www.sciencemag.org/content/355/6331/1324/suppl/DC1

Supplementary Materials for PI3K pathway regulates ER-dependent transcription in breast cancer

through the epigenetic regulator KMT2D Eneda Toska, Hatice U. Osmanbeyoglu,* Pau Castel,* Carmen Chan, Ronald C

Hendrickson, Moshe Elkabets, Maura N. Dickler, Maurizio Scaltriti, Christina S. Leslie, Scott A. Armstrong,† José Baselga†

*These authors contributed equally to this work.†Corresponding author. Email: scott_armstrong@dfci.harvard.edu (S.A.A.); baselgaj@mskcc.org (J.B.)

Published 24 March 2017, Science 355, 1324 (2017) DOI: 10.1126/science.aah6893

This PDF file includes:

Materials and Methods Figs. S1 to S8 Tables S1 to S3 References

Materials and Methods

Human cell lines, transient transfection assays, and lentiviral production

All cell lines were obtained from ATCC and used at low passages. T47D and ZR751

breast cancer cells were maintained in RPMI 1640 with 10% FBS, 1% L-glutamine, and 1%

penicillin-streptomycin. MCF7 and CAMA-1 breast cancer cells were maintained in DF12

DMEM Dulbecco medium with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin.

HEK 293T cells were maintained in DMEM Dulbecco medium supplemented with 10% FBS,

1% L-glutamine and 1% penicillin-streptomycin. For RNA analysis, MCF7 and T47D cells

underwent hormonal starvation in estrogen-deprived media. MCF7 and T47D cells were cultured

for 3 days in phenol-red-free DF12 DMEM and phenol-red-free RPMI respectively, and media

was supplemented with 5% charcoal/dextran-treated FBS. Cells were induced with E2 (estrogen)

(100nM), BYL719 (1µM) or vehicle (DMSO) for 24 hours.

When indicated, MCF7 and T47D cells were transduced with lentiviruses expressing

empty vector (pLKO.1) or shRNA against KMT2D (pLKO.1) from Sigma-Aldrich: shKMT2D

#1: TRCN0000013140; shKMT2D #2: TRCN0000013139; shKMT2D #3 TRCN0000013138; or

shRNA against FOXA1: shFOXA1 #1: TRCN0000014882; shFOXA1 #2: TRCN0000014880;

or shRNA against PBX1: shPBX1 #1: TRCN0000020390; shPBX1 #2: TRCN0000020391.

When indicated, MCF7 cells were transduced with pTRIPZ vectors (TRE-RFP-miR30/shRNA-

UBC-IRES-PURO) targeting PBX1 or FOXA1 knockdown upon doxycycline administration.

When indicated, MCF7 cells were transduced with LT3REPIR (pRRL) vector (T3G-dsRED-

mirE/shRNA-PGK-PURO-IRES-rtTA3) targeting KMT2D knockdown upon doxycycline

administration. For lentiviral production, 293T cells were transfected with pCMV-VSVG,

pCMV-dR8.2, and the plasmid of interest using Lipofectamine 3000 according to the

manufacturer’s protocol.

For transient transfections, HEK 293T cells were transfected with equimolar amounts 1

(µg/ml) of wild type or mutant pCMV-HA-KMT2D plasmids using polyethylenimine. Cells

were collected 48 h after transfection and used for protein extraction or for isolation of

semipurified HA-KMT2D proteins as described below. MCF7 and T47D cells were transiently

transfected with pCMV3-FLAG-KDM1 or pCMV-HA-KMT2D plasmids using either

Lipofectamine 3000 according to the manufacturer’s protocol or Amexa nucleofector (Lonza)

according to the manufacturer’s protocol. T47D cells were transfected with two pooled KMT2D

3’UTR siRNAs (Ambion) using Lipofectamine 3000 according to the manufacturer’s protocol.

Reagents and cell viability

BYL719 was obtained from the Stand Up to Cancer (SU2C) pharmacy. BYL719 was

dissolved in DMSO for in vitro experiments. The MTT assay was used to measure cell viability.

Briefly, 5000 cells were seeded in 96 well plates, treated with BYL719 for 5 days and assayed

using 0.25% MTT (Sigma-Aldrich) and 50 (mM) sodium succinate (Sigma-Aldrich) for 3 hours.

After the formazan crystals were dissolved with DMSO, the absorbance was measured at 570

(nm) of wavelength. Doxycycline was purchased from Sigma-Aldrich.

Immunoblot, immunoprecipitation, and in vitro kinase assay

For immunoblot analysis, RIPA buffer supplemented with protease and phosphatase

inhibitors (Roche) was used to lyse the cell pellets. The supernatant was collected by

centrifugation for 10 minutes at 12,000 (g). Protein lysates were separated using SDS-PAGE

gradient gels (4-12%). The KMT2D probed gels were separated using low percentage SDS-

PAGE gradient gels (3-8%). The gels were transferred to a PVDF membrane for 2 hours at 70

(V). The membranes were probed using specific antibodies. Actin, pAKT (S473), H3 and

phospho-RXRXX(S/T) were from Cell Signaling Technology (CST), HA (clone 3F10) was from

Roche, and V5 was from GenScript. The rabbit KMT2D and pKMT2D (S1331) antibodies were

generated by Eurogentec (third bleed) and affinity purified against GRARLKSTASSIC and

GRARLKS(PO3H2)TASSIC peptides respectively. Histone H3 (mono methyl K4) (ab8895),

histone H3 (di methyl K4) (ab7766), and FOXA1 (ab5089) were from Abcam. PBX1 antibody

(H00005087-MO1) was from Abnova, and ER alpha antibody was (sc-543) from Santa Cruz.

For immunoprecipitation assays, cells were transfected with appropriate plasmids and

48h post transfection, cells were lysed using NP-40 buffer [150 (mM) NaCl, 10 (mM) Tris pH 8,

1% NP-40, 10% glycerol]. Lysates were incubated at 4 (°C) overnight with EZviewTM RED

Anti-HA agarose beads (Sigma-Aldrich) or protein G agarose beads (Thermo Scientific)

followed by the appropriate antibody. The immunocomplexes were washed three times using

NP-40 buffer. For in vitro kinase assay, immunoprecipitated V5-KMT2D (1222-1819 amino

acids) or HA-KMT2D (full length) were used as a substrate in a reaction with recombinant His-

AKT (MRC-PPU Reagents DU1850) and ATP (Signalchem) in kinase buffer [25 (mM) MOPS,

pH 7.2, 12.5 (mM) β-glycerolphosphate, 25 (mM) MgCl2, 5 (mM) EGTA, 2 (mM) EDTA, and

0.25 (mM) DTT] at 30 (°C) for 30 minutes.

In vitro histone methyltransferase (KMT) assay

Partially purified HA-KMT2D wild-type and mutant derivative proteins (S1331A and

S1331D) were obtained from transfecting the appropriate plasmids into HEK 293T cells,

followed by immunoprecipitation assays using HA beads. Briefly, the cells were lysed in IP

buffer [50 (mM) Tris, pH 7.5, 250 (mM) NaCl, 1% TritonX-100, 1 (mM) EDTA], the

supernatants were collected by centrifugation for 10 minutes at 12,000 (g) and the lysates were

incubated overnight with EZview Red Anti-HA Affinity beads (Sigma-Aldrich) at 4 (°C). After

three washes, the beads were eluted in BC100 buffer [20 (mM) Tris pH 7.5, 10% Glycerol, 0.2

(mM) EDTA, 1% TritonX-100, 100 (mM) NaCl) containing 100 (µM) HA peptide (Sigma-

Aldrich)]. Vivaspin Sample Concentrators [GE Healthcare, 10 (kDa) cutoff] were used to

concentrate the samples at 12,000 (g) for 10 minutes. KMT2D protein amounts were quantified

by Coomassie staining and western blot analysis using rat monoclonal antibody to HA (clone

3F10, Roche).

KMT activity against an artificial H3 peptide was measured by the EpiQuik Histone

Methyltransferase Activity-Inhibition Assay Kit (H3K4) (Epigentek), following the

manufacturer’s protocol. Relative activity was calculated as the fold change in OD450nm over the

mean reading of control IgG samples (IgG, Immunoglobin G). Experiments were performed in

triplicate and repeated independently three times.

Plasmids and generation of pCMV-HA-KMT2D mutant expression constructs

The pCMV-HA-KMT2D plasmid was a gift from Dr. Laura Pasqualucci from Columbia

University. The KMT2D mutants (S1331A and S1331D) were generated from the wild-type

pCMV-HA-KMT2D using a PCR-based site-directed mutagenesis approach. All plasmids were

verified for integrity by diagnostic restriction enzyme digestions followed by Sanger sequencing

of the full-length KMT2D coding sequence. pCMV3-FLAG-KDM1 plasmid was obtained from

Sino Biological Inc.

Animal studies

Animals were maintained based on the institutional guidelines of Memorial Sloan

Kettering Cancer Center (Protocol number 12-10-019). 6 x 106 MCF7 cells in 1:1 DF12

media/Matrigel (Corning) were injected subcutaneously into four to six week old female athymic

Foxn1nu mice maintained in the presence of exogenous estradiol added into the drinking water.

Mice were randomized when tumors reached ~130 (mm3) of volume. 5 mice (10 tumors) per

group were then treated with BYL719, 25 (mg x kg-1) in 0.5% in carboxymethylcellulose

(Sigma-Aldrich) daily or vehicle for the indicated times. Tumors were collected at the end of the

experiments, two to four hours after the last treatment.

Mass spectrometry

Proteins were resolved using SDS-polyacrylamide gel electrophoresis, stained with

SimplyBlue SafeStain (Life Technologies), and gel sections excised with in situ trypsin digestion

of polypeptides in each gel slice performed as described (34). The tryptic peptides were desalted

using a 2 (µl) bed volume of Poros 50 R2 (Applied Biosystems) reversed-phase beads packed in

Eppendorf gel-loading tips and eluted with 40% acetonitrile. The purified peptides were diluted

with 0.1% formic acid and each gel section was analyzed separately by microcapillary liquid

chromatography with tandem mass spectrometry using the NanoAcquity (Waters) with a 100-

µm-inner-diameter ×  10-cm-length C18 column [1.7 (µm) BEH130, Waters] configured with a

180-µm x 2-cm trap column coupled to an OrbiElite mass spectrometer (Thermo Fisher

Scientific) scanning 300-1650 (m/z) at 120000 resolution with AGC set at 1 x 106. Peptides were

eluted with a linear gradient of 0-50% acetonitrile (0.1% formic acid) in water (0.1% formic

acid) over 90 minutes with a flow rate of 300 (nL/min). Key parameters for the data dependent

MS were top 10 DDA, AGC 104, and CID ms/ms collected in the linear ion trap. Key parameters

for the targeted MS/MS were isolation width 2, and for ETD anion target 5 x 105, reaction time

150 (ms), with product ions collected in the ion trap using enhanced resolution scan mode from

50 to 2000 (m/z). Initial protein/peptide identifications from the LC-MS/MS data were

performed using the Mascot search engine (Matrix Science, version 2.3.02;

www.matrixscience.com) with the Uniprot human protein database (downloaded on Feb 23,

2015). The search parameters were as follows: (i) two missed cleavage tryptic sites were

allowed; (ii) precursor ion mass tolerance 10 (ppm); (iii) fragment ion mass tolerance 0.8 (Da);

and (iv) variable protein modifications were allowed for methionine oxidation, deamidated (NQ),

protein N-terminal acetylation, phosphoserine, phosphothreonine, and phosphotyrosine.

RNA extraction, cDNA synthesis, quantitative real-time PCR

Total RNA was extracted from patient biopsy tissue using TRIzol (Life Technologies),

treated with DNAse, and sent for sequencing. Total RNA was extracted from cells using RNeasy

Mini kit (Qiagen). cDNA synthesis was performed using the Bio-Rad iScript cDNA synthesis kit

according to the manufacturer's instructions. The qPCR SYBR green master mix (Applied

Biosystems) was used to amplify specific cDNA fragments with the oligonucleotides listed

below using the ViiATM Real Time PCR system (Applied Biosystems). The data was analyzed

by the change-in-threshold (2−ΔΔCT) method using Actin or GAPDH as housekeeping genes to

obtain relative RNA expression. Primers used for mRNA expression were:

GREB1: 5’-GTGGTAGCCGAGTGGACAAT-3’; 5’-ATTTGTTTCCAGCCCTCCTT-3’

PGR: 5’-GGCATGGTCCTTGGAGGT-3’; 5’-CCACTGGCTGTGGGAGAG-3’

cFOS: 5’-TGATGACCTGGGCTTCCCAG-3’; 5’-CAAAGGGCTCGGTCTTCAGC-3’

EGR3: 5’-GGAGCAAATGAAATGTTGGTG-3’; 5’-AGGAAAACCTATGGGGAATG-3’

ERBB3: 5’-CTGATCACCGGCCTCAAT-3’; 5’-GGAAGACATTGAGCTTCTCTGG-3’

SERPINA1: 5’-AATGGGGCTGACCTCTCC-3’; 5’-GTCAGCACAGCCTTATGCAC-3’

MYC: 5’-GCTGCTTAGACGCTGGATTT-3’; 5’-TAACGTTGAGGGGCATCG-3’

IFRD1: 5’-TTTGGCACTTCTCTTTGAATTG-3’; 5’-CGTCAAGGACTCCATGTCTTC-3’ AKAP1: 5’-AGGCTCCAACCCTAAGAAGG-3’; 5’-AGCCGACCGACTAAGTGCT-3’ TPM3: 5’-GAGCTGAGCTGGCAGAGTCTA-3’; 5’-GTTGTTGGTGACATTCTTCAGC-3’

ACTIN: 5’-CGTCTTCCCCTCCATCGT-3’; 5’-GAAGGTGTGGTGCCAGATTT-3’

GAPDH: 5’-ACAGTCAGCCGCATCTTCTT-3’; 5’-ACGACCAAATCCGTTGACTC-3’

RNA-seq analysis and gene set enrichment analysis (GSEA)

Reads were first processed with Trimmomatic (35) to remove adaptor sequences and

bases with quality scores below 20, and reads with less than 30 remaining bases were discarded.

Trimmed reads were then aligned to hg19 human genome with the STAR spliced-read aligner

(36). For each gene from the RefSeq annotations, the number of uniquely mapped reads

overlapping with the exons was counted with HTSeq (http://www-

huber.embl.de/users/anders/HTSeq/). The reads per kilobase per million (RPKM) value was

calculated for each gene, and RPKM values were quantile normalized across all samples to

obtain gene expression levels.

Using the normalized RPKM counts for each sample, we first added a pseudo count to

the data which was chosen to be the smallest non-zero value and then transformed the data to log

base 2. We then did a paired moderated t-test for differential analysis using the Bioconductor

package LIMMA. The t-score for from this analysis was then used to do a pre-ranked GSEA

analysis on a subset of pathways that included transcription factor binding sites from Transfact

(MSigDB sets C3:TFT) plus pathways with the search terms: ER (Estrogen Receptor), RAR

(Retinoic Acid Receptor), and RXR (Retinoid X Receptor). For the study of patients treated with

the PI3K inhibitors, pre treatment and on treatment biopsies from patients enrolled in the clinical

trial NCT01870505 conducted at MSKCC or a multicentric clinical trial also conducted at

MSKCC NCT02340221 were used for RNA-seq analysis as described above. The MSKCC

Institutional Review Board approved the study and informed consent was obtained from all

patients.

Chromatin immunoprecipitation (ChIP)

Cells were crosslinked with paraformaldehyde added directly to the culture medium at a

final concentration of 1%, and incubated at room temperature for 15 minutes. The crosslinked

cells were quenched with ice-cold glycine for 5 minutes, washed with PBS and collected. The

cells were then lysed with SDS lysis buffer [10 (ml) of 1% SDS, 10 (mM) EDTA, 50 (mM) Tris-

HCl, pH 8.1) containing protease and phosphatase inhibitors (Roche) for 15 minutes prior to

sonication. Cells were sonicated in 10 second pulses for a total of 10 minutes. The sheared

chromatin was diluted with ChIP Dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 (mM)

EDTA, 16.7 (mM) Tris-HCl, pH 8.1, 167 (mM) NaCl] and incubated overnight with Protein G

Dynabeads (Thermo Fisher Scientific), which were pre-incubated with specific antibodies. ChIP

antibodies for the following proteins were used: FOXA1 (ab5089) from Abcam, PBX1

(H00005087-MO1) from Abnova, ER alpha (sc-543) from Santa Cruz, KMT2D (HPA035877)

from Sigma-Aldrich. The immunecomplexes were washed twice with low salt wash buffer [0.1%

SDS, 1% Triton X-100, 2 (mM) EDTA, 20 (mM) Tris-HCl, pH 8.1, 150 (mM) NaCl], high salt

wash buffer [0.1% SDS, 1% Triton X-100, 2 (mM) EDTA, 20 (mM) Tris-HCl, pH 8.1, 500

(mM) NaCl], LiCl wash buffer [0.25 (M) LiCl, 1% NP40, 1% doexycholate, 1 (mM) EDTA, 10

(mM) Tris-HCl, pH 8.1] and 1x TE buffer [10 (mM) Tris-HCl, 1 (mM) EDTA pH 8.0]. The

complexes were eluted with elution buffer [1% SDS, 0.1 (M) NaHCO3]. The eluates were

reverse crosslinked at 65 (°C) for 4 hours by adding 20 (µM) of 5 (M) NaCl followed by

proteinase K treatment for one hour at 4 (°C). The DNA was purified using QIAquick PCR

purification kit (Qiagen). Primers used for ChIP-qPCR analysis were:

GREB1: 5’-GAAGGGCAGAGCTGATAACG-3’; 5’-GACCCAGTTGCCACACTTTT-3’

PGR promoter: 5’-AGGGAGGAGAAAGTGGGTGT-3’;

5’-GGAGAACTCCCCGAGTTAGG-3’

PGR enhancer: 5’-CTGGGAGTTCAGAGCAGGAC-3’; 5’-TCAGCTTTGCTTGGACCTTT-3’ cFOS: 5’-AATGCAAACAGGACCAAAGG -3’; 5’-TGGCAGTGTCAGGACAGAAG-3’

EGR3: 5’-ACCTCCAAGAGGGAGAGGAG-3 -3’; 5’-CTGTCCAGCCGGAGTTAGAG-3’

SERPINA1: 5’-AGGTATGGGCACAAGACCTG-3’; 5’-TCAGGGGAAAATTGTCTTCG-3’

MYC: 5’-GTCAGCCAATCTTCGCACTT-3’; 5’-TGCCAGAGGAAGCTACTGGT-3’

ERBB3: 5’-GAGACCTGGGTGAAGAGCTG-3’; 5’-GGACCAAGCAGTCATTTGGT-3’

IFRD1: 5’-TTCGATCACAGCTCTTCACG-3’; 5’-GTTCCGCTTCTTGTTCTTCG-3’ AKAP1: 5’-TGGCAAGTGTATTCGCTGAG-3’; 5’-CACGGTCCACCAAACTTTCT-3’ TPM3: 5’-TCCATCAGGCTTCCCTACAC-3’; 5’-TTTTCTCCATGCCTCTGCTT-3’ ACTIN: 5’-TGTTCCAGGCTCTGTTCCTC-3’; 5’-AGAAAAGAACGCAGGCAGAA-3’

ChIP-seq library preparation, Illumina sequencing, ChIP-seq analysis

ER and FOXA1 ChIP-seq was performed as above with the exception that 10 million

T47D cells were used for each ChIP. Single ends 36bp sequencing was performed at the

Genomics Core of Memorial Sloan Kettering Cancer Center using HiSeq (Illumina). Reads were

first processed with Trimmomatic to remove the adaptor sequences and bases with quality scores

below 20, and reads with less than 30 remaining bases were discarded (35). Trimmed reads were

then aligned to hg19 human genome with the bowtie aligner (37). ER (antibody, Santa Cruz, sc-

543) and FOXA1 (antibody, Abcam, ab5089) peaks were called using MACS2 using P value

cut-off of 0.01 (38). To find a set of peaks that are reproducible across the two biological

replicates of a given cell condition, we calculated the per-replicate P value of each peak using

only the read count at the peak from the individual replicate, and estimated the irreproducible

discovery rate (IDR) (39) from the two sets of P values that the replicates produced. Only peaks

with an IDR of 0.05 or less were kept for downstream analyses. The same analysis was also

performed for KMT2D ChIP-seq (antibody, Sigma-Aldrich, HPA035877) with the exception that

60 million T47D cells were used per ChIP. Due to antibody issues, the enrichment of peaks for

KMT2D ChIP-seq using MACS2 was lower than expected.

Differentially accessible peaks from combined atlases were identified with DESeq (40)

by counting all read ends overlapping peaks in each condition. DESeq was run with a fold-

change threshold of 1.5, and FDR  <  0.1 (FDR, false discovery rate). The distribution of the peaks

around the TSS (transcription start site) was calculated using the ChIPpeakAnno package (41).

DNA motif analysis was performed with HOMER (42).

Transposase-accessible chromatin using sequencing (ATAC-seq) and analysis

Starting from fastq files containing ATAC-seq paired-end reads, sequencing adaptors

were removed using Trimmomatic (35). Trimmed reads were mapped to the hg19 human

genome using Bowtie2 (37) allowing at most 1 seed mismatch and keeping only uniquely

aligned reads. Duplicates were removed using Picard (http://picard.sourceforge.net). For peak-

calling the read start sites were adjusted (reads aligning to the +/- strand were offset by +4bp/-

5bp, respectively) to represent the center of the transposon binding-event, as described in

Buenrostro et al. (17). For each sample, ATAC-seq was run on two biological replicates. Peak

calling was performed on each condition individually by pooling reads from biological

replicates, and using MACS2 (38) with a P value threshold (-P 1e-2). To find a set of peaks that

are reproducible across the two biological replicates of a given cell condition, we calculated the

per-replicate p value of each peak using only the read count at the peak from the individual

replicate, and estimated the irreproducible discovery rate (IDR) (39) from the two sets of P

values that the replicates produced. Only peaks with an IDR of 0.05 or less were kept for

downstream analyses. This procedure generated a set of reproducible accessible sites for each

condition. To create a single atlas of accessible sites for the before and after treatment, we

merged peaks from two conditions if their overlap was 75% or more; if they overlapped by 25%

or less, two peaks were kept separate by removing the overlapping region. In this way, we

created an atlas of 52070 accessible sites (or peaks) that were reproducible in at least one

condition. To link site accessibility to regulation of gene expression, we associated each peak to

its nearest gene in the human genome using ChIPpeakAnno package (41). Differentially

accessible peaks from this atlas were identified with edgeR (40) by counting all read ends

overlapping peaks in each condition. edgeR was run with default settings, a fold-change

threshold of 2, and FDR  <  0.01. For patient ATAC-seq samples, peak calling was performed by

pooling reads from pre and on treatment samples using MACS2 (38) with a q value threshold

(1e-3). The Bioconductor (43) and deepTools (44) were used for visualization. For the study of

patients treated with BYL719, pre treatment and on treatment biopsies from patients enrolled in

the clinical trial NCT01870505 conducted at MSKCC were used for ATAC-seq analysis as

described above. The MSKCC Institutional Review Board approved the study and informed

consent was obtained from all patients.

Statistical analyses

Two-way t-tests were performed using GraphPad Prism (GraphPad Software), and P

values are indicated in the respective graphs. Asterisks are also shown in the respective graphs,

*P<0.05, **P<0.01, ***P<0.001. All cellular experiments were repeated at least three times.

The in vivo experiments contained 10 tumors for each treatment group and the sample size was

chosen to reflect a difference in means of 20% with a power of 90%. Before the commencement

of the in vivo experiments, animals were measured and randomized in groups with similar

average tumor volume. Bioinformatic statistics are indicated in the respective method

descriptions.

ER

FOXA1

GREB1

C

E

D

EGR3

ER

FOXA1

*

*

*

*

*

*

*

*

*

*

*

**

*

*

* * * *

**

* *

*

*

* *

*

* *

*

*

*

* *

A

Name Motif P-value

ERE (Nuclear

Receptor) 1e-70

FOXA1 (Forkhead)

1e-27

AP-2 gamma

1e-21

Homeobox

1e-20

GATA3

1e-20

RUNX

1e-18

TEAD

1e-13

!!!!!!

Name Motif P-value

FOXA1

(Forkhead) 1e-260

Nuclear Receptor

1e-59

SMAD3

1e-30

Homeobox

1e-29

EGR1

1e-19

PDX1

1e-17

GATA3

1e-17

!

FOXA1 (Forkhead)

Homeobox

Nuclear Receptor

Enriched Motifs

FOXA1 (Forkhead)

Homeobox

Nuclear Receptor

Enriched Motifs

Name Motif P-value

ERE (Nuclear

Receptor) 1e-70

FOXA1 (Forkhead)

1e-27

AP-2 gamma

1e-21

Homeobox

1e-20

GATA3

1e-20

RUNX

1e-18

TEAD

1e-13

!!!!!!

Name Motif P-value

FOXA1

(Forkhead) 1e-260

Nuclear Receptor

1e-59

SMAD3

1e-30

Homeobox

1e-29

EGR1

1e-19

PDX1

1e-17

GATA3

1e-17

!

FOXA1 (Forkhead)

Homeobox

Nuclear Receptor

Enriched Motifs

FOXA1 (Forkhead)

Homeobox

Nuclear Receptor

Enriched Motifs B

Fig. S1. FOXA1, PBX1 and ER recruitment to shared target genes is enhanced upon PI3K

inhibition. (A, B) The top enriched motifs observed at the gained ER or FOXA1 binding sites

upon BYL719 treatment. (C) Examples of ER and FOXA1 binding sites presented as read per

million (RPM) that are enhanced upon BYL719 treatment (1µM) for 24h in T47D breast cancer

cells. (D) ChIP-qPCR for ER, FOXA1, PBX1 and IgG occupancy in T47D cells treated with

DMSO or BYL719 (1µm) for 24h. Values are represented as relative enrichment; i.e., the ratio of

mean percentage of input enrichment of the candidate gene over the mean percentage of input

enrichment of a control gene. n=3; mean ±SD is shown as fold enrichment compared to the

DMSO treatment group. *P<0.05, **P<0.01. (E) Similar assay was performed in MCF7 breast

cancer cells.

Fig. S2. FOXA1 regulates the activation of ER upon PI3K inhibition. (A) ChIP-qPCR to test

ER occupancy in the enhancer or promoter regions of indicated target genes when FOXA1 is

silenced by two distinct shRNAs in T47D breast cancer cells treated with BYL719 (1µM) for

24h. Error bars are the ±SD of n=3 shown as fold enrichment compared to the shGFP control

group. *P<0.05, Student’s t test. (B) mRNA levels were measured by RT-qPCR in shGFP

(control) or shFOXA1 T47D cells maintained in estrogen-depleted for 3 days followed by

treatment with DMSO, E2 (estrogen) (100nm), BYL719 (1µM) or E2 plus BYL719 for 24h.

A

B

C

D

* * *

* * * * *

* *

* * *

* ** * *

*

* * *

* * *

* *

* *

*

**

**

**

*

* *

**

**

** **

** *

**

*

* * * *

** ** * * *

*

n=3; mean ±SD is shown as relative expression compared to the shGFP control group. *P<0.05,

**P<0.01 by Student’s t test. (C, D) The same as above but in MCF7 breast cancer cells.

Fig. S3. PBX1 regulates the activation of ER upon PI3K inhibition. (A) ChIP-qPCR for ER

occupancy in shGFP or shPBX1 (#1, #2) T47D cells treated with BYL719 (1µM) for 24h. Error

bars are the ±SD of n=3 shown as fold enrichment compared to the shGFP control group.

T47D

B

D

A

C

*

* *

*

* * * *

* * * *

* *

* * * * * * *

* **

* * * * *

* * * *

* *

* * * *

* *

* * * *

*

* *

*

* *

* *

**

** * * *

* *

* * *

*

** ** * * *

*

*

*

* *

*P<0.05 by Student’s t test. (B) RT-qPCR was used to measure mRNA levels of shGFP or

shPBX1 T47D cells maintained in estrogen-free media for 3 days followed by treatment with

DMSO, E2, BYL719, or E2 plus BYL719 for 24h. Error bars represent the ±SD of n=3 shown as

relative enrichment compared to the shGFP control group. *P<0.05, **P<0.01 by Student’s t

test. (C, D) The same as above but in MCF7 breast cancer cells.

Fig. S4. FOXA1 or PBX1 silencing augments the clinical activity of BYL719. (A) Dose

response cell proliferation curves of T47D breast cancer cells transduced with shGFP, shFOXA1

(#1, #2) and treated with increasing concentrations of BYL719 for 5 days. Also shown is the

western blot demonstrating the knockdown of FOXA1. (B) The same as (A) but in MCF7 breast

cancer cells. (C) Cell viability assays in MCF7 breast cancer cells transduced with doxycycline

(DOX) inducible FOXA1 shRNA (shFOXA1+DOX) and treated with increasing concentrations

of BYL719 for 5 days. Also shown is the western blot demonstrating knockdown of FOXA1

0 5 10 15 20 25 30 35 400

40

80

120

160

200

240

280

320

360

Days of treatment

Tum

or V

olum

e (m

m3)

MCF7 shPBX1

VEHICLE

BYL719

DOX VEHICLEDOX BYL719

p=0.0016

p=0.0109

A C

D E F

G H

B

PBX1

pAKT (S473)

RFP

Actin

BYL719: !"""""""!"""""""+""""""+""""""!""""""!"""""""+"""""""+"shPBX1: !"""""""!"""""""!"""""""!""""""+""""""+""""""+"""""""+"

- DOX + DOX

shG

FP

shF

OX

A1-

1

shF

OX

A1-

2

FOXA1

Actin

MCF7

FOXA1

Actin

shG

FP

shF

OX

A1-

1

shF

OX

A1-

2

T47D

Ac#n%

PBX1

shG

FP

shP

BX

1-1

shP

BX

1-2

T47D

PBX1

shG

FP

shP

BX

1-1

shP

BX

1-2

PBX1

Actin

MCF7

RFP

- + DOX:

FOXA1

Actin

MCF7

PBX1

RFP

Actin

- + DOX:

MCF7

I

0.0

0.5

1.0

1.5

2.0

2.5

Fol

d E

nric

hmen

t

MYC enhancer

BYL719: - + - +

+ DOX

p<0.01

- DOXshPBX1:

ER

0

2

4

6

8

Fol

d E

nric

hmen

tPGR promoter

BYL719: - + - +

+ DOX

p=0.01

- DOXshPBX1:

ER

upon doxycycline administration. (D, E) Proliferation curves of T47D and MCF7 breast cancer

cells transduced with shGFP, shPBX1 (#1, #2) and treated with BYL719 for 5 days. Also shown

is the western blot demonstrating the knockdown of PBX1. (F) Proliferation curves of MCF7

breast cancer cells transduced with doxycycline inducible shPBX1 and treated with BYL719 for

5 days. Also shown is the western blot demonstrating the knockdown of PBX1 upon doxycycline

administration. (G) MCF7 shPBX1 in vivo xenograft activated in the presence of doxycycline

and treated with vehicle or BYL719 daily (25mg/kg) (n=10/group). (H) Western blot analysis of

tumors collected at the end of the experiment 4h after the last dosage. (I) Tissue ChIP-qPCR to

test the occupancy of ER in each tumor group. Student’s t test was used to calculate the indicated

P values. Error bars, ±SEM.

Fig. S5. Inhibition of PI3K alters the chromatin landscape towards an active ER-dependent

signature. (A) Example of ER, FOXA1 ChIP-seq binding region, and open chromatin region of

PGR in T47D cells treated with DMSO and BYL719 (1µM) for 24h. (B) Heat map of gained

accessible sites upon BYL719 treatment within 1kb distance, and the corresponding gained

accessible sites that also have an enhanced ER or FOXA1 binding site upon treatment. 881 out of

4693 gained accessible sites correspond with enriched ER peaks upon treatment, and 2117 out of

4693 gained accessible regions match with enriched FOXA1 peaks upon treatment. (C) Clinical

A

ER

FOXA1

ATAC- seq

PGR

DMSO BYL719 DMSO BYL719

B

C

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−0.05

0.00

0.05

0.10

0.15

0.20

0 5000 10000 15000rank

enric

hmen

t sco

re

DUTERTRE ESTRADIOL RESPONSE 24HR DN

Enr

ichm

ent s

core

(ES

)

Enrichment after treatment

Dutertre estradiol response 24hr

NES= 1.4 P= 0.004

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●0.0

0.1

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0.4

0.5

0 5000 10000 15000rank

enric

hmen

t sco

re

YANG BREAST CANCER ESR1 UP

Enr

ichm

ent s

core

(ES

)

Enrichment after treatment

Yang Breast Cancer ESR1 up

NES= 2.13 P= 0.001

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0.00

0.05

0.10

0.15

0.20

0 5000 10000 15000rank

enric

hmen

t sco

re

DUTERTRE ESTRADIOL RESPONSE 24HR DN

Enr

ichm

ent s

core

(ES

)

Enrichment after treatment

Dutertre estradiol response 24hr

NES= 1.4 P= 0.004

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●0.0

0.1

0.2

0.3

0.4

0.5

0 5000 10000 15000rank

enric

hmen

t sco

re

YANG BREAST CANCER ESR1 UP

Enr

ichm

ent s

core

(ES

)

Enrichment after treatment

Yang Breast Cancer ESR1 up

NES= 2.13 P= 0.001

D

!3kb Peak Center +3kb !3kb Peak Center +3kb !3kb Peak Center +3kb

ATAC-seq

ER FOXA1

T47D: BYL719

Gained accessible sites overlapped with gained ER/FOXA1 sites

Pt 1 Pt 2 Pt 3 Pt 4 Pt 5 Pt 6 Pt 7 Pt 8 Pt 9 Pt 10

Pt 11

Pt 12

Pt 13

Pt 14

Pt 15

ESR1 1.7 1.7 1.1 2.1 0.7 1.0 0.0 1.1 0.8 1.4 0.7 0.8 7.5 1.8 0.5

PGR 0.8 7.4 0.4 0.7 0.3 1.2 0.7 1.2 1.3 1.3 0.8 1.0 6.6 4.5 0.2

GREB1 0.2 ## 0.6 0.7 1.1 1.0 0.7 0.9 0.9 3.9 0.7 0.6 2.4 1.8 1.0

MYC 1.2 ## 0.4 0.4 1.5 1.0 ## 2.4 1.0 3.0 1.0 1.1 0.2 4.2 2.6

SLC44A1 1.1 1.8 0.3 1.3 0.9 1.2 1.6 0.9 0.8 1.6 1.0 1.2 0.8 1.2 0.8

0.8

0.2

1.2

No change!

>1.25 !

<0.75!

E

and pathological features of breast cancer patients treated with PI3K inhibitors enrolled in a trial

of BYL719 in combination with aromatase inhibitors. The lesion biopsied column specifies the

site of the metastatic disease that was biopsied pre-treatment and on-treatment. IDC: infiltrating

ductal carcinoma. (D) Gene set enrichment analysis (GSEA) of ER-associated signatures

corresponding to 15 paired pre treatment and PI3K inhibitor treatment tumor samples (FDR <

0.05). (E) Differential ESR1 expression and target gene examples: PGR, GREB1, MYC, and

SLC44A1 in 15 patients treated with PI3Kα inhibitors.

A

B

C

F G

EV

Flag-KDM1

Actin

KD

M1

MCF7

D

* * * *

* *

* *

* *

* *

* *

* *

*

* * *

*

*

* *

* * * *

* *

* * * *

* *

*

* *

* *

*

*

* * * *

* *

* *

* * * *

* * * *

*

* *

*

*

*

* * *

*

*

*

* *

*

*

*

*

*

*

*

*

* *

* *

* *

HA-KMT2D: EV

WT

N54

37S

R54

32W

T47D IP: HA

HA

ER

FOXA1PBX1

IgG

0

2

4

6

8

10

12

Fold

Enr

ichm

ent

FOS enhancer

EV + siControlEV + siKMT2D (3'UTR)siKMT2D (3'UTR) + KMT2DsiKMT2D (3'UTR) + N5437S KMT2DsiKMT2D (3'UTR) + R5432W KMT2D

ER

FOXA1PBX1

IgG

0

2

4

6

8

10

12

Fold

Enr

ichm

ent

MYC enhancer

EV + siControlEV +

siKMT2D (3'UTR)

siKMT2D (3'UTR) + KMT2DsiKMT2D (3'UTR) + N5437S KMT2D

siKMT2D (3'UTR) + R5432W KMT2D

ER

FOXA1PBX1

IgG

0

1

2

3

4

5

6

7

8

9

Fold

Enr

ichm

ent

EGR3 enhancer

EV + siControlEV +

siKMT2D

(3'UTR)

siKMT2D (3'UTR) + KMT2DsiKMT2D (3'UTR) + N5437S KMT2D

siKMT2D (3'UTR) + R5432W KMT2D

* *

*

*

* *

*

*

*

siC

ontr

ol

siK

MT

2D

3’U

TR

KMT2D

Actin

T47D

E

*

Fig. S6. KMT2D is required for FOXA1-PBX1-dependent ER activation upon PI3K

blockage. (A) ChIP-qPCR for ER, FOXA1, PBX1, and control IgG, occupancy in MCF7 breast

cancer cells depleted of KMT2D by two distinct shRNAs (#1 and #2) and treated with BYL719

(1µM) for 24h. (B) ChIP-qPCR to test H3K4me1 and H3K4me2 binding in control cells or cells

depleted of KMT2D by two distinct shRNAs (#1 and #2) and treated with BYL719 (1µM) for

24h. (C) Estrogen depleted shGFP or shKMT2D (#1, #2) MCF7 cells were subjected to

treatment with DMSO, E2 (100nM), BYL719 (1µM), or E2 plus BYL719 for 24h and mRNA

levels were measured by RT-qPCR. (D) ChIP-qPCR analysis of ER, FOXA1, PBX1, and control

IgG in control cells or cells depleted of endogenous KMT2D and transfected with wild-type

KMT2D or the SET domain mutants of KMT2D: N5437S, R5432W. Also shown are the western

blots showing expression of KMT2D mutants and knockdown of shKMT2D 3’UTR. (E) ChIP-

qPCR analysis to test the binding of ER, FOXA1, PBX1, and control IgG in the regions of the

specified ER target genes after overexpression of the H3K4me1/2 demethylase, KDM1, or empty

vector (EV) in MCF7 cells and upon treatment with BYL719 (1µM) for 24h. Also shown is the

western blot showing overexpression of FLAG-tagged KDM1 in MCF7 cells. (F) ChIP-qPCR

analysis for H3K4me1/2 and IgG occupancy in the cells overexpressed with KDM1 or empty

vector. (G) mRNA levels were measured by RT-qPCR in MCF7 cells maintained in estrogen-

free media for 3 days, transfected with empty vector or KDM1 and treated with DMSO, E2

(100nM), BYL719 (1µM), or E2 plus BYL719 for 24h. Error bars represent the ±SD of n=3,

*P<0.05 by Student’s t test.

0 2 4 8 24 0 2 4 8 240.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Fol

d E

nric

hmen

t

SERPINA1 enhancer

KMT2D IgG

BYL719 (h):

A B C

0 2 4 8 24 0 2 4 8 240

1

2

3

4

Fol

d E

nric

hmen

t

SERPINA1 enhancer

H3K4me1 IgG

BYL719 (h): 0 2 4 8 24 0 2 4 8 240

2

4

6

8

10

12

Fol

d E

nric

hmen

t

SERPINA1 enhancer

H3K4me2 IgG

BYL719 (h):

0 2 4 8 24 0 2 4 8 240

2

4

6

8

10

12

Fol

d E

nric

hmen

t

cFOS enhancer

H3K4me1 IgG

BYL719 (h): 0 2 4 8 24 0 2 4 8 240

5

10

15

20

25

30

35

Fol

d E

nric

hmen

t

cFOS enhancer

H3K4me2 IgG

BYL719 (h):0 2 4 8 24 0 2 4 8 240

2

4

6

8

10

12

Fol

d E

nric

hmen

t

cFOS enhancer

KMT2D IgG

BYL719 (h):

shG

FPsh

KMT2

D-1sh

KMT2

D-2

0.0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e E

xpre

ssio

n

KMT2D

shG

FPsh

KMT2

D-1sh

KMT2

D-2

0.0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e E

xpre

ssio

n

KMT2D

0.0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e E

xpre

ssio

n

KMT2D

DOX: - +

DMSO

BYL719 (1µM) 24h

0 4 8 240.0

0.5

1.0

1.5

2.0

Rel

ativ

e E

xpre

ssio

n

IFRD1

BYL719 (1µM) (h): 0 4 8 240.0

0.5

1.0

1.5

2.0

Rel

ativ

e E

xpre

ssio

n

AKAP1

BYL719 (1µM) (h): 0 4 8 240.0

0.5

1.0

1.5

2.0

Rel

ativ

e E

xpre

ssio

n

TPM3

BYL719 (1µM) (h):

E

*

* *

* *

* *

*

*

*

*

*

*

*

**

** **

**

** **

*

*

**

** **

D

F

*

*

*

*

ER DMSO

ER BYL719

FOXA1 DMSO

FOXA1 BYL719

KMT2D BYL719

KMT2D DMSO

ER DMSO

ER BYL719

FOXA1 DMSO

FOXA1 BYL719

KMT2D DMSO

KMT2D BYL719

GREB1 cFOS SERPINA13P SERINC2

ER DMSO

ER BYL719

FOXA1 DMSO

FOXA1 BYL719

KMT2D DMSO

KMT2D BYL719

GREB1 cFOS SERPINA13P SERINC2

ER DMSO

ER BYL719

FOXA1 DMSO

FOXA1 BYL719

KMT2D DMSO

KMT2D BYL719

GREB1 cFOS SERPINA13P SERINC2 GREB1 cFOS CUL2 EPHA6 TRPC6

ER DMSO

ER BYL719

FOXA1 DMSO

FOXA1 BYL719

KMT2D DMSO

KMT2D BYL719

GREB1 cFOS SERPINA13P SERINC2 SERPINA13P SERINC2 DDX11L5

Fig. S7. KMT2D silencing augments the activity of BYL719. (A, B, C) Dose response curves

of T47D or MCF7 cells transduced with shGFP, shKMT2D (#1, #2) and treated with BYL719

(1µM) for 5 days. Also shown are the dose response proliferation curves from MCF7 cells

transduced with doxycycline inducible shKMT2D and treated with BYL719 (1µM) for 5 days.

Moreover, RT-qPCR analysis demonstrating knockdown of KMT2D (#1, #2) is also shown.

Error bars represent the ±SD of n=3, *P<0.05 by Student’s t test. (D) ChIP-qPCR for KMT2D,

H3K4me1 and H3K4me2, and IgG, Immunoglobin G control upon treatment with BYL719

(1µM) for 2, 4, 8, 12, and 24h. Error bars represent the ±SD of n=3, *P<0.05, **P<0.01. (E)

Expression analysis of ER, FOXA1, and PBX1 target genes IFRD1, AKAP1, and TPM3 whose

expression is not affected by BYL719 (1µM) treatment. Also shown is KMT2D ChIP-qPCR

analysis in these loci upon treatment with BYL719 (1µM) for 24h. Error bars represent the ±SD

of n=3. (F) Examples of ER, FOXA1, and KMT2D ChIP-seq binding regions in T47D breast

cancer cells treated with BYL719. Data are presented as read per million (RPM).

Fig. S8. Activation of PI3K results in phosphorylation of KMT2D. (A, B) CAD and ETD

mass spectra respectively recorded on the (M+3H)3+ ions at m/z = 776.72 and retention time 51.8

HA

H3K4m

e1/2 Ig

G0

1

2

3

4

Fold

Enr

ichm

ent

EGR3 enhancer

EVWT KMT2DS1331A KMT2DS1331D KMT2D

HA

H3K4m

e1/2 Ig

G0

1

2

3

4

Fold

Enr

ichm

ent

MYC enhancer

EVWT KMT2DS1331A KMT2DS1331D KMT2D

HA

H3K4m

e1/2 Ig

G0

1

2

3

4

5

6

7

Fold

Enr

ichm

ent

GREB1 promoter

EVWT KMT2DS1331A KMT2DS1331D KMT2D

HA

H3K4m

e1/2 Ig

G0.0

0.5

1.0

1.5

2.0

Rel

ativ

e E

nric

hmen

t

GREB1 promoter

EVWT KMT2DS1331A KMT2DS1331D KMT2D

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

D

In vitro kinase assay

His-AKT: - + - + - +

IP: V5-KMT2D

EV WT S1331A

V5 WCL

pKMT2D (S1331)

pRXRXX(S/T)

V5

KMT2D (1222-1819) C

E

pKMT2D S1331

WCL

Con

trol

T47D

pKMT2D S1331 + Blocking Peptide

460

460

kDA

BY

L719

F

G

A

400 600 800 1000 1200 1400 1600 1800 2000 m/z

20 40 60 80

100

Rel

ativ

e A

bund

ance

(%) 848.4

919.5

764.8 620.3 1117.6 418.2 733.4

331.2 597.9 1310.7 1409.8

y3 y4

y5

y6

y7

y8 y9

y10

y11

b11 b13

b12

A"

L""K""pS""T""A""S""S""I""E""T""L""V""V""A""D""I""D""S""S""P""S""K"" 2328 2215 2086 1919 1818 1747 1660 1573 1460 1331 1230 1117 1018 919 848 733 620 505 418 331 234 147

"114 242 409 510 581 668 755 868 997 1098 1211 1310 1409 1480 1595 1708 1824 1911 1998 2095 2182 2328

yx

bx

200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

0 1 2 3 4 5 6

Rel

ativ

e A

bund

ance

(%) 776.5 1164.6 1552.8

1115.6

1726.0 1497.9 1035.0 1214.6 1928.0 604.3 999.6

1426.8 832.4 402.2 772.4 1841.0 1612.8 1294.5 489.3 214.1 351.1

B"

c18

c17

c16

c15

c14

c13

c11

c10

c8 c7

c6

z4 z5

z6

z7

z8

z9

z12

z16

+++" ETnoD"

L""K""pS""T""A""S""S""I""E""T""L""V""V""A""D""I""D""S""S""P""S""K"" 2328 2199 2070 1903 1802 1731 1644 1557 14441315 1214 1101 1002 903 832 717 604 489 402 ---- 218 131

"131 259 426 527 598 685 772 885 1014 1115 1228 1327 1426 1497 1612 1725 1840 1927 ---- 2112 2199 2328

zx

cx

400 600 800 1000 1200 1400 1600 1800 2000 m/z

20 40 60 80

100

Rel

ativ

e A

bund

ance

(%) 848.4

919.5

764.8 620.3 1117.6 418.2 733.4

331.2 597.9 1310.7 1409.8

y3 y4

y5

y6

y7

y8 y9

y10

y11

b11 b13

b12

A"

L""K""pS""T""A""S""S""I""E""T""L""V""V""A""D""I""D""S""S""P""S""K"" 2328 2215 2086 1919 1818 1747 1660 1573 1460 1331 1230 1117 1018 919 848 733 620 505 418 331 234 147

"114 242 409 510 581 668 755 868 997 1098 1211 1310 1409 1480 1595 1708 1824 1911 1998 2095 2182 2328

yx

bx

200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

0 1 2 3 4 5 6

Rel

ativ

e A

bund

ance

(%) 776.5 1164.6 1552.8

1115.6

1726.0 1497.9 1035.0 1214.6 1928.0 604.3 999.6

1426.8 832.4 402.2 772.4 1841.0 1612.8 1294.5 489.3 214.1 351.1

B"

c18

c17

c16

c15

c14

c13

c11

c10

c8 c7

c6

z4 z5

z6

z7

z8

z9

z12

z16

+++" ETnoD"

L""K""pS""T""A""S""S""I""E""T""L""V""V""A""D""I""D""S""S""P""S""K"" 2328 2199 2070 1903 1802 1731 1644 1557 14441315 1214 1101 1002 903 832 717 604 489 402 ---- 218 131

"131 259 426 527 598 685 772 885 1014 1115 1228 1327 1426 1497 1612 1725 1840 1927 ---- 2112 2199 2328

zx

cx B

pKMT2D S1331

KMT2D

T47D

Con

trol

BK

M12

0

BY

L719

MK

2206

pAKT S473

Actin

Con

trol

BK

M12

0

MK

2206

pKMT2D S1331

CAMA1

KMT2D

pAKT S473

Actin

Con

trol

BK

M12

0

MK

2206

pKMT2D S1331

KMT2D

ZR751

pAKT S473

Actin

H

T47D IP: HA

HA-KMT2D: EV

WT

S13

31A

S13

31D

pKMT2D (S1331)

KMT2D

pAKT (S473)

Actin

MCF10A

WT H1047R PIK3CA:

minutes. Tandem mass spectra recorded during targeted analyses (nHPLC-ESI-MS/MS) of

peptides generated in an in gel tryptic digest of human KMT2D protein expressed and

immunoprecipitated from 293T cells. (A) CAD spectrum dominated by fragment ions

corresponding to the low mass y-type ions and high mass b-type ions. (B) ETD spectrum

containing 22 of 41 possible c- and z-type product ions. (C) Due to the large size of KMT2D

(~593 kDa), the in vitro kinase assay was also performed using recombinant AKT and the

KMT2D (1222-1819 amino acids) fragment containing the S1331 phosphorylation site

immunoprecipitated from 293T cells. (D) Isogenic mammary epithelial MCF10A cells

expressing wild type (WT) or an activating mutation (H1047R) of PIK3CA were subjected to

western blot with the indicated antibodies. (E) Western blots with the indicated antibodies of

whole cell lysates of ER-positive breast cancer cell lines; T47D, ZR751, and CAMA1. (F)

Western blot of T47D cell lysates blotted with pKMT2D (S1331) antibody or the antibody pre-

incubated for 2h with KMT2D S1331 phospho-specific blocking peptide. (G) ChIP-qPCR

analysis of HA, H3K4me1/2, and control IgG binding in ER target genes regions in T47D cells

treated with empty vector control (EV), wild type KMT2D, S1331A KMT2D, or S1331D

KMT2D. Error bars are the ±SD of n=3, *P<0.05 by Student’s t test. Also shown is the

immunoprecipitated HA western blot showing equal HA expression of these plasmids in T47D

cells. (H) Proposed model: Upon activation of PI3K pathway, activated AKT phosphorylates

KMT2D at S1331. Phosphorylation of KMT2D attenuates its activity, leading to loss of

H3K4me1/2 and loss of binding of FOXA1-PBX1-ER transcriptional network and target gene

expression off (left). Blockage of the PI3Kα pathway by BYL719 inhibits AKT, leading to an

increase of KMT2D activity and H3K4me1/2 methylation that facilitates the recruitment of

FOXA1-PBX1 to allow subsequent binding of ER transcription factor and target gene expression

on (right).

Table S1. GSEA (gene set enrichment analysis) in MCF7 cells treated with BYL719. GSEA

was performed on a subset of pathways that includes transcription factor binding sites of ER

(estrogen receptor), FOXA1 (gene name HNF3ALPHA), PBX1, PR (progesterone receptor), GR

(glucocorticoid receptor), AR (androgen receptor), VDR (vitamin D receptor), TR (thyroid

receptor), PPARα (peroxisome proliferator-activator receptor alpha), PPARγ, (peroxisome

proliferator-activator gamma), and pathways with the search terms: ER, RAR (retinoic acid

receptor), and RXR (retinoid x receptor). GSEA was performed in MCF7 cells treated with

BYL719 for 8h versus the control cells (FDR < 0.05). P adj, adjusted P value. ES, enrichment

score. NES, normalized enrichment score. Size represents the number of genes enriched in the

dataset.

!

Pathway P value P adj ES NES Size BHAT_ESR1_TARGETS_VIA_AKT1_DN 2.021E-06 5.395E-06 0.75 3.13 82 BHAT_ESR1_TARGETS_NOT_VIA_AKT1_DN 2.018E-06 5.395E-06 0.72 3.01 87 DUTERTRE_ESTRADIOL_RESPONSE_6HR_DN 2.020E-06 5.395E-06 0.68 2.85 90 DUTERTRE_ESTRADIOL_RESPONSE_24HR_DN 2.049E-06 5.395E-06 0.53 2.77 494 V$HNF3ALPHA_Q6 2.031E-06 5.395E-06 0.40 1.93 202 V$ER_Q6 2.037E-06 5.395E-06 0.39 1.91 265 V$ER_Q6_01 2.036E-06 5.395E-06 0.36 1.76 262 V$PR_02 2.025E-06 5.395E-06 0.51 2.30 128 V$PR_01 2.025E-06 5.395E-06 0.49 2.21 139 V$GR_01 2.030E-06 5.395E-06 0.45 2.13 194 V$VDR_Q6 2.036E-06 5.395E-06 0.41 2.02 256 V$GR_Q6_01 2.037E-06 5.395E-06 0.38 1.86 267 V$PBX1_02 4.254E-05 7.838E-05 0.41 1.83 124 V$VDR_Q3 7.713E-05 0.000133 0.35 1.67 218 YANG_BREAST_CANCER_ESR1_LASER_UP 8.452E-05 0.000144 0.61 2.07 31 V$AR_03 0.000141 0.000228 0.50 1.94 57 YANG_BREAST_CANCER_ESR1_UP 0.000181 0.000289 0.52 1.96 48 DELACROIX_RARG_BOUND_MEF 0.000414 0.000610 0.29 1.48 357 V$AR_02 0.000544 0.000784 0.54 1.91 38 DELACROIX_RAR_BOUND_ES 0.000769 0.001080 0.27 1.42 443

Table S2. GSEA (gene set enrichment analysis) in T47D cells treated with BYL719. GSEA

was performed in a subset of pathways that includes transcription factor binding sites of ER,

FOXA1 (gene name HNF3ALPHA), PBX1, PR, GR, AR, VDR, TR, PPARα, PPARγ, and

pathways with the search terms: ER, RAR, and RXR. GSEA was performed in T47D cells

treated with BYL719 for 8h versus the control cells (FDR < 0.05). P adj, adjusted P value. ES,

enrichment score. NES, normalized enrichment score. Size represents the number of genes

enriched in the dataset.

!

Pathway P value P adj ES NES Size DUTERTRE_ESTRADIOL_RESPONSE_24HR_DN 1.440E-06 3.814E-06 0.51 2.54 494 V$PBX1_01 1.560E-06 3.814E-06 0.45 2.10 246 V$HNF3ALPHA_Q6 1.591E-06 3.814E-06 0.46 2.09 202 V$ER_Q6_01 1.551E-06 3.814E-06 0.41 1.90 262 V$GR_Q6_01 1.548E-06 3.814E-06 0.47 2.18 267 V$AR_Q6 1.562E-06 3.814E-06 0.47 2.14 244 DELACROIX_RAR_BOUND_ES 1.459E-06 3.814E-06 0.36 1.75 443 BHAT_ESR1_TARGETS_VIA_AKT1_DN 1.713E-06 3.829E-06 0.71 2.80 82 BHAT_ESR1_TARGETS_NOT_VIA_AKT1_DN 1.706E-06 3.829E-06 0.69 2.73 87 DUTERTRE_ESTRADIOL_RESPONSE_6HR_DN 1.701E-06 3.829E-06 0.61 2.45 90 YANG_BREAST_CANCER_ESR1_LASER_UP 1.807E-06 4.001E-06 0.73 2.36 31 V$PR_Q2 3.110E-06 6.431E-06 0.39 1.80 256 YANG_BREAST_CANCER_ESR1_UP 3.537E-06 6.995E-06 0.60 2.14 48 V$AR_02 7.161E-06 1.326E-05 0.62 2.12 38 DELACROIX_RARG_BOUND_MEF 1.499E-05 2.658E-05 0.34 1.63 357 MASSARWEH_RESPONSE_TO_ESTRADIOL 2.100E-05 3.622E-05 0.55 2.04 58 V$PPARA_01 3.231E-05 5.363E-05 0.62 2.07 36 YANG_BREAST_CANCER_ESR1_BULK_UP 0.000166 0.000246 0.65 2.03 26 BIOCARTA_RARRXR_PATHWAY 0.001446 0.001887 0.70 1.89 15

Table S3. GSEA (gene set enrichment analysis) in MCF7 xenografts tumors treated with

BYL719. GSEA was carried out on a subset of pathways that includes transcription factor

binding sites of ER, FOXA1 (gene name HNF3ALPHA), PBX1, PR, GR, AR, VDR, TR,

PPARα, PPARγ, and pathways with the search terms: ER, RAR, and RXR. GSEA was

performed in MCF7 xenografts tumors treated with BYL719 for 7 days versus the control tumors

(FDR < 0.05). P adj, adjusted P value. ES, enrichment score. NES, normalized enrichment score.

Size represents the number of genes enriched in the dataset.

!

Pathway P value P adj ES NES Size DUTERTRE_ESTRADIOL_RESPONSE_6HR_DN 4.100E-06 9.270E-05 0.67 2.94 90 BHAT_ESR1_TARGETS_VIA_AKT1_DN 3.930E-06 9.270E-05 0.66 2.85 82 BHAT_ESR1_TARGETS_NOT_VIA_AKT1_DN 4.030E-06 9.270E-05 0.62 2.70 87 V$HNF3ALPHA_Q6 6.690E-06 9.810E-05 0.37 1.80 202 V$GR_01 1.300E-05 0.000141 0.35 1.72 194 V$PR_01 4.120E-05 0.000321 0.38 1.76 139 YANG_BREAST_CANCER_ESR1_UP 0.000995 0.003757 0.47 1.82 48 V$AR_03 0.000995 0.003757 0.44 1.78 57 YANG_BREAST_CANCER_ESR1_BULK_UP 0.001545 0.005441 0.57 1.89 26 V$PPARA_01 0.001901 0.006446 0.50 1.82 36

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