes Yes

CAMK2N1 0.274 NA Yes NA

BLVRB 0.273 0.121 Yes No

GULP1 0.269 NA Yes NA

JAZF1 0.269 0.363 Yes Yes

DDIT3 0.231 -0.652 Yes No

STX3 0.196 0.051 Yes No

ST3GAL1 0.195 0.432 Yes Yes

RAB32 0.183 0.081 Yes No

TMEM140 0.169 0.226 Yes Yes

CDH7 0.162 0.287 Yes Yes

RHOQ 0.154 0.062 Yes No

PTPRR 0.150 0.051 Yes No

FUCA1 0.149 0.162 Yes No

GATM 0.085 0.238 No Yes

MAF 0.050 0.354 No Yes

HMGCS1 -0.105 0.314 No Yes

LSS -0.179 0.477 No Yes

COBLL1 NA 0.525 NA Yes

OAS1 NA 1.298 NA Yes

PTGS2 NA 0.572 NA Yes

TMEM47 NA 0.746 NA Yes


KDM6C/UTY copy number in urothelial bladder carcinoma patients

No Sample ID Tumor type Gender

KDM6C/UTY

copy number

KDM6A

mutation type

1 17475125 NMI UBC M 0 Indel


3 85262131 NMI UBC M 0 Nonsense


5 Z1750 MI UBC M 0 Nonsense

6 00929697 NMI UBC M 0 Wildtype

7 03721368 MI UBC M 0 Wildtype




11 92130677 MI UBC M 1 Indel


13 61487606 MI UBC M 1 Nonsense



16 31085175 NMI UBC M 1 Missense

17 *Z1229 MI UBC M 1 Missense

Indel


















No Sample ID Tumor Type Gender KDM6C/UTY

copy number

KDM6A

mutation type


36 Z1128 MI UBC M 1 Wildtype

37 Z1360 NMI UBC M 1 Wildtype



40 Z2492 NMI UBC M 1 Wildtype


42 Z1658 MI UBC F 0 Indel

43 43368963 NMI UBC F 0 Nonsense

44 23182620 MI UBC F 0 Missense

45 01828434 MI UBC F 0 Wildtype

46 00980134 MI UBC F 0 Wildtype

47 81878157 NMI UBC F 0 Wildtype






Primers for PCR amplification of KDM6A full-length coding sequence

Gene ID Location Primer sequence (5' → 3')

KDM6A Exon 1-2 F: GAGGAGGAGGCGGCGATAAAGTTG

R: GAGAGCAGAGGCAGGAAATAGTTTC

Exon 3 F: GACTTCTTAGGTGATCGAATGGAG

R: GTTTGCTCATGCACTTACAATTTC

Exon 4 F: CTAGATGCTGTTCAAGTGCAGATC

R: CAAGGAGAGTCATACAAGACAGCAC

Exon 5 F: GTTGATTGGAATCTGTGCCATCTG

R: CACCCTTCAAGGAACAATCTAATC

Exon 6 F: GATTAAACCATGAAATACTGTCTGTTTG

R: CAGTTGATAAACAGTTCTGTCACCTG

Exon 7 F: CAAAGTATTTCTAGTTGGTAGGCTAGTG

R: CATGGCAACCAATACAGTTACATTAAAC

Exon 8 F: CATGATTGTGTTCTTTAGTGTGTTTG

R: CAAGCACTCCTTGGAATAATTG

Exon 9 F: GAAACATTCAATAATGGAATCAGCAC

R: GAGGATACAAATATGCTGGGTTC

Exon 10 F: CTCTTGAATGAAAGCCCTTTGTAG

R: CACCATATTTGGAAACTTTATCTTGTTG

Exon 11 F: CAAGCTGTATAAGAAGAGTGGAAGAGTG

R: CTGCTGATATAGCACACTGGAAAG

Exon 12 F: GTGTTCAATAGGGTAAGCCTTTG

R: CACAACTAGACAAACTTTCCAGCCAC

Exon 13 F: CACAAAGGTTTATATTCCGGTTAC

R: GCCTCCTGTGCTTAGTAAATTAAG

Exon 14 F: CATAGTCATTTGGCCTCCTCTAAC

R: GAGGCTGAGGCACAAGAATAAC

Exon 15 F: GTTTGACCAGATAGTGGTTCTGAG

R: CTAAATTCTGTCAAATACCAGTAGAAAG

Exon 16 F: CTCAACTTAGAGAAATTAAGCATTTG

R: CAATGTAGTAACATTGTAGTGCCAC

Exon 17 F: CTTGGGTCAAATTATCTTATACAGTTAG

R: CTGGGTGGATGTTATTGACTTTG

Exon 17 F: CACTGGAGAGACACCTAACAGCAC

R: CAAACTCTTAGATGAATGACTACACCATC

Exon 18 F: CATGGACTTGTGCAAATGCCTAG

R: CTGCTGCCAATAATTGTAATGTTTC


Exon 19 F: GAAAGGAGAAATGTATGTGGTTACTATCTG

R: GTGATACACACACTCTCTCTCATTCTTAG

Exon 20 F: CTCAGGTTGTGCAGAGGCCCTAG

R: CATACGAGACAACTGGAGAACAC

Exon 21-22 F: CAACAGGAGTCAAACCTTGTCTC

R: GACAATAAATGAAACAGAAGAGGAAATTG

Exon 23 F: GAAGAAATGGTAAACTTCCACAGGTATTTG

R: GAACTTATTTCCCAGTGGTGCTCC

Exon 24 F: GTGTTCTCTGTTGAGCATTTGTAAG

R: CTTCGCTGAATGGTAAGTGAATAC

Exon 25 F: GAGCTTCTTAATGTAGTTGATCCATTTG

R: CAATTCTATGCAAGGAGTCATTCTTCTTAC

Exon 26 F: CACCTGAGCAGGTGATAATGGTTATC

R: GAAAGAAGCACAGGTCTGTGACTC

Exon 27 F: GAAGTCATAGACATTAGAATCAAGTCTC

R: CACAGTGAAATATCATTATTATCACACTG

Exon 28 F: CTCTTAACCAGAGATCACTGTCCAC

R: GAATGATATGAATATACCCTCATGC

Exon 29 F: GATTCTTAGGAAGATTGGCTGAATG

R: CTGAAGTACAGCTCATCAGCTTTG

F, forward; R, reverse.


Primers for Sanger sequencing of KDM6A full-length coding sequence


KDM6A Exon 1-2 R: GAGAGCAGAGGCAGGAAATAGTTTC

Exon 3 F: GACTTCTTAGGTGATCGAATGGAG

R: GTTTGCTCATGCACTTACAATTTC

Exon 4 F: CTAGATGCTGTTCAAGTGCAGATC

R: CAAGGAGAGTCATACAAGACAGCAC

Exon 5 F: GTTGATTGGAATCTGTGCCATCTG

R: CACCCTTCAAGGAACAATCTAATC

Exon 6 F: GATTAAACCATGAAATACTGTCTGTTTG

R: CAGTTGATAAACAGTTCTGTCACCTG

Exon 7 F: CAAAGTATTTCTAGTTGGTAGGCTAGTG

R: CATGGCAACCAATACAGTTACATTAAAC

Exon 8 F: CATGATTGTGTTCTTTAGTGTGTTTG

R: CAAGCACTCCTTGGAATAATTG

Exon 9 F: GAAACATTCAATAATGGAATCAGCAC

R: GAGGATACAAATATGCTGGGTTC

Exon 10 F: CTCTTGAATGAAAGCCCTTTGTAG

R: CACCATATTTGGAAACTTTATCTTGTTG

Exon 11 F: CTTCCCTTCTCAGGTGCTATTC

R: GTCAACTCAGAAGAACTGCTTGG

Exon 12 F: GTGTTCAATAGGGTAAGCCTTTG

R: CACAACTAGACAAACTTTCCAGCCAC

Exon 13 F: CACAAAGGTTTATATTCCGGTTAC

R: GCCTCCTGTGCTTAGTAAATTAAG

Exon 14 R: CTTGTTTGCTACCTCTACTCC

Exon 15 F: GTTTGACCAGATAGTGGTTCTGAG

R: CTAAATTCTGTCAAATACCAGTAGAAAG

Exon 16 F: CTCAACTTAGAGAAATTAAGCATTTG

R: CACTTCTCTCTTCTTCTCTCAAAGTG

Exon 17 F: CTTGGGTCAAATTATCTTATACAGTTAG

R: CTGGGTGGATGTTATTGACTTTG

Exon 17 F: CACTGGAGAGACACCTAACAGCAC

R: CAAACTCTTAGATGAATGACTACACCATC

Exon 18 F: CATGGACTTGTGCAAATGCCTAG

R: CTGCCAATAATTGTAATGTTTCCTAAAG

Exon 19 F: GAAAGGAGAAATGTATGTGGTTACTATCTG

F: GTCACAGTGATAAGATACTGTCAAATAG


Exon 20 F: CTCAGGTTGTGCAGAGGCCCTAG

R: CGAGACAACTGGAGAACACTTAC

Exon 21-22 F: CAACAGGAGTCAAACCTTGTCTC

R: CAATAAATGAAACAGAAGAGGAAATTG

Exon 23 R: GACAAGATTCTGGCTGTCTTTG

R: GTCTTATAACAGGAACAACTCTCAG

Exon 24 F: GTGTTCTCTGTTGAGCATTTGTAAG

R: GAATGGTAAGTGAATACTGGCAATG

Exon 25 F: GAGCTTCTTAATGTAGTTGATCCATTTG

R: CTATGCAAGGAGTCATTCTTCTTACAAC

Exon 26 F: CACCTGAGCAGGTGATAATGGTTATC

R: GTGGATTCTCATAATACATTCTGCTAGAC

Exon 27 F: GAAGTCATAGACATTAGAATCAAGTCTC

R: CACAGTGAAATATCATTATTATCACACTG

Exon 28 F: CTCTTAACCAGAGATCACTGTCCAC

R: GAATGATATGAATATACCCTCATGC

Exon 29 F: GATTCTTAGGAAGATTGGCTGAATG

R: CTGAAGTACAGCTCATCAGCTTTG

F, forward; R, reverse.

supplementary materials for · 2017-02-17 · supplementary materials for loss of tumor suppressor...

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