HOXB7 is an ERα cofactor in the activation of HER2 and multiple ER target leading to endocrine resistance

Kideok Jin, Sunju Park, Wei Wen Teo, Preethi Korangath, Sean Soonweng Cho, Takahiro

Yoshida, Balázs Győrffy, Chirayu Pankaj Goswami, Harikrishna Nakshatri, Leigh-Ann Cruz,

Weiqiang Zhou, Hongkai Ji, Ying Su, Muhammad Ekram, Zhengsheng Wu, Tao Zhu, Kornelia

Polyak, and Saraswati Sukumar

Supplemental Methods

Plasmids

Full-length promoter region including ER and one of two different HOXB7 binding sites of CA12 was cloned into the pGL3 promoter luciferase construct. Full length miR196a cDNA was cloned into the pcDNA3.1 vector. Full-length promoter region including binding region of miR-196a was cloned into pGL3 promoter luciferase plasmid. 3’UTR region of HOXB7 was cloned into pGL2 promoter luciferase plasmid. The full-length human HER2 plasmid was a generous gift from Daniel

Leahy (Johns Hopkins University, Baltimore, MD) and the HER2 cDNA was cloned to pLPCX vector.

EGFR WT, pRetrosuper Myc shRNA, Lenti-sh1368 knockdown c-myc were purchased at Addgene

(1-3). siRNAs against HOXB7 were purchased at Invitrogen and shRNAs against HOXB7 was purchased at Thermo scientific.

Immunohistochemistry

Immunohistochemical analysis of HOXB7 and HER2 expression was performed using monoclonal antibodies against HOXB7 (Santa Cruz Biotechnology) and HER2 (Cell signaling). After blocking with 5% goat serum in PBST for 1 hour at room temperature, the sections were treated with the HOXB7 or HER2 antibodies overnight at 4°C, then the peroxidase conjugated streptavidin

1 complex method was performed, followed by the 3, 3' diaminobenzidine (DAB) procedure according to manufacturer’s protocols (VECTASTAIN Elite ABC Kit, Vector Lab).

Site-directed mutagenesis

3’UTR-HOXB7-Luciferase mutant and pcDNA3.1-MYC mutants (S62A and T58A) were generated using a QuikChange site-directed mutagenesis kit (Stratagene, Santa Clara, California). The sequences were confirmed by automated DNA sequencing. Primer sequences were: MYC (T58A),

Forward (5’-GAG CTG CTG CCC GCC CCG CCC CTG TC-3’), Reverse (5’-GAC AGG GGC GGG

GCG GGC AGC AGC TC-3’); MYC (S62A), Forward (5’-CAC CCC GCC CCT GAC CCC CTA GCC

GCC-3’), Reverse (5’-GGC GGC TAG GGG TCA GGG GCG GGG TG-3’).

Analysis of the Metabric dataset

Illumina microarray data generated by the Metabric consortia were downloaded from EGA (4). The database contains 1,988 patients, the average overall survival is 8.07 years; 76% of the patients are ER positive and 47.3% are lymph node positive. The raw Illumina microarray files were imported into R and summarized using the beadarray package (5). Follow-up was censored after 15 years.

For annotation, the illuminaHumanv3 database of bioconductor was used

(http://www.bioconductor.org). Quantile normalization was performed using the preprocessCore package (6).

ChIP-PED analysis

The ChIP-PED data set is publicly available from http://www.biostat.jhsph.edu/~gewu/ChIPPED/

(7), which contains 13,182 human samples from Affymetrix

U133A Array in NCBI Gene Expression Omnibus (GEO). The data set is a matrix with columns corresponded to samples and rows corresponded to genes. The cross-sample gene expression correlation between two genes was calculated by the Pearson correlation coefficient between the

2 corresponding rows in the data set. The density curves (Figure 1B) for the distributions of correlations from HOXB7 versus 144 ER target genes and that from HOXB7 versus 144 random genes were estimated using kernel density estimation. Two tailed t-test was performed to evaluate the significance of difference between these two distributions (P<10-15). We observed that there are two groups of samples separated by the diagonal line y=x, one group (y

S1D) showed good correlation between ER and HOXB7 gene expression and the other group (blue circles in Figure S1D) did not. To rank the ER target genes, 8,425 samples from the group with good correlation between ER and HOXB7 were used to compute the cross-sample correlations between ER target genes and HOXB7 (or ER). To estimate the significance of correlation, given a target gene, HOXB7 (or ER) gene expression data was permuted 10,000 times and a null distribution was constructed by the correlations between the target gene and each permuted

HOXB7 (or ER) gene expression. Then, the density curve of the null distribution was estimated by kernel density estimation and the p-value was calculated as the area under the curve past the observed correlation. The ER target genes were ranked according to their average cross-sample correlations with HOXB7 and ER.

In Figure 1B, the p-value was calculated as follows: Given a gene t, let x_t=(x_t1,〖 …,x〗 _tN ) be a vector consisting of its expression values in N different samples. Let y=(y_1,〖 …,y〗 _N ) be a vector consisting of expression values of HOXB7 in the same N samples. The correlation (r_t) between gene t and HOXB7 was calculated as the Pearson correlation coefficient between x_t and y. To determine whether HOXB7 is significantly correlated with ER target genes, we first calculated the correlation between each of the 144 ER target genes and HOXB7, yielding a set of 144 correlation coefficients A={r_t }. Next, we randomly selected 144 genes and calculated the correlation between each of the random genes and HOXB7, yielding another set of 144 correlation coefficients B={r_t^' } which served as the control. Finally, a two-sample t-test was performed to

3 evaluate whether the two sets of correlation coefficients A and B have the same mean or not. The two-sided p-value (P<10-15) was reported.

In Figure S1E to K, the p-values for the correlation between HOXB7 (or ER) and ER target genes were calculated as follows: To rank the ER target genes, 8,425 samples from the group with good correlation between ER and HOXB7 were used to compute the cross-sample correlations between

ER target genes and HOXB7 (or ER). To estimate the significance of correlation between HOXB7 and a given ER target gene, HOXB7 (or ER) gene expression data was permuted 10,000 times and a null distribution was constructed by the correlation between the target gene and permuted HOXB7

(or ER) gene expression. For example, consider HOXB7 and a target gene t. Let 〖 x〗 _t=(x_t1,〖 …,x〗 _tN ) be a vector consisting of expression values of gene t in N microarray samples (N=8,425). Let y=(y_1,〖 …,y〗 _N ) be a vector consisting of expression values of HOXB7 in the same N samples. Let r_t be the Pearson correlation coefficient between 〖 x〗 _t and y. To determine the p-value of r_t , we randomly permuted y 10,000 times. In each permutation i

(i=1,…,10000), the order of the N numbers in y was shuffled to create a permuted vector y^((i)) (e.g., y=(2,3,6,5,…,10) may become y^((i))=(6,10,3,2,…,5) after permutation). This process broke the association between HOXB7 and the target gene t. The correlation coefficient between 〖 x〗 _t and each permuted y^((i)) was computed and denoted as r_t^((i)). This produced 10000 correlation coefficients. We then applied kernel density estimation (using R function density) to these 10000 r_t^((i))s to estimate the probability density function of the correlation coefficients of permuted data.

The estimated density function was used as the null distribution for evaluating the significance of the observed correlation r_t. The p-value of r_t was calculated as the area under the density curve with x-axis values larger than the observed correlation r_t (Figure 1B).

4

DNase-seq analysis

DNase I hypersensitive sites sequencing (DNase-seq) (8) is a state-of-the-art technology to identify accessible chromatin regions genome-wide. DNase I hypersensitive sites are proved to be robust marker (9) of regulatory DNA regions which enables one to screen candidate binding sites (TFBSs) through DNase-seq experiment. The ENCODE project has performed

DNase-seq experiments on a number of human cell types which provide a good resource to study

TFBSs. In this study, we obtained the DNase hotspot dataset (downloaded from http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeUwDnase/, the DNase hotspots were generated by ENCODE using the hotspot algorithm of estradiol treated MCF7 cell line. (10) Because the data contained two replicates, we first screened for DNase hotspots which were overlapped between the two replicates. Then, we searched for DNase I hypersensitive

(DNase-seq) hotspots in the proximal promoter region (15,000 base pairs upstream of transcript start site) for MYC gene and found five different DNase peaks, (1) chr8: 128,735,412-128,735,765

(2) chr8: 128,737,633-128,738,395 (3) chr8: 128,738,780-128,739,066 (4) chr8: 128,739,355-

128,740,018 (5) chr8: 128,746,080-128,746,796. We selected two peaks close to the MYC transcription starting site. Using the regions between the peaks, we chose three putative HOXB7 binding sites based on the “TAAT” consensus HOX-binding motif. We created MYC luciferase constructs containing an ER enhancer region tagged to three different putative HOXB7 binding sites

(MYC-B7-1, MYC-B7-2, and MYC-B7-3) using previously studied MYC enhancer/promoter luciferase constructs containing ER-binding site (11).

Supplemental References

1. Lin CH, Jackson AL, Guo J, Linsley PS, Eisenman RN. Myc-regulated microRNAs attenuate embryonic stem cell differentiation. The EMBO journal. 2009;28:3157-70.

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2. Popov N, Wanzel M, Madiredjo M, Zhang D, Beijersbergen R, Bernards R, et al. The ubiquitin-specific protease USP28 is required for MYC stability. Nature cell biology. 2007;9:765-

74.

3. Greulich H, Chen TH, Feng W, Janne PA, Alvarez JV, Zappaterra M, et al. Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants. PLoS Med. 2005;2:e313.

4. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature.

2012;486:346-52.

5. Dunning MJ, Smith ML, Ritchie ME, Tavare S. beadarray: R classes and methods for

Illumina bead-based data. Bioinformatics (Oxford, England). 2007;23:2183-4.

6. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics (Oxford,

England). 2003;19:185-93.

7. Wu G, Yustein JT, McCall MN, Zilliox M, Irizarry RA, Zeller K, et al. ChIP-PED enhances the analysis of ChIP-seq and ChIP-chip data. Bioinformatics (Oxford, England). 2013;29:1182-9.

8. Crawford GE, Holt IE, Whittle J, Webb BD, Tai D, Davis S, et al. Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome

Res. 2006;16:123-31.

9. Gross DS, Garrard WT. Nuclease hypersensitive sites in chromatin. Annual review of biochemistry. 1988;57:159-97.

10. John S, Sabo PJ, Thurman RE, Sung MH, Biddie SC, Johnson TA, et al. Chromatin accessibility pre-determines glucocorticoid binding patterns. Nature genetics.

2011;43:264-8.

11. Wang C, Mayer JA, Mazumdar A, Fertuck K, Kim H, Brown M, et al. Estrogen induces c- myc gene expression via an upstream enhancer activated by the and the AP-

1 transcription factor. Molecular endocrinology. 2011;25:1527-38.

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Supplemental Figures Supplemental B Figure S1 A

Vector.1

Vector.2

B7-1.1

B7-1.2

Up Down ER target ER target

Vector.1 genes genes ( C Vector.2 Responseto hormone Systemdevelopment Dutertre Signaltransduction Metabolicprocess B7-1.1 Cell migration et al, 2010 et al, B7-1.2 0 ) 5

% Gene/Term Vector.1

Vector.2 10 ** ** B7-1.1 * ** B7-1.2 ** FOS MMP9 CKB ERBB2 NCOR1 MTA1 HSP90AA1 SAFB RARA S100A6 PELP1 NR3C1 BRCA1 NR2F6 LGALS1 MYC VEGFA NCOA2 FOXA1 MED1 JUNB RPLP0 PHB2 BCAR1 EBAG9 BDNF RALA L1CAM NAB2 SOCS3 BCL2L1 PDZK1 15 Figure S1 D E CA12

R=0.552936, P=6.97e-17 R=0.506948, P=6.78e-17

1 1 0 1 2

-

0 0 2 4 6

ER expression ER

HOXB7 expression HOXB7 HOXB7 HOXB7 expression -1 0 1 2 3 -1 0 1 2 3

CA12 expression CA12 expression

2 2 0 2 4 6 8 - -2 0 2 4 6 8 ER expression

RET TFF1 F G

R=0.519612, P=7.01e-17 R=0.505052, P=8.08e-17 R=0.523147, P=7.20e-17 R=0.519696, P=7.29e-17

1 1 0 1 2 1 0 1 2

- -

0 0 2 4 6

0 0 2 4 6

ER expression ER

ER expression ER

HOXB7 HOXB7 expression HOXB7 expression 0 2 4 6 8 0 2 4 6 8 -1 0 1 2 3 -1 0 1 2 3 RET expression RET expression TFF1 expression TFF1 expression

H GREB1 I PGR

R=0.440489, P=1.20e-16 R=0.40374, P=1.21e-16 R=0.392191, P=1.78e-16 R=0.337965, P=1.40e-16

1 1 0 1 2 1 0 1 2

- -

0 0 2 4 6 0 2 4 6

ER expression ER expression ER

HOXB7 HOXB7 expression HOXB7 expression 0 2 4 6 0 2 4 6 0 2 4 6 8 10 0 2 4 6 8 10 GREB1 expression GREB1 expression PGR expression PGR expression

J CCND1 K RARA

R=0.392028, P=1.20e-16 R=0.341632, P=1.11e-16 R=0.295250, P=1.53e-16 R=0.325925, P=1.40e-16

1 1 0 1 2 1 0 1 2

- -

0 0 2 4 6 0 2 4 6

ER expression ER expression ER

HOXB7 HOXB7 expression HOXB7 expression -1 0 1 2 3 -1 0 1 2 3 -3 -2 -1 0 1 2 3 4 -3 -2 -1 0 1 2 3 4 CCND1 expression CCND1 expression RARA expression RARA expression Figure S1 (related to Figure 1): HOXB7 microarray and ChIP-PED analysis. A, Identification of ER target genes using microarray analysis and comparisons with those identified in (Dutertre et al., 2010). Expression values are shown as a ratio relative to vector control cells and represented in a red-green color scale (red = high expression; green = low expression). B, MR-GSE was used to test for enrichment of gene sets in both up- and down-regulated genes by HOXB7 compared to vector control. As expected, there was no significant enrichment of the Hsiao_Housekeeping gene set in either direction (FWER > 0.05). However, there were significant overlaps of upregulated or downregulated genes by HOXB7 with the Dutertre Estradiol Response 24hr Up or down gene sets (FWER < 0.001). C, Gene Ontology analysis of HOXB7-overexpressing MCF-7 cells compared to vector control cells. D, 8,425 samples with good correlation between ER and HOXB7 gene expression were selected (red) for ranking of ER target genes from ChIP-PED dataset. E-K, Examples are shown of highly ranked target genes, CA12, RET, TFF1, GREB1, PGR, CCND1 and RARA. Scatter plots of gene expression show significant correlation between the expression of ER or HOXB7 and each target gene. Figure S2

A B C T47D ERIN T47D 12 ERIN 14 * Vector Vec B7 Vec B7 12 Vector * * * 10 * HOXB7 10 HOXB7 8 MYC * * 8 6 * * * Bcl-2 6 4 * * 4 * * * * * * 2 * * 2 * * * CyclinD1 0 0

HOXB7

Relative mRNA levels mRNA Relative

FOS

PGR

MYC

TFF1

Relative mRNA levels mRNA Relative

FOS

CA12

XBP1 SDF1

PGR

MYC

HER2

RAPA

TFF1

CA12 SDF1

XBP1 β-Actin

HER2

RAPA

NRIP-1

GREB1

NRIP-1

CCND-1

GREB1 CCND1

RT-qPCR of ER target genes with Vehicle, E , and TAM in MCF-7-HOXB7-1 D 2

XBP1 CXCL12 FOS CCND1

* * 30 15 5 * 5 Veh * 25 E2 4 4 20 10 3 3 15 2 2 10 5

5 1 1

Relative Relative expression Relative expression Relative expression Relative Relative expression 0 0 0 0

Vector HOXB7 Vector HOXB7 Vector HOXB7 Vector HOXB7 *

20 4 8 6 *

Veh * TAM * 15 3 6 4 10 2 4 2

5 1 2

Relative Relative expression Relative expression Relative expression Relative Relative expression 0 0 0 0 Vector HOXB7 Vector HOXB7 Vector HOXB7 Vector HOXB7 RT-qPCR of ER target genes with Vehicle, E , and TAM in T47D cells E 2 RARA GREB1 XBP1 CA12 CCND1 TFF1 0 5 10 0 25 500 2 4 0 2 4 0 2.5 5 0 5 10 15

Veh Vector HOXB7 E2 * * * * * *

TAM * * * * * *

CXCL12 MYC NRIP1 PGR FOS BCL-2 0 2 4 6 0 2.5 5 0 5 10 0 10 20 0 5 10 0 2.5 5

Veh

E2 * * * * * *

TAM * * * * * *

F RT-qPCR of ER target genes with Vehicle, E2, and TAM in ERIN cells

RARA MYC TFF1 CCND1 CXCL12 0 2 4 6 0 2 4 6 0 2 4 6 0 5 10 0 5 10

Veh Vector HOXB7 E2 * * * * *

TAM * * * * * Figure S2

ERIN G ERIN H 8 * Vector Vehicle E2 TAM 7 HOXB7 6 Vector 5 4 * 3 2 HOXB7 1 Relative cellular viability cellular Relative 0 Veh E2 TAM

I HMEC J TMR 2 * Vector HOXB7 Veh E2 Tam GST-HOXB7 1.5 WB: ERα Pull down

1 Lysate WB: ERα * GST-HOXB7 0.5

Ponceau Relative cellular viability cellular Relative 0 GST Veh E2 TAM

Figure S2 (related to Figure 1): HOXB7 enhances expression of ER target genes. A, Real time RT-qPCR analysis of ER target genes in stable T47D-HOXB7 and B, ERIN-HOXB7 cells. C, Immunoblot analysis of ER target genes in T47D-HOXB7 and ERIN-HOXB7 cells. D, RT-qPCR analysis of ER target genes in MCF-7-HOXB7, E, T47D-HOXB7 and F, ERIN-HOXB7 cells treated with Vehicle, E2, and TAM. G, Cellular viability assay and H, Crystal violet staining of ERIN-Vec and ERIN-HOXB7 cells treated with Vehicle, E2 and TAM in absence of EGF for 72 hours. I, Cellular viability assay of HMEC cells co-transfected transiently with ER and either Vector or HOXB7 treated with Vehicle, E2 and TAM for 72 hours. J, GST-HOXB7 pull down assay with lysates from TMR cells treated with Vehicle, E2 and TAM and Western blot analysis of ER. Gel stained with Ponceau for loading controls.. Mean ± s.d. for three independent replicates. (*P<0.001). Figure S3

A

RARA

GREB1

MYC

XBP1

FOS

CCND1

CXCL12

NRIP1

CA12

B C HOXB7 ChIP to ER binding sites ER ChIP to ER sites in MCF-7-HOXB7 6 Vehicle E2 TAM Input IgG GREB1 Input IgG MYC Input IgG 5 * * XBP1 * 4 * * 3 * Input IgG CCND1 Input IgG CXCL12 Input IgG * * * * * * * * * NRIP1 2 * * * 1

Input IgG FOS Input IgG CA12 Input IgG RARA Vehicleover fold Relative 0 D MCF-7-HOXB7 Figure S3 Pol II ChIP TFIIβ Re-ChIP 1.2 1 0.8 0.6 0.4 vehicle 0.2

0 Relative fold over over fold Relative Vehicle E2 TAM E MCF-7-HOXB7 F MCF-7-HOXB7 5 HOXB7 ChIP HOXB7 Re-ChIP Vehicle IgG ChIP HOXB7 Re-ChIP Vehicle 1.4 4.5 E2 E2 * * 4 * 1.2 TAM * TAM 3.5 * * 1 3 * * * * * * * * 0.8 2.5 * * * 2 * 0.6 1.5 0.4 1 0.5 0.2

Relative fold over Vehicleover fold Relative 0 Relative fold over Vehicleover fold Relative 0

TMR MCF-7 H G Cofactor ChIPs to ER site at CA12 6 Cofactor ChIPs to ER site at CA12 10 * * 9 5 8 Vector Control si 7 E treatment 4 2 HOXB7 E treatment HOXB7 si 6 2 * * 3 5 * * 4 2 * 3 * * * 2 * 1 * 1 * * * * * *

Relative fold over vector over fold Relative * * si conrol over fold Relative * 0 * 0

I Cofactor ChIPs to ER sites in MYC J 12 5 Cofactor ChIPs to ER sites in MYC

* si 4.5 * Vector 10 4 Control si E2 treatment HOXB7 3.5 E treatment 8 control 2 HOXB7 si 3 * * 6 * 2.5 * * 2 * * 4 * 1.5 * 1 2 * * 0.5 * * * Relative fold over Vectorover fold Relative * * * * *

* over fold Relative 0 0

K Cofactor ChIPs to ER site in MYC L Cofactor ChIPs to ER site in MYC 2.5 4 Vector Control si * * 3.5 HOXB7 si HOXB7 2 3 * TAM treatment 2.5 1.5 TAM treatment * * 2 * * * * 1 1.5 * * * * 1 0.5 * * * * * * 0.5 * * Vectorover fold Relative * Relative fold over Vectorover fold Relative * 0 0 Figure S3 (related to Figure 2): Validation of HOXB7 binding events to ER target genes. A, Schematic representation of know ER binding sites at RARA, GREB1, MYC, XBP1, FOS, CCND1, CXCL12, NRIP1, and CA12 gene loci. Red box indicates the ER binding site selected for ChIP analysis (Hurtado et al., 2011). B, Representative images of PCR amplification products resolved on 2% agarose gel following HOXB7 ChIP analysis of ER binding site at each gene loci in MCF-7- HOXB7 cells. C, ER ChIP assay of known ER-binding sites of ER target genes in MCF-7-HOXB7 cells treated with vehicle, estrogen and tamoxifen. D, TFIIβ–re-ChIP following RNA Polymerase II ChIP to GAPDH promoter and E, HOXB7-re-ChIP following HOXB7 ChIP factor to the ER binding site in the CA12 gene locus was performed as positive control in MCF-7-HOXB7 cells treated Vehicle, E2 and TAM. F, HOXB7-re-ChIP following IgG ChIP was performed as a negative control in the same condition as (F). G, ChIP analysis of each indicated cofactor to ER binding site in the CA12 gene locus in MCF-7-HOXB7 cells compared to MCF-7-vector cells treated with estrogen. H, Same ChIP analysis of each cofactor was performed as (G) in HOXB7-depleted TMR-siHOXB7 cells compared to TMR-si-Scr. I, Same ChIP analysis of each cofactor was performed as (G and H) at the MYC gene locus in MCF-7-HOXB7 cells or J, HOXB7-depleted TMR-siHOXB7 cells treated with estrogen or K and L, tamoxifen. RNA Polymerase II ChIP analysis was performed to CA12 or MYC promoter region as a positive control. Mean ± s.d. for three independent replicates. (*P<0.001). Figure S4

A ChIPs to ER site at CA12 in MCF-7 B ChIPs to ER site at MYC in MCF-7 7 NCOR AIB1 6 NCOR AIB1 * * 6 * * 5 * 5 * * * 4 4 3 * *

3 enrichment fold 2 2

1 Relative fold enrichment fold Relative 1 Relative 0 0 0 0.5 ug 1 ug 2 ug 0 0.5 ug 1 ug 2 ug

HOXB7 expression HOXB7 expression

C

1.7 kb 3’ 5’

D ER HOXB7-1 HOXB7-2 pGL3 promoter LUC CA12 WT

ER HOXB7-2 pGL3 promoter LUC CA12-HOXB7-1Δ

ER HOXB7-1 pGL3 promoter LUC CA12-HOXB7-2Δ

Vector E MCF-7 HOXB7-WT G HOXB7-ΔH3 9 * 8 * 7 6 5

RLU 4 3 2 1 0 WT HOXB7-2Δ ER HOXB7-1 C-MYC prom LUC MYC-B7-1 F MCF-7 1.2 Veh Fulv * * ER HOXB7-2 C-MYC prom LUC MYC-B7-2 1

0.8 ER HOXB7-3 C-MYC prom LUC MYC-B7-3

0.6 RLU 0.4

0.2

0 WT HOXB7-2Δ H MCF-7 I MCF-7-TMR Figure S4 3000 Vector * 1500 Control sh HOXB7 2500 HOXB7 sh * * 2000 1000 *

1500

RLU RLU 1000 500 500

0 0 B7-1 B7-2 B7-3 B7-1 B7-2 B7-3 J P4 PstI PstI PstI PstI PstI PstI

P5 ER P1 P2 P3 HOXB7 P6 binding binding site site M coactivators 65 kb

NCOR TAM K 1 2 3 4 5 ERα MYC M - + - + - + - + - + : E2 TAM sensitive cell 1: P1 – P3 2: P2 – P3 1000 bp 3: P1 – P4 500 bp 4: P2 – P4 5: P5 – P6 L 35 * N Ligase - 30 Ligase + NCOR 25 coactivators 20 TAM ERα 15 HOXB7 * MYC 10

Relative fold change fold Relative 5 TAM Resistant cell 0 Veh E2 TAM Figure S4 (related to Figure 2): HOXB7 is required for ER target gene expression. A, NCOR and AIB1 ChIP assay at ER binding site in CA12 or B, MYC locus using MCF-7 cells transiently transfected with HOXB7 plasmid at different doses. C, Schematic representation of ER binding site at CA12 gene loci. D, Schematic representation of CA12 luciferase constructs that include both ER binding site and two putative candidates of HOXB7-binding sites (CA12-WT), or deletion of HOXB7-binding site 1 (CA12-HOXB7-1Δ), or deletion of HOXB7-binding site 2 (CA12-HOXB7-2Δ). E, Luciferase assay was performed in MCF-7 cells transiently co-transfected with each HOXB7 WT or MT (ΔH3: deletion of Helix domain 3 of homeodomain) plasmids along with each CA12-WT and CA12- HOXB7-2Δ luciferase construct compared to Vector control. F, The same assay was performed as in (K) in MCF-7 cells treated with vehicle and fulvestrant. G. Schematic representation of MYC promoter luciferase constructs including both ER binding site and three HOXB7 binding sites (MYC-B7-1, MYC- B7-2, MYC-B7-3). ). H, Luciferase assay was performed in MCF-7 cells transiently co-transfected with HOXB7 plasmid and each of the luciferase constructs and compared to vector control. I, The same luciferase assay in HOXB7-depleted TMR cells using HOXB7-shRNA. J, Schematic of MYC gene region depicting the location of the PstI sites and the primers used in the 3C assays. K, 3C assays were performed with MCF-7-HOXB7 cells treated with or without estrogen for 45 minutes. Ligation products were detected by PCR using the primers indicated. L, Real time quantitative RT-PCR analysis of region P1-P3. 3C assay was performed with and without ligase treatment in MCF-7-HOXB7 cells after vehicle, estrogen, and tamoxifen treatment. M, The inhibition model of MYC transcription by the interaction between ER bound TAM and NCOR, a corepressor in TAM-sensitive cells. N, The activation model of MYC transcription by the interaction between the HOXB7-ER complex and coactivators in TAM-resistant cells. Mean ± s.d. for three independent replicates. (*P<0.001). Figure S5

A ChIP-PED B C R=0.432938 R=0.467089 MCF-7 TMR Vec H12 P=1.45e-16 P=1.10e-16 HER2 EGFR P-AKT(S473) HER2

ER HER3 T-AKT

HOXB7

1 1 0 1 2 0 0 2 4 6 - HER4 P-4EBP1 β-Actin 0 2 4 6 8 0 2 4 6 8 T-4EBP1 HER2 HER2 Actin

T47D ERIN D Vec B7 Vec B7 E F BT474 TMR Control HER2 HOXB7 siRNA siRNA P-AKT(S473) Control shRNA HOXB7 shRNA

T-AKT Veh

P-4EBP1 Veh T-4EBP1 TAM Actin TAM H G TMR 120 Control sh 120 TMR 100 B7 sh#1 100 80 * 80 B7 sh#2 60 * 60 * 40 40 * * K MCF-7-Vec MCF-7-HOXB7

20 20 * * Cellular viability (%) viability Cellular Cellular viability (%) viability Cellular 0 * * 0 * TAM TtzmT LapaL

HER2

3’ UTR-HOXB7-Luc I J 800 TMR 600

Veh Ttzm Veh Lapa 400

RLU HOXB7 HOXB7 200 * β-Actin * 0 Veh Ttzm Veh Lapa Figure S5 (related to Figure 3): Correlation between HOXB7 and HER2 expression. A, Scatter plots of HER2 gene expression show a significant correlation between the expression of ER or HOXB7 and HER2 by ChIP-PED analysis. B, Immunoblot analysis of the HER family. HER1 (EGFR) and HER2 are overexpressed, while HER3 and HER4 show no change in TAM resistant cell lines, TMR1 and TMR2 cells compared to MCF-7. C, Immunoblot analysis of HER2, P-AKT (ser473), and P-4EBP1 in TMR and H12 cells. D, Immunoblot analysis of HER2, P-AKT, and P-4EBP1 in T47D-HOXB7 and ERIN-HOXB7 cells. E, Crystal violet staining of HOXB7-depleted TMR-shHOXB7 after 7 days in culture with and without TAM treatment. F, Representative phase contrast photomicrograph images of HOXB7-depleted BT-474 -HOXB7siRNA. G, Cellular viability assay was performed in TMR cells or H, in HOXB7-depleted TMR-shHOXB7 compared to control shRNA cells after treatment with 2 uM tamoxifen (TAM), 100 ug/ml trastuzumab (Ttzm) and 1 uM Lapatinib (Lapa). I, Western blot analysis of HOXB7 in TMR cells treated as in (G). J, Luciferase assay was performed in TMR cells transiently transfected with 3’UTR-HOXB7-Luciferase construct and grown in Vehicle, Trastuzumab or Lapatinib containing medium. K, Representative photomicrograph of immunohistochemical analysis of HOXB7 and HER2 in MCF-7-vector and MCF-7-HOXB7 xenograft tumors from Fig. 3F. Mean ± s.d. for three independent replicates. (*P<0.001). Figure S6

A ERBB2

MCF-7 TMR B C ER binding site of HER2 ER binding site of HER2 6 6 Vector Control si * * HOXB7 si 5 HOXB7 5 * * * 4 4 * * 3 3 * * 2 * 2 * 1 1 * * Relative fold over control si * * Relative fold over Vector over fold Relative * * * * * 0 0

TCGA data set D E F ** HER2 mRNA

4 Veh

TAM * 3

2 HOXB7 HOXB7 expression

1 HOXB7 expression Relative expression Relative

0 ***P<0.001 **P<0.01 Vector HOXB7 Her2 LO HER2 MED Her2 HI 2 2 4 6 8 10 12 *P<0.05 Basal Her2 LumA LumB n= 75 46 187 104

Figure S6 (related to Figure 3): HOXB7 regulates HER2 expression. A, Schematic representation of ER binding site at ERBB2/HER2 gene locus. B, ChIP analysis of ERα, HOXB7, and cofactors at ER binding site of the HER2 gene locus in MCF-7-HOXB7 cells compared to MCF-7-vector cells. C, Same ChIP analysis of ERα, HOXB7, and cofactors was performed as (B) in HOXB7-depleted TMR cells. RNA Polymerase II ChIP analysis at HER2 promoter region. D, RT-qPCR analysis of HER2 genes in MCF-7-HOXB7 cells treated with Vehicle and TAM. E, Box plots displaying HOXB7 mRNA levels in 48 breast cancer cell lines. Her2Lo contained 16 cell lines with HER2 expression (1 to 163, relative HER2 expression); Her2MED contained 16 cell lines with HER2 expression (194 to 2011). Her2HI contained 16 cell lines with HER2 expression (2230 to 154612) and F, HOXB7 expression in subtypes of breast cancer in the TCGA data set. Basal-like (n=75), HER2 (n=46), Luminal A (LumA) (n=187), Luminal B (LumB) using Mean ± s.d. for three independent replicates. (*P<0.001, **P<0.0001). Figure S7

A B

3’UTR-HOXB7-Luc 3’UTR-HOXB7-Luc 1200 450 MCF-7 1000 * 400 Vector miR-196a 350 800 300

250 600 RLU

RLU 200 400 150 * 100 200 50 0 0 MCF-7 WT MutMT Vector H12 C D 3’UTR-HOXB7-Luc 3’UTR-HOXB7-Luc MCF-7 1600 600 * * 1400 500 1200 400 1000

800 300

RLU RLU 600 200 400 J 100 200 0 0 MCF-7 TMR Control miR196 inh.

E ERE-Luc 1.2 1 0.8 0.6 RLU 0.4 0.2 0

TMR MCF-7

Figure S7 (related to Figure 4): miRNA-196a regulates HOXB7 expression. A, Luciferase assay was performed in MCF-7 cells transiently co-transfected with miR-196a and 3’UTR-HOXB7-Luciferase construct WT or Mut (10 bp deletion of miR-196a target sequence). The same assay was performed in B, H12 (MCF-7-HER2) or C, TMR cells transiently transfected only with WT- 3’UTR-HOXB7-Luciferase construct. D, Luciferase assay was performed in MCF-7 cells transiently co-transfected with miR-196a inhibitor and 3’UTR-HOXB7-Luciferase construct WT. E, ERE-Luciferase assay was performed in TMR cells or MCF-7 cells transiently co-transfected with miR-196a or in miR-196a-inhibitor and ERE-Luciferase construct. Mean ± s.d. for three independent replicated. (p<0.001). Figure S8

A B 120 Chase time (min) Vector 100 H12 0 10 20 30 40 60 80 80 Vector MYC 60 H12 MYC 40

20 Relative protein level protein Relative 0 0 10 20 30 40 60 80 minute C D 120 Chase time (min) 100 0 10 20 30 40 60 80 Vector 60 40 Vector HOXB7 20 HOXB7

Relative MYC protein MYC Relative 0 0 10 20 30 40 60 minutes

E F G MCF-7 TMR BT474 Cont Vector HOXB7 MCF-7 TMR Vec H12 Cont B7 B7 : ShRNA P-MYC (Ser62) MYC (S62) MYC (S62)

P-MYC (Thr58) MYC (T58) MYC (T58)

Total MYC Total MYC Total MYC HOXB7 Actin Actin Actin

miR-196a in TMR H I 2.5 TMR 3.5 * * Control shRNA 3 2 MYC shRNA 2.5 1.5 2 1 1.5 * 0.5 * * 1

Relative expression Relative 0.5 Relative mRNA levels mRNA Relative 0 0 Veh 10058-F4

J 3’UTR-HOXB7-Luc K 3’UTR-HOXB7-Luc 600 800 * 700 500 600 400 500

400 300 RLU RLU 300 200 200 * * 100 100 0 0 Cont sh MYC MYC Vector MYC-TA MYC-SA sh#1 sh#2 Figure S8

L HOXB7 promoter M HER2 promoter N -1802 +90 -533 +38 4 E box Luc E box Luc MCF-7 * TMR

200 400 3 * 7

350 - 2 150 300 250 MCF 1 *

100 200 * *

RLU RLU 150 over fold Relative 0 50 100 ChIP 50 0 0 Cont.sh MYC sh Cont.sh MYC sh O P Q MYC pGL3 prom LUC miR-196a-Luc 6 4 Contol sh Vehicle * * MCF-7 TMR 5 MYC sh 10058-F4 3 10074-G5 100 * 200 4 * 80 * 150 * 3 2 60 100 2 RLU 40 1

1 * 20 50 Relative occupancy Relative * occupancy Relative * 0 0 0 0 ChIP MYC Pol2 ChIP MYC Pol2 Vector MYC MYC Cont MYC MYC S62A T58A sh sh1 sh2

R S T 120 TMR TMR 10058-F4 BT474 10074-G5 100 Control MYC Veh 10 100 Veh 10 100 : uM shRNA shRNA 80 HER2 60 Veh HOXB7 Control shRNA 40 * MYC shRNA * MYC

Cellular viability (%) viability Cellular 20 TAM Bcl-2 0 Actin TAM (uM)

U V W BT474 10058-F4 10074-G5 TMR 120 - + + - + + : MYC inhib. 3’UTR-HOXB7-Luc BT474 Vehicle 600 100 Trastuzumab - - + - - + : HOXB7 500 * 80 HER2 400 * 60 * 300 MYC RLU * 200 40 20 * HOXB7 100 *

* (%) viability Cellular 0 0 Veh 10058-F4 10074-G5 Vehicle 1 5 10 50 Actin 10074-G5 (uM) Figure S8 (related to Figure 5): MYC and miRNA-196a regulates HOXB7 expression. A, Cycloheximide (CHX) chase examining the MYC protein stability in MCF-7-Vector cells compared to H12 cells by Immunoblot analysis. B, Quantification of MYC decay following CHX treatment based on densitometric scanning of the immunohybridization signals. C, The same experiment as (A) in MCF-7-Vector and MCF-7-HOXB7 cells. D, The same analysis for (C). E, Immunoblot analysis of phosphorylation of MYC at serine 62 and threonine 58 residues in MCF-7-HOXB7 or, F, TMR, or G, in HOXB7 depleted TMR and BT474 cells. H, Real time quantitative RT-PCR analysis of miR-196a, HOXB7, and HER2 in MYC depleted TMR cells by MYC shRNA or I, in BT474 treated with 10058- F4. J, Luciferase assay was performed in MCF-7 cells transiently co-transfected with 3’UTR- HOXB7-Luciferase construct and MYC shRNAs, or K, mutant MYC (S62A or T58A) compared to vector control. L, Luciferase assay was performed in TMR cells transiently co-transfected with HOXB7 promoter or M, Her2 promoter Luciferase construct including E box sites and MYC shRNAs. N, ChIP analysis of cofactors at MYC binding site in the miR-196a gene in TMR cells. O, ChIP analyses for a putative MYC binding site and RNA Polymerase II in the miR-196a promoter region in MYC-depleted TMR cells, or P, in TMR cells treated with MYC inhibitor (10058-F4 and 10074-G5). Q, Luciferase assay was performed in MCF-7 cells transiently co-transfected with miR-196a luciferase construct and mutant MYC (S62A or T58A) compared to vector control (Left). Same assay performed in MYC-depleted TMR cells using MYC shRNAs (Right). R, Cellular viability assay in MYC-depleted TMR cells compared to vector control with TAM treatment at different doses. S, Crystal violet staining of MYC depleted TMR cells using MYC shRNA after 7 days in culture with and without TAM treatment. T, Immunoblot analysis of HER2, HOXB7, MYC, and BCL-2 in BT474 cells after treatment with 10058-F4 or 10074-G5 at indicated concentrations. U, Luciferase assay in TMR cells transiently transfected with 3’UTR-HOXB7-Luciferase construct after treatment with 10058-F4 and 10074-G5. V, Cellular viability of BT474 cells treated with 1, 5, 10, 50 uM of 10074-G5 plus 100 ug/ml of trastuzumab for 48 hours. W, Immunoblot showing rescue of HER2 and MYC expression upon enforced HOXB7 overexpression in BT474 cells treated with 10058-F4 or 10074-G5. Mean ± s.d. for three independent replicates. (*P<0.001). Figure S9

A B 40

Vehicle 30 10058-F4 20 Vehicle Trastuzumab Trastuzumab

Body weight (g) weight Body 10 10058-F4 10058-F4 +Trastuzumab T+F4 0 0 1 2 3 4 5 C D BT474 xenografts; HER2 IHC 120 Vehicle Fulvestrant 100 80 * 60 * * Veh. 40 20

0 Cellular viability (%) viability Cellular

F4 E 600

Vehicle ) 3 500 Fulvestrant

400

300 Ttzm 200 *

Tumor volume (mmTumor 100

0 0 1 2 3 4 5 6 7 8 9 Week Ttzm+F4

Tumor #1 Tumor #2 Tumor #3

Figure S9 (related to Figure 5): In vivo analysis of effects of inhibiting MYC and HER2 to overcome TAM-resistance in BT474 cells A, Size of BT474 xenografts in NSG mice after 5 weeks of treatment with vehicle, 10058-F4, trastuzumab, or a combination of 10058-F4 and trastuzumab. B, Mice remained healthy through treatment schedule. No significant weight loss was observed with treatment. C, Representative photomicrograph of IHC analysis of HER2 in three different BT474 xenograft tumors treated with Vehicle, 10058-F4 (F4), or Trastuzumab (Ttzm) alone or in combination (Ttzm+F4). D, Cellular viability assay in TMR, BT474, and MCF-7-HOXB7 cells treated with 1uM Fulvestrant for 72 hours. E, Tumor growth curve of MCF-7-HOXB7 cells implanted s.c. in athymic Swiss female mice and treated with either vehicle or Fulvestrant (10 mg/week) in the absence of an exogenous estrogen supplementation (n=8 tumors per group). Mean ± s.d. for three independent replicates. (*P<0.001). Figure S10

Endocrine therapy (n=1388)

A B

Low HOXB7 Low HOXB7+HER2

High HOXB7+HER2 Probability; RFS Probability; High HOXB7 RFS Probability; (Cox HR=1.54, p=0.0003) (Cox HR=2.43, p=9.4E-06)

van der Vijver dataset (n=295) C D

Low HOXB7+Low MYC Low HOXB7+High MYC

Low HOXB7 OS Probability; High HOXB7+Low MYC Probability; OS Probability; High HOXB7 High HOXB7+High MYC (p=0.0085) (p=0.0083)

NKI dataset (n=288) E F

Low HOXB7 survival Overall Low HOXB7+HER2+MYC Overall survival Overall High HOXB7 High HOXB7+HER2+MYC

(Cox HR=2.089, p=0.018) (Cox HR=1.117, p=0.003)

0.0 0.2 0.4 0.6 0.8 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.8 0.6 0.4 0.2 0.0 0 1000 2000 3000 4000 5000 6000 0 1000 2000 3000 4000 5000 6000 (days) (days) Figure S10

G H

fee survival fee - HOXB7/HER2 Overall survival Overall HOXB7/HER2 -/- -/- -/+ -/+ +/- Relapse P < 0.001 +/- P < 0.001 +/+ +/+

Months after surgery Months after surgery

I J freesurvival HOXB7/MYC - HOXB7/MYC Overall survival Overall -/- -/- -/+ -/+ +/- +/- P < 0.004 Relapse +/+ +/+ P < 0.004

Months after surgery Months after surgery

K

HOXB7 HER2 c-MYC Figure S10 (related to Figure 6): MYC-HOXB7-HER2 predicts clinical outcome of breast cancer in women treated with endocrine therapy. Kaplan-Meier plots of A, recurrence free survival in endocrine therapy treated women (RFS, n=1388) with tumors expressing HOXB7 or B, co-expression of HOXB7 and HER2. Kaplan-Meier plot of C, overall survival probability of ER+ breast cancer patients with tumors expressing HOXB7 or D, co- expression of HOXB7 and MYC in van der Vijver dataset (n=295). Kaplan-Meier plot of E, overall survival of ER+ breast cancer patients with tumors expressing HOXB7 or F, tumors that show co- expression of HOXB7, HER2, and MYC in NKI dataset (n=288). Kaplan–Meier plots of G, overall survival and H, relapse-free survival survival of ER+ breast cancer patients with tumors co- expressing HOXB7 and HER2. Kaplan–Meier plots of I, overall survival and J, relapse-free survival of ER+ breast cancer patients with tumors co-expressing HOXB7 and MYC by IHC in tissue microarray (TMA) of TAM monotherapy treated breast cancer patients (n =72). K, Representative photomicrograph of IHC analysis of HOXB7, HER2 and MYC expression in primary breast cancers in the TMA described in J. Table S1 (related to Figure 1): Family wise error rate (FWER) for MR-GSE of genes differentially expressed between MCF-7-HOXB7 and MCF-7-Vector cells.

Family-wise Error Rate P-value Up-regulated Down-regulated genes genes Dutertre_Estradiol_Response_24hrs_Up 3.1 x 10-9 1.0 Dutertre_Estradiol_Response_24hrs_Down 1.0 1.1 x 10-25 Hsiao_Housekeeping_Genes 1.0 0.11

Table S2 (related to Figure 1): ER target genes ranked in order of the average correlation with HOXB7 or ER. Entrezgene HOXB7 hgnc_symbol Ranking HOXB7 p-value ER correlation ER p-value Average correlation id correlation 771 CA12 1 0.506947746 6.78E-17 0.5529362 6.97E-17 0.529941973 7031 TFF1 2 0.519695768 7.29E-17 0.523146576 7.20E-17 0.521421172 5979 RET 3 0.505052321 8.08E-17 0.519611522 7.01E-17 0.512331921 2064 ERBB2 4 0.467089303 1.10E-16 0.432938331 1.45E-16 0.450013817 8614 STC2 5 0.428390984 1.49E-16 0.458151033 1.11E-16 0.443271009 9080 CLDN9 6 0.446237579 1.17E-16 0.431541285 1.29E-16 0.438889432 56937 PMEPA1 7 0.426677736 1.18E-16 0.431916603 9.57E-17 0.429297169 9687 GREB1 8 0.403739587 1.21E-16 0.440489258 1.20E-16 0.422114422 3280 HES1 9 0.396798498 8.67E-17 0.443733275 1.13E-16 0.420265887 4488 MSX2 10 0.403557133 1.48E-16 0.417336633 1.56E-16 0.410446883 2674 GFRA1 11 0.377742876 9.77E-17 0.419335885 1.24E-16 0.398539381 23612 PHLDA3 12 0.398894771 1.07E-16 0.3723579 1.27E-16 0.385626336 2886 GRB7 13 0.418260633 1.18E-16 0.34502162 1.36E-16 0.381641127 9088 PKMYT1 14 0.413630549 1.29E-16 0.346312743 1.43E-16 0.379971646 2288 FKBP4 15 0.350018946 1.52E-16 0.396417878 1.00E-16 0.373218412 1999 ELF3 16 0.375897935 1.17E-16 0.360766677 1.15E-16 0.368332306 7164 TPD52L1 17 0.351228495 1.25E-16 0.383654692 1.21E-16 0.367441593 595 CCND1 18 0.341631666 1.11E-16 0.39202841 1.20E-16 0.366830038 5241 PGR 19 0.337964716 1.40E-16 0.39219082 1.78E-16 0.365077768 655 BMP7 20 0.375434248 1.15E-16 0.353230142 1.25E-16 0.364332195 6548 SLC9A1 21 0.365270036 1.30E-16 0.334522683 1.55E-16 0.349896359 3487 IGFBP4 22 0.349727858 1.30E-16 0.349689646 1.44E-16 0.349708752 9518 GDF15 23 0.395289611 1.12E-16 0.298493464 1.20E-16 0.346891537 1942 EFNA1 24 0.33005682 1.52E-16 0.346078514 1.76E-16 0.338067667 7286 TUFT1 25 0.29745676 1.21E-16 0.334249901 1.33E-16 0.31585333 4886 NPY1R 26 0.274518323 1.38E-16 0.355128902 1.29E-16 0.314823613 29995 LMCD1 27 0.317673665 1.45E-16 0.304048586 1.35E-16 0.310861126 5914 RARA 28 0.325924652 1.40E-16 0.295249658 1.53E-16 0.310587155 6387 CXCL12 29 0.317935028 1.28E-16 0.285055639 1.16E-16 0.301495333 11045 UPK1A 30 0.279898001 1.46E-16 0.273342272 1.44E-16 0.276620137 1826 DSCAM 31 0.282811737 1.27E-16 0.252017795 1.26E-16 0.267414766 5493 PPL 32 0.263898651 1.37E-16 0.267306852 1.37E-16 0.265602752 55723 ASF1B 33 0.298510262 1.16E-16 0.232327469 1.16E-16 0.265418865 7494 XBP1 34 0.236815529 1.54E-16 0.269529529 1.17E-16 0.253172529 26503 SLC17A5 35 0.242307708 1.53E-16 0.259140611 1.48E-16 0.25072416 64858 DCLRE1B 36 0.225589961 1.48E-16 0.23940445 1.55E-16 0.232497205 8714 ABCC3 37 0.269703468 1.53E-16 0.183372294 1.94E-16 0.226537881 23677 SH3BP4 38 0.198533641 1.64E-16 0.238611311 1.21E-16 0.218572476 2175 FANCA 39 0.255464377 1.51E-16 0.175121477 1.79E-16 0.215292927 2491 CENPI 40 0.204691108 1.41E-16 0.222102005 1.62E-16 0.213396556 10626 TRIM16 41 0.246584894 1.37E-16 0.178988815 1.89E-16 0.212786854 9619 ABCG1 42 0.211362853 1.75E-16 0.189829136 1.82E-16 0.200595994 25800 SLC39A6 43 0.191904962 1.76E-16 0.207367901 1.60E-16 0.199636431 578 BAK1 44 0.249487489 1.64E-16 0.140597797 1.62E-16 0.195042643 23397 NCAPH 45 0.213070033 1.66E-16 0.176117159 1.65E-16 0.194593596 3897 L1CAM 46 0.180134314 1.94E-16 0.206132799 1.69E-16 0.193133556 2535 FZD2 47 0.21038141 1.68E-16 0.152477612 1.67E-16 0.181429511 1543 CYP1A1 48 0.181050565 1.69E-16 0.176887211 2.00E-16 0.178968888 90362 FAM110B 49 0.167488299 1.59E-16 0.184126871 1.76E-16 0.175807585 Table S2 (related to Figure 1):

64798 DEPTOR 50 0.159220457 1.55E-16 0.186613796 1.85E-16 0.172917126 23649 POLA2 51 0.213309655 1.68E-16 0.125033373 1.64E-16 0.169171514 983 CDK1 52 0.165758996 1.70E-16 0.163211643 1.69E-16 0.16448532 23138 N4BP3 53 0.128414509 2.14E-16 0.194808977 1.81E-16 0.161611743 2034 EPAS1 54 0.135852564 1.75E-16 0.165391993 1.81E-16 0.150622278 4173 MCM4 55 0.138528968 1.93E-16 0.145457647 1.82E-16 0.141993307 51706 CYB5R1 56 0.165350821 1.66E-16 0.116294574 2.17E-16 0.140822697 81620 CDT1 57 0.152703384 1.77E-16 0.125916343 1.80E-16 0.139309863 55165 CEP55 58 0.144769954 1.41E-16 0.12981088 1.70E-16 0.137290417 27289 RND1 59 0.110460981 2.14E-16 0.162684289 1.66E-16 0.136572635 25788 RAD54B 60 0.133865163 2.07E-16 0.135081603 1.81E-16 0.134473383 55872 PBK 61 0.135247514 1.81E-16 0.130935213 1.89E-16 0.133091363 55765 C1orf106 62 0.15964133 1.80E-16 0.104337 2.48E-16 0.131989165 9133 CCNB2 63 0.129225616 1.85E-16 0.121854493 1.81E-16 0.125540055 10615 SPAG5 64 0.100558565 2.52E-16 0.142371505 1.84E-16 0.121465035 9833 MELK 65 0.125638197 1.75E-16 0.113431994 2.02E-16 0.119535096 3554 IL1R1 66 0.114204739 2.34E-16 0.107535938 2.21E-16 0.110870338 6241 RRM2 67 0.118185058 1.83E-16 0.098225135 2.63E-16 0.108205097 55215 FANCI 68 0.110002969 1.72E-16 0.099804931 2.15E-16 0.10490395 9837 GINS1 69 0.101901444 2.56E-16 0.102459133 2.60E-16 0.102180289 7153 TOP2A 70 0.106267536 2.09E-16 0.097112109 2.44E-16 0.101689822 10057 ABCC5 71 0.099001217 2.59E-16 0.102584167 2.04E-16 0.100792692 64785 GINS3 72 0.083679191 2.85E-16 0.114210011 2.11E-16 0.098944601 11130 ZWINT 73 0.095092309 2.64E-16 0.10141199 2.50E-16 0.098252149 990 CDC6 74 0.095917537 2.62E-16 0.092220901 2.48E-16 0.094069219 29028 ATAD2 75 0.082879733 2.77E-16 0.10492106 2.29E-16 0.093900397 7298 TYMS 76 0.082811877 2.66E-16 0.095664854 2.59E-16 0.089238366 9055 PRC1 77 0.082123226 2.49E-16 0.088961839 2.58E-16 0.085542532 10184 LHFPL2 78 0.104931803 2.17E-16 0.064250239 8.32E-15 0.084591021 55568 GALNT10 79 0.107967312 2.16E-16 0.060131416 2.75E-16 0.084049364 9124 PDLIM1 80 0.109071952 2.06E-16 0.057515273 2.77E-16 0.083293612 55055 ZWILCH 81 0.083976394 2.85E-16 0.081900132 2.54E-16 0.082938263 4522 MTHFD1 82 0.082382069 2.64E-16 0.074429214 2.76E-16 0.078405641 5983 RFC3 83 0.089487635 2.83E-16 0.067307308 2.63E-16 0.078397471 8458 TTF2 84 0.077421911 2.71E-16 0.076890137 3.00E-16 0.077156024 84187 TMEM164 85 0.121146374 1.92E-16 0.021674866 0.027602295 0.07141062 6482 ST3GAL1 86 0.071329352 2.76E-16 0.068801129 2.63E-16 0.07006524 3399 ID3 87 0.074364194 2.80E-16 0.058705654 2.80E-16 0.066534924 890 CCNA2 88 0.064254188 2.82E-16 0.068641586 2.59E-16 0.066447887 2237 FEN1 89 0.079913783 2.58E-16 0.050252624 1.68E-09 0.065083203 10105 PPIF 90 0.07634615 2.72E-16 0.048438082 3.78E-08 0.062392116 6324 SCN1B 91 0.020846728 0.02586113 0.099891246 2.51E-16 0.060368987 54892 NCAPG2 92 0.070322623 2.65E-16 0.049009538 2.85E-06 0.059666081 5985 RFC5 93 0.066031926 2.95E-16 0.047788076 3.91E-09 0.056910001 2131 EXT1 94 0.015686586 0.076338568 0.090498339 2.55E-16 0.053092462 3070 HELLS 95 0.045505766 1.73E-05 0.053281464 6.20E-16 0.049393615 9134 CCNE2 96 0.029354598 0.004944699 0.046068052 4.86E-06 0.037711325 64151 NCAPG 97 0.037040954 0.000204774 0.030002511 0.003284109 0.033521732 1512 CTSH 98 0.084688357 2.71E-16 -0.022849312 0.981304017 0.030919522 4171 MCM2 99 0.036674198 0.000601732 0.019792748 0.034895108 0.028233473 51514 DTL 100 0.029598877 0.003185306 0.017447038 0.051819617 0.023522957 7374 UNG 101 0.033388695 0.001506054 0.003301302 0.374123406 0.018344999 29127 RACGAP1 102 -0.003664834 0.619861083 0.027767093 0.005147499 0.012051129 6491 STIL 103 0.006005197 0.293218942 -0.000955553 0.531354492 0.002524822 3832 KIF11 104 0.004765633 0.331191373 -0.005966026 0.701843966 -0.000600197 3978 LIG1 105 0.022282815 0.020504527 -0.029335462 0.996878711 -0.003526324 65018 PINK1 106 0.000194559 0.485514125 -0.008693714 0.779483579 -0.004249577 80010 RMI1 107 -0.001651688 0.548211909 -0.007804951 0.766117199 -0.00472832 6240 RRM1 108 -0.003883144 0.633647544 -0.009080596 0.791967033 -0.00648187 9735 KNTC1 109 -0.020524928 0.970118749 -0.005797673 0.690764953 -0.0131613 Table S2 (related to Figure 1):

79682 MLF1IP 110 -0.022431271 0.980456001 -0.006889153 0.722489596 -0.014660212 4001 LMNB1 111 -0.001832298 0.562318951 -0.036528349 0.999794713 -0.019180323 8537 BCAS1 112 -0.064341427 1 0.020086863 0.038189599 -0.022127282 4176 MCM7 113 -0.0085062 0.780972472 -0.037010478 0.999765228 -0.022758339 3300 DNAJB2 114 -0.033605493 0.999136876 -0.016267296 0.92794139 -0.024936394 7045 TGFBI 115 0.025374709 0.010861346 -0.076324518 1 -0.025474905 79980 DSN1 116 -0.049485507 1 -0.002201976 0.566044856 -0.025843741 4172 MCM3 117 -0.020551659 0.967899501 -0.065154976 1 -0.042853317 79801 SHCBP1 118 -0.040506432 0.999919264 -0.049099938 1 -0.044803185 4739 NEDD9 119 -0.029407412 0.996308704 -0.060258639 1 -0.044833025 51053 GMNN 120 -0.0577466 1 -0.044853205 1 -0.051299903 580 BARD1 121 -0.060961672 1 -0.04309056 0.999943997 -0.052026116 5121 PCP4 122 -0.09909732 1 -0.007404717 0.737568365 -0.053251018 8204 NRIP1 123 -0.096674452 1 -0.026356938 0.992516666 -0.061515695 55625 ZDHHC7 124 0.000739249 0.472362967 -0.125642492 1 -0.062451622 3726 JUNB 125 -0.066180311 1 -0.076205756 1 -0.071193033 26271 FBXO5 126 -0.086050191 1 -0.065456796 1 -0.075753494 901 CCNG2 127 -0.086957197 1 -0.0770802 1 -0.082018699 4175 MCM6 128 -0.08109993 1 -0.089237448 1 -0.085168689 54541 DDIT4 129 -0.101125055 1 -0.087837368 1 -0.094481211 1836 SLC26A2 130 -0.082398733 1 -0.107092049 1 -0.094745391 7112 TMPO 131 -0.088366353 1 -0.104077398 1 -0.096221876 5984 RFC4 132 -0.098897628 1 -0.09576198 1 -0.097329804 5422 POLA1 133 -0.107397042 1 -0.093834131 1 -0.100615586 55323 LARP6 134 -0.154667813 1 -0.054164426 1 -0.10441612 2289 FKBP5 135 -0.096760697 1 -0.145916628 1 -0.121338663 1831 TSC22D3 136 -0.13562762 1 -0.159458516 1 -0.147543068 1501 CTNND2 137 -0.217192134 1 -0.106674288 1 -0.161933211 2353 FOS 138 -0.171924394 1 -0.174080245 1 -0.173002319 64761 PARP12 139 -0.142368964 1 -0.222398871 1 -0.182383918 3251 HPRT1 140 -0.219256509 1 -0.173551777 1 -0.196404143 1786 DNMT1 141 -0.196864927 1 -0.233104536 1 -0.214984731 1992 SERPINB1 142 -0.186359879 1 -0.245605423 1 -0.215982651 23108 RAP1GAP2 143 -0.324649366 1 -0.283323493 1 -0.30398643 56271 BEX4 144 -0.441319922 1 -0.329211556 1 -0.385265739