CAL120 MDAM134 SUM149 BT20 BT474 CAL51 CAMA1 HCC1143 HCC202 JIMT1 MCF7 MDAMB157 MDAMB231 MDAMB453 MDAMB468 SKBR3 SUM44 T47D VP229

Gene essential Z score A A Z score gene Loss of viability Z score threshold

     BT20 BT474 CAL120 CAL51 CAMA1 HCC1143 HCC202 JIMT1 MCF7 MDAMB157 MDAMB231 MDAMB453 MDAMB468 SKBR3 SUM149 SUM44 T47D VP229 MDAM134

Z score gene B Gene not essential

     BT20 BT474 CAL120 CAL51 CAMA1 HCC1143 HCC202 JIMT1 MCF7 MDAMB157 MDAMB231 MDAMB453 MDAMB468 SKBR3 SUM149 SUM44 T47D VP229 MDAM134

     Z score gene C      

34 breast cell lines

 Optimizationation for high- throughput RNAi screening      

3 replica screens per cell lines

LOG transform data

Normalize tto platel median

Z-score standardize datat using median absolute deviations !    !   Quality control (1) Z’ values calculated per plate using siCON and siPLK1 (2) confirm normal distribution

Combine replicate data

Functional viabilityib profiles

Integration with other profiling platforms "   "    #

                        *,$+..( $%&% $%&% '() *()( $() ,) '. 2",$ 3+ 4!((/ $%&% ,( *%+ -,./ -,%/ , ,%.+ , ,%01 , ,(+ , ,+% $%&  , ,.& -,%% , ,& *.&1$

#(

#

!

!$#

$"      (   !"3+ 5 !"3+  !"3+ ( !"3+ +   !"3+

$"

 PIK3CA Pool PIK3CA oligo 1

6 6

PIK3CA oligo 2 PIK3CA oligo 3

6 6

 PIK3CA mutant PTEN mutant/null , ,(+ !# , ,+%  $ *%+    "    4!((/  -,%%  '() 2",$ $%&% 3+ "

!"+3) 

!$#

3$

3$(

3$+

$"

siRNA BT474 CAL120 HCC1143 HCC202 BT20 MDAMB468 SUM149 CAL51 JIMT SKBR3 VP229 MDAMB453 MDAMB231 MDAMB134 SUM44 CAMA MCF7 T47D AKT1 z score -0.5 -0.4 -0.2 -1.8 -1.1 -0.5 -3.2 -0.3 -0.1 -3.6 -1.6 -2.4 0.6 -0.8 0.7 -3.0 0.0 -0.4 AKT2 z score -2.5 0.1 -0.5 -3.2 -0.6 1.4 0.3 -3.0 -0.3 -1.1 -0.4 -1.7 -1.9 0.6 0.1 -1.5 -1.2 -1.2 AKT3 z score -0.2 -0.2 -0.2 -1.4 -0.8 0.3 0.2 -2.1 0.5 -0.1 0.1 -0.8 0.1 0.6 -0.9 0.1 -3.6 -1.1 #    5 #( #(  #( ( #( + #( %

#(

$"

 ERBB2 oligo 1 ERBB2 oligo 2 ERBB2oligo 3

ERBB2oligo 4 ERBB2 Pool      +  

      %

 11q13.3 AMP dependencies

 MYC 8q24.21 AMP dependencies

 FGFR1 8p12 AMP dependencies  AURKA 20q13 AMP dependencies

# CDK4 12q14.1 AMP dependencies  PPM1D 17q21-q23 AMP dependencies  MDM2 12q15 AMP dependencies      .  PTEN mutant cell line dependencies

 CDKN2A mutant dependencies

 KRAS mutant dependencies  P53 mutant dependencies

# BRCA1 mutant dependencies

 RB mutant dependencies Supplementary Figure 6. A.                        *()( , ,%01 $%&% 2",$ -,%% ,( $%&% '() *%+ $() ,) -,./ -,%/ '. 3+ 4!((/ , ,%.+ *,$+..( $%&% , ,.& , ,(+ , ,+% , ,& $%&  *.&1$

!$#

$"

B. '. *#!$#7$

  #

PTEN WT PTEN MUT PTEN WT PTEN MUT

PTEN WT PTEN MUT PTEN WT PTEN MUT

PTEN WT PTEN MUT PTEN WT PTEN MUT #   $$3 ** 5$5'

$$3 *** 

AZ3146: - + - +

PTEN WT PTEN MUT *

!$#8  !$#  

10 μm

!$#8 9(,$$3 !$#  9(,$$3

10 μm 10 μm      &  

 CAMA1 T47D MDAMB134 MCF7

ZR75.1 SUM44 S68

MDAMB361 BT474 ZR75.30 Normalised Percent Inhibition (NPI) *   

'.

-,%%

5  3( -,%/

***  3()+ '() p <0.0001 , ,.&  3(). 3+  3(! Increasing sensitivityto ADCK2 siRNA

, ,%.+  3( $"

*%+ *()( # 4!((/ ** p p =0.0075

=0.0341 *.&1$ $() ,(

$%& 

, ,%01 2",$ $%&% , ,(+ , ,+% ,& , $" ESR1  3( ER+       1  

CAMA1 MDAMB134 SUM44 '  HCC202 SKBR3 MCF7 MDAMB453 T47D BT474 VP229 BT20 JIMT1 MDAMB468    SUM149 HC1143 HS578T MDAMB157 CAL51 MDAMB231 CAL120



Group 1 Group 2

Low expression High expression 

Z>-2 DEL/lossD Low expression Z<-2 AMP/gainA High expression E. Group 1 Group 2 Functional Breast Cancer Profiles Sup.Info.1

Supplementary Figures

Supplementary Figure 1. Functional profiling of a breast cancer tumour cell line panel. a. Cell lines were profiled with a siRNA library targeting 714 and - related . Shown here are the effects on cellular viability in the tumour cell panel of siRNAs targeting one of three genes A, B or C. Z score plots for each example are shown, where a Z score of less that -2 signifies that an siRNA is having a significant effect of cell viability. An siRNA causing significant loss of cell viability in all of the cell lines assayed (as for gene A) likely targets a gene that has an essential and ubiquitous function in both normal and tumour cells. Similarly, an siRNA that had no significant effect on viability in any of the cell lines is either not functional or targets a non-essential gene (as for gene B). Finally, an siRNA that caused significant lethality in only some but not all cell lines (as for gene C), identifies a gene that represents a candidate tumour-specific dependency and a candidate therapeutic target. b. Data processing procedure for RNA interference screens. Raw luminescence readings were log2 transformed and then median centred per plate (excluding control wells) and standardised per screen using a robust Z score. Scores from replicate screens were combined using the mean. The separation of positive and negative controls (Z’- factor) was used to estimate screen quality. c. Reproducibility of RNAi viability profiles in breast tumour cell panel. Plot of R2 values for the three screening replicates of each cell line is shown. d. and e. Distribution of viability effects in each cell line. Normalised distributions of RNAi Z scores for each cell line. In this analysis, median Z scores from triplicate screens were used. f. Western blot of lysates from cell line panel showing expression of ERBB2, ER, PR, PTEN and .

Supplementary Figure 2. PIK3CA and ERBB2 addiction effects in breast tumour cells. a. Heat map showing the results of a supervised clustering of siRNA Z scores. Breast tumour cell lines were clustered according to PIK3CA gene status and differential effects between PIK3CA mutant and wild type groups identified using the median permutation test. Statistically significant effects (p<0.05) are shown. siRNA targeting PIK3CA is marked by the arrow. b. Western blot of total cell lysates from MCF7 cells 48 hours after transfection with individual and pooled PIK3CA siRNA. c. Functional Breast Cancer Profiles Sup.Info.2

Box/whiskers plots of individual and pooled PIK3CA siRNA effects on cell lines characterised by PIK3CA (*=p<0.05 using Student’s t-test). d. Western blot of total cell lysates from the breast cell line panel showing expression of PI3K p110a, PTEN, AKT1, AKT2, AKT3 and ACTIN. PIK3CA mutant cell lines are indicated in red whilst PTEN deficient cell lines are indicated in blue and double mutants are boxed. Z scores of AKT1, 2 and 3 are shown in a table. e. Heat map showing the results of a supervised clustering of siRNA Z scores. Breast tumour cell lines were clustered according to ERBB2 gene mutation status and differential effects between ERBB2 mutant and wild type groups identified using the median permutation test. Statistically significant effects (p<0.05) are shown. f. Western blot of total cell lysates from BT474 cells 48 hours after transfection with individual and pooled ERBB2 siRNA. g. Box/whiskers plots of individual and pooled ERBB2 siRNA effects. *p<0.05 measured by t-test. NPI = normalised percent inhibition

Supplementary Figure 3. a. Waterfall plot of BRAF Z scores in the breast tumour cell line panel. The breast tumour cell line MDAMB231 containing a BRAF mutation is highlighted in red. b. Z score plot of the BRAF mutant COLO-829 cell line profiled with an siRNA viability screen targeting 714 kinases and kinase-related genes.

Supplementary Figure 4. Candidate genetic dependencies for amplification events commonly found in breast cancer. Waterfall plots of kinases that selectively kill breast cancer cell lines containing a. an amplification of 11q13.3. b. MYC amplified breast cancer cell lines. c. FGFR1 amplified breast cancer cell lines. d. AURKA amplified breast cancer cell lines. e. CDK4 amplified breast cancer cell lines. f. PPM1D amplified breast cancer cell lines. g. MDM2 amplified breast cancer cell lines. p<0.05 for a-g, using the Student’s t-test.

Supplementary Figure 5. Candidate tumour suppressor and oncogene mutation dependencies. Waterfall plots of kinases that selectively kill breast cancer cell lines containing a. PTEN mutations. b. CDKN2A mutations. c. KRAS mutations. d. p53 mutations. e. BRCA1 mutation. f. RB mutation. p<0.05 for a-f, using the Student’s t-test.

Functional Breast Cancer Profiles Sup.Info.3

Supplementary Figure 6. Inhibition of TTK is PTEN selective. a. Western blot of total cell lysates of the breast cell line panel showing the expression of PTEN. b. PTEN FISH analysis of CAL51 and HEC1b (PTEN wildtype) cells using the Vysis LSI PTEN (10q23) (red) and CEP 10 (green) dual colour probes. Red arrows indicate PTEN probe hybridization in the HEC1b cell line. The Cal51 image shows a signal pattern indicative of a PTEN (10q23) deletion. c. Waterfall plots of mitotic/checkpoint kinases that selectively inhibit PTEN mutant/null cell lines (p<0.05). d. Heat map showing the results of a supervised clustering of siRNA Z scores. Breast tumour cell lines were clustered according to PTEN gene mutation status and differential effects between PTEN mutant and wild type groups identified using the median permutation test. Statistically significant effects (p<0.05) are shown. e. Box/whiskers plots showing the effect of pooled siRNA of non-silencing siRNAs (siControl 1-3) and other non-mitotic/checkpoint kinases in PTEN mutant and wild type breast cancer cell lines. f. Western blot of total cell lysates from MCF7 cells 48 hours after transfection with individual and pooled TTK siRNA. g. Histograms plot showing the percentage of abnormal for HCT116 PTEN wt and HCT116 PTEN null cells with and without 2uM AZ3146 treatment for 24 hours. counts from 60 metaphase spreads for each treatment were analysed (**p=0.003, ***p<0.0001, fishers exact test). h. Metaphase spread images of HCT116 PTEN wt/null cells with and without 2uM AZ3146 treatment for 24 hours.

Supplementary Figure 7. ADCK2 is a candidate estrogen (ER) selective target. a. Heat map showing the results of a supervised clustering of siRNA Z scores. Breast tumour cell lines were clustered according to ER status and differential effects between ER+ve and ER-ve groups identified using the median permutation test. Statistically significant effects (p<0.05) are shown. b. Box/whiskers plots of pooled siRNA of non-silencing siRNAs (siControl 1-3) and other control kinases in ER-ve or ER+ve breast cancer cell lines. c. Histograms showing breast cell line dependency on ESR1 and ADCK2. Cell lines were transfected with two individual and pooled siRNAs against ADCK2 and 3 individual siRNA silencing ERα (ESR1), siCONTROL (siCON) and siPLK1 (negative and positive controls, respectively). Cell viability was measured 5 days after transfection using Cell Titre Glo reagent (Promega). NPI = normalised percent inhibition. d. Western blot of total cell lysates from MCF7 cells 48 hours after transfection with individual and pooled ADCK2 siRNA. e. Correlation of Functional Breast Cancer Profiles Sup.Info.4

ADCK2 Z score and ADCK2 mRNA expression in breast tumour cell lines (p=0.0015, Pearson correlation). f. Western blots of total cell lysates of the breast cell line panel showing expression of ADCK2 and ERα (ESR1). g. Box/whiskers plots of ADCK2 expression in ADCK2 targeting resistant/sensitive and ER-/+ve breast tumour cell lines (p=0.0001 and p=0.0075 respectively). h. Correlation of average ADCK2 siRNA effects with average ERα (ESR1) siRNA effects (p=0.0341 using Student’s t test).

Supplementary Figure 8. Hierarchical clustering of Z scores. a. and b. Dendrogram showing hierarchical clustering of expression data which clusters cell lines into luminal and basal subtype. c. Hierarchical clustering of z scores as triplicate values. d. Hierarchical clustering of the siRNAs that are a hit in 2 or more cell lines with corresponding expression and aCGH heatmaps.                 !"  #$ #$% &  %  

Functional Breast Cancer Profiles Sup.Info.5

Supplementary Tables

Supplementary Table 1: R2 values for triplicate screens in 20 breast cell lines.

Cell Line Rep1 v Rep2 Rep 1v Rep3 Rep2 vRep 3 Mean BT20 0.92 0.93 0.93 0.93 BT474 0.88 NA NA 0.88 CAL120 0.79 0.77 0.80 0.78 CAL51 0.92 0.87 0.90 0.90 CAMA1 0.93 0.93 0.95 0.94 HCC1143 0.67 0.64 0.71 0.68 HCC202 0.92 0.91 0.89 0.91 HS578T 0.90 0.92 0.93 0.92 JIMT1 0.93 0.91 0.93 0.92 MCF7 0.89 0.83 0.79 0.84 MDAMB134 0.93 0.94 0.93 0.94 MDAMB157 0.87 0.87 0.89 0.88 MDAMB231 0.90 0.89 0.87 0.89 MDAMB453 0.80 0.78 0.85 0.81 MDAMB468 0.67 0.69 0.75 0.70 SKBR3 0.90 0.83 0.85 0.86 SUM149 0.93 0.95 0.93 0.93 SUM44 0.84 0.75 0.81 0.80 T47D 0.93 0.92 0.91 0.92 VP229 0.93 0.94 0.94 0.93

Functional Breast Cancer Profiles Sup.Info.6

Supplementary Table 2: Z’-factor for the triplicate screens for the selected 20 breast cancer cell lines.

Screen Z'-factor Rep1 Z'-factor Rep2 Z'-factor Rep3 Mean CAMA1 0.78 0.79 0.82 0.80 JIMT1 0.65 0.73 0.72 0.70 MDAMB453 0.72 0.70 0.66 0.69 BT474 0.64 0.72 NA 0.68 BT20 0.72 0.57 0.74 0.68 SKBR3 0.74 0.66 0.61 0.67 SUM149 0.64 0.60 0.73 0.66 HCC202 0.71 0.64 0.61 0.65 MDAMB157 0.66 0.57 0.73 0.65 VP229 0.54 0.68 0.62 0.61 HS578T 0.58 0.58 0.67 0.61 MDAMB468 0.48 0.63 0.59 0.57 MDAMB231 0.56 0.51 0.60 0.56 T47D 0.62 0.46 0.54 0.54 CAL51 0.51 0.45 0.52 0.49 CAL120 0.61 0.41 0.44 0.49 MDAMB134 0.65 0.37 0.42 0.48 HCC1143 0.51 0.46 0.37 0.45 MCF7 0.29 0.29 0.38 0.32 SUM44 0.41 0.28 0.21 0.30 Functional Breast Cancer Profiles Sup.Info.7

Supplementary Table 3: ERBB2, ER and PR status are shown as are the top 30 hit genes for each of the 20 cell lines with robust RNAi screening data.

Known Top 30 Hit Genes (Z score) Mutation Cell line ERBB2 ER PR Subtype (gene name) -8.347, AURKA -6.773, -5.574, COASY -4.315, PRKAG3 -4.061, CHEK1 - 3.46, EEF2K -3.426, CDC2 -3.282, PNKP - 3.158, AURKB -3.001, STK38L -2.965, CDKN2A, GUCY2D -2.93, CIT -2.92, PANK4 -2.911, BT20 Not AMP NEG NEG BASAL PIK3CA, PRKAB1 -2.906, PIK3CA -2.524, PTK7 - TP53 2.465, MYLK2 -2.428, MAPK4 -2.412, SNRK -2.378, KSR -2.302, PAK6 -2.302, CSNK2A2 -2.299, MARK3 -2.277, MAP2K7 -2.276, KCNH2 -2.273, CALM2 -2.266, CDC2L2 - 2.231, STK11 -2.186, STK39 -2.168 COASY -9.297, PLK1 -9.204, HCK -7.798, PTK7 -7.032, ERBB2 -6.95, ERBB3 -6.58, CAMK2B -6.411, SSTK -6.344, RPS6KA4 - 5.69, PIK3CA -5.465, CDK10 -5.238, GUCY2D -5.124, STK11 -4.912, MYLK2 - PIK3CA, BT474 AMP POS POS Luminal 4.721, MAP2K3 -4.095, DAPK3 -3.747, GCK TP53 -3.747, MAP4K1 -3.684, SSTK -3.432, FASTK -3.425, EPHA6 -3.324, MAP3K9 - 3.24, SGKL -3.153, AURKA -3.139, BRD3 - 3.131, PCTK3 -3.111, RELA -3.109, FN3KRP -3.095, CSNK2A2 -3.087, IKBKB -3.074 PLK1 -5.062, PRPF4B -4.991, PRKAB1 - 4.491, ILK -4.29, PRKDC -3.923, GUCY2D - 3.794, PRPS1L1 -3.567, PIK3C2A -3.497, CIB2 -3.435, RPS6KL1 -3.124, MAP3K4 - 3.02, TRIO -2.976, MAP4K4 -2.967, CAMK1 -2.942, PLK4 -2.934, MAP2K1 -2.922, CAL120 Not AMP NEG NEG BASAL TP53 PIK4CA -2.78, PFKFB1 -2.714, RPS6KA4 - 2.606, NRBP -2.545, CDC42BPA -2.53, MAPK14 -2.363, RELA -2.348, LTK -2.306, PRKCL2 -2.296, PIK3C3 -2.295, RP6- 213H19.1 -2.271, CSNK2B -2.197, NEK1 - 2.155, IHPK2 -2.152 PLK1 -7.797, GUCY2D -6.449, PIK3C2A - 5.731, WEE1 -3.706, PRKAG3 -3.239, CSNK2A2 -3.165, PRKCL2 -3.145, TTK - 3.099, AKT2 -3.031, TTBK1 -3.016, PFKFB1 -2.985, AURKB -2.902, CAMKIINALPHA - 2.879, PIP5K2B -2.851, PRKCN -2.813, CAL51 Not AMP NEG NEG BASAL PIK3CA FRAP1 -2.779, KCNH8 -2.773, KIT -2.77, KSR -2.718, PTK7 -2.693, PANK4 -2.66, CSF1R -2.543, NEK7 -2.509, C9ORF12 - 2.493, EFNA4 -2.467, BCKDK -2.459, CDC2L2 -2.457, PRKAB1 -2.428, PIM1 - 2.378, P101-PI3K -2.302 Functional Breast Cancer Profiles Sup.Info.8

PLK1 -14.256, AURKA -12.135, GUCY2D - 11.391, CIT -10.068, JAK2 -10.021, CDC2L2 -8.602, TTK -7.864, ADCK2 -6.906, NUP62 -6.621, CDC42BPA -6.01, GRK4 - E- 5.865, EPHA6 -5.778, PCTK3 -5.371, PLK4 - Cadherin, CAMA1 Not AMP POS POS Luminal 5.282, CDC2 -5.207, PASK -5.064, CDK4 - PTEN, 4.69, STK35 -4.18, AURKB -4.098, BRD4 - TP53 4.081, PRKAG3 -3.983, MARK3 -3.97, VRK1 -3.784, BUB1B -3.651, ZAP70 -3.625, ADRBK1 -3.607, NEK7 -3.401, HUNK - 3.354, FRAP1 -3.334, PRKY -3.269

CHEK1 -7.137, GUCY2D -6.552, PLK1 -5.53, CDC2L2 -4.663, FLT4 -4.563, AURKA - 4.495, PRKAG3 -4.432, PKN3 -4.125, CSNK1G1 -3.945, MYLK2 -3.511, MAP4K5 - 3.379, STK35 -3.352, FRAP1 -3.301, FGR - 3.247, EPHB6 -3.247, EEF2K -3.239, HCC1143 Not AMP NEG NEG BASAL TP53 ALS2CR2 -3.233, DAPK3 -3.225, SPHK1 - 3.116, CIT -3.106, TRIB3 -3.005, MAP3K8 - 2.985, TGFBR1 -2.971, WEE1 -2.888, PCTK3 -2.884, JAK2 -2.876, BUB1B -2.825, PDGFRB -2.8, CIB2 -2.686, PRKAR2B - 2.675 PIK3CA -6.845, PLK1 -6.31, CHEK1 -6.096, MAP2K3 -5.444, DAPK3 -5.364, SSTK - 5.174, WEE1 -5.12, AURKA -4.199, CIT - 3.946, SRMS -3.919, PFKFB1 -3.899, PRKAB1 -3.893, EPHA6 -3.588, SYK -3.32, HCC202 AMP NEG NEG Luminal TP53 TYRO3 -3.275, RPS6KA4 -3.207, AKT2 - 3.187, EPHB1 -3.164, CAMK2B -3.03, COASY -3.02, GUCY2D -2.816, MARK4 -2.8, STK38L -2.771, TLK1 -2.634, PRKAG3 - 2.629, SGKL -2.567, MYLK2 -2.515, ADP- GK -2.504, PHKG1 -2.432, PTK7 -2.425 PLK1 -4.671, PIK3C2A -4.058, GUCY2D - 4.027, WEE1 -3.803, STK39 -3.776, JAK2 - 3.713, FN3KRP -3.701, AURKA -3.623, STK11 -3.53, PRKAB1 -3.508, CDK10 - CDKN2A, 3.397, MAP3K7 -3.377, EEF2K -3.357, PLK4 HRAS, -3.317, PRKACB -3.255, PRKAG3 -3.051, HS578T Not AMP NEG NEG BASAL PIK3R1, MYLK2 -3.019, CALM2 -2.956, CHEK1 - TP53 2.873, MAPK3 -2.851, PFKFB4 -2.822, ALS2CR2 -2.747, CDC42BPA -2.741, CAMK2A -2.646, IHPK3 -2.646, STK33 - 2.521, CSNK2B -2.499, MAPKAPK3 -2.476, PFKP -2.457, MATK -2.445 CHEK1 -14.69, MAP3K4 -13.122, AURKA - 11.848, DCK -10.008, CAMK1 -7.824, JAK2 -7.593, CDC2L2 -7.253, MAP2K7 -6.044, STK38L -5.63, MYLK2 -5.62, PRKAG3 - 4.669, WEE1 -4.66, PRKY -4.468, PTK7 - 4.423, PIM2 -4.379, PCTK1 -3.856, PCTK3 - JIMT1 AMP NEG NEG 3.409, AURKB -3.391, CDC2L1 -3.304, PFKP -3.135, CSNK2A2 -3.096, CAMKIINALPHA -2.895, TTBK1 -2.847, PI4K2B -2.822, STK22C -2.791, MERTK - 2.758, NAGK -2.63, EEF2K -2.532, RPS6KA2 -2.511, GALK1 -2.492 Functional Breast Cancer Profiles Sup.Info.9

PLK1 -10.556, GUCY2D -5.963, PCTK3 - 5.963, KCNH2 -5.382, RIOK1 -5.183, CDC2 -4.857, CIT -4.821, AURKA -4.536, EGFR - 4.282, PRKD2 -4.117, PRKAG3 -3.92, CSNK1G1 -3.915, PLK4 -3.859, SRMS - 3.784, CDC2L2 -3.755, PRPF4B -3.645,

MCF12A Not AMP NEG NEG BAIAP1 -3.583, CHEK1 -3.367, WEE1 - 3.343, GSG2 -3.318, CARKL -3.253, NUP62 -3.133, LMTK3 -3.048, RPS6KA5 -3.039, CDK5R1 -3.02, TTK -2.927, PACE-1 -2.914, RPS6KA4 -2.905, PIK3C2A -2.848, AURKC - 2.79 FYN -9.868, PLK1 -6.28, PAK1 -5.358, CALM2 -3.859, CSNK1E -3.827, AKT3 - 3.614, ADCK2 -3.472, PFKFB1 -3.409, CSNK2A2 -3.227, CDC42BPA -3.188, FLJ10761 -3.041, EPHA6 -3.026, KCNH8 - CDKN2A, 3.018, MAPK1 -2.996, AURKB -2.742, MCF7 Not AMP POS POS Luminal PIK3CA PRKCN -2.712, EPHA4 -2.672 KSR -2.632, KHK -2.608, MAP3K11 -2.598, MAP3K3 -2.551, MAPKAPK3 -2.535, RPS6KA2 -2.533, NEK3 -2.465, PRKD2 - 2.457, PFKFB3 -2.436, GSG2 -2.428, STK33 -2.36, PIK3C2A -2.339, TTK -2.276 AURKA -10.58, PLK1 -9.326, CHEK1 -8.052, GUCY2D -5.693, CDC42BPA -5.233, WEE1 - 5.224, CSNK2A2 -3.624, PTK7 -3.575, NEK7 -3.564, MAPKAPK3 -3.497, AURKB - CDH1, 3.497, IHPK3 -3.412, MYLK2 -3.34, MDAMB MAP2K4, PIK3C2A -3.203, RPS6KA2 -3.148, MARK3 - Not AMP POS NEG Luminal 134 KRAS, 3.071, ADCK2 -2.865, STK22D -2.841, TP53 LMTK3 -2.732, CDC2 -2.726, STK11 -2.636, ERK8 -2.636, MAPK4 -2.606, PFKFB1 - 2.553, CIT -2.514, RAGE -2.388, PRKAB1 - 2.34, MAPK3 -2.332, PRKCN -2.27, HIPK3 - 2.261 PLK1 -16.644, GUCY2D -7.668, WEE1 - 6.844, CHEK1 -4.245, MUSK -3.731, AURKA -3.673, RPS6KA2 -3.637, MYLK2 -3.604, PIK3C2A -3.14, ADRBK1 -3.106, CDC42BPA -3.042, GSK3A -2.989, RELA -2.988, CDKL4 NF1, MDAMB -2.859, PLK4 -2.791, EPHA3 -2.755, IRAK1 Not AMP NEG NEG BASAL MSH6, 157 -2.656, SYK -2.476, FLJ23074 -2.469, TP53 BUB1B -2.466, SPHK1 -2.463, STK22B - 2.448, NEK3 -2.445, MAPKAPK3 -2.439, C9ORF96 -2.408, SSTK -2.399, KCNH8 - 2.341, PDGFRB -2.328, KHK -2.327, MAPK1 -2.294 Functional Breast Cancer Profiles Sup.Info.10

FN3KRP -10.414, MPP3 -10.002, PKLR - 9.669, WEE1 -7.915, NME6 -6.733, PLK1 - 5.408, STK11 -4.985, FLJ10761 -4.851, BRAFM FGR -4.626, MAP3K7 -4.557, C9ORF96 - CDKN2A, 4.223, PTK7 -3.712, EPHA6 -3.511, CDKL4 MDAMB Not AMP NEG NEG BASAL KRS, -3.478, GUCY2D -3.339, CDKN2D -3.194, 231 NF2, CSNK2A2 -3.167, PIK3C2A -3.13, PNKP - TP53 3.05, MAPK1 -2.995, NEK8 -2.993, CALM2 - 2.966, MGC4796 -2.948, MAPK4 -2.904, PDK4 -2.851, PI4K2B -2.797, SNRK -2.765, PTK2B -2.705, BRAF -2.691, RELA -2.672

PLK1 -19.118, AURKA -13.261, PIK3CA - 10.492, CIT -9.446, CDK4 -7.966, AURKB - 7.803, GUCY2D -6.595, WEE1 -6.478, PLK4 -6.302, TTK -6.282, EPHB6 -5.864, BUB1B - E- 5.659, CDC2 -5.598, CHEK1 -5.562, IPMK - MDAMB Cadherin, AMP NEG NEG Luminal 5.025, CDC42BPA -4.734, DYRK4 -4.583, 453 PTEN, FRDA -4.408, CDKL5 -4.074, FRAP1 -4.066, TP53 PCTK3 -4.015, SMG1 -3.737, STK22C - 3.344, ERBB4 -3.325, ATR -3.307, STK35 - 3.184, RPS6KA5 -3.146, JAK2 -3.079, MKNK2 -2.882, FGFR4 -2.832 PLK1 -10.654, RPS6KA2 -10.099, AURKA - 8.277, DCK -7.049, PRKCG -5.196, AURKB - 4.922, EFNA5 -4.764, MAP3K10 -4.414, SYK -4.275, STK11 -4.205, CSNK1G2 - CDH1, 4.021, FGFRL1 -4.005, GUCY2D -3.742, MDAMB RB1, Not AMP NEG NEG BASAL TJP2 -3.479, MAP3K4 -3.461, JAK2 -3.321, 468 PIK3CA, PTK7 -3.246, CDC42BPA -3.215, SMG1 - PTEN 3.172, GCK -3.091, CIT -3.067, STK35 - 2.976, FRAP1 -2.908, MYLK2 -2.844, GRK4 -2.782, WEE1 -2.509, LAK -2.404, MAPK11 -2.219, PCTK2 -2.101, NEK3 -2.095 PLK1 -15.147, GUCY2D -10.871, AURKA - 8.246, CHEK1 -6.999, ERBB2 -6.397, PFKP -5.962, CSNK1E -5.752, FLJ10761 -5.075, MAP2K3 -4.96, EPHA6 -4.764, CDC2L2 - 4.212, BUB1B -3.99, PLK4 -3.798, HK2 - E- 3.727, PAK2 -3.704, MGC8407 -3.663, Cadherin, SKBR3 AMP NEG NEG Luminal PCTK2 -3.618, AKT1 -3.584, PFKFB1 - TP53 3.523, MYLK2 -3.476, MAGI-3 -3.471, COASY -3.428, PIK3CA -3.183, PIP5K1A - 3.168, STK35 -3.093, ULK1 -2.916, CDC42BPA -2.827, CAMK1 -2.823, PCTK3 - 2.812, PDPK1 -2.81 JAK2 -15.53, CHEK1 -13.067, PLK1 - 12.369, GUCY2D -11.417, WEE1 -9.967, DCK -9.208, CSNK2A2 -5.529, AURKA - 4.883, PIK3C2A -4.76, PLK4 -4.383, PTK7 - BRCA1, 4.304, NUP62 -4.181, CDC2L2 -3.784, CDKN2A, CDC42BPA -3.47, MAPK8 -3.465, CSNK1G1 SUM149 Not AMP NEG NEG BASAL PTEN, -3.455, AKT1 -3.25, MYLK2 -3.176, NAGK - TP53 3.027, MAP3K9 -2.942, CIB2 -2.907, SMG1 -2.903, GALK1 -2.84, PFKFB1 -2.729, MAPK1 -2.702, SNRK -2.65, MARK3 -2.615, STK22C -2.61, BUB1B -2.556, CDK10 - 2.489 Functional Breast Cancer Profiles Sup.Info.11

STK11 -5.233, PLK1 -4.636, CHEK1 -4.261, PFKFB1 -3.956, RPS6KA4 -3.9, WEE1 - 3.528, AURKA -2.997, MYLK2 -2.835, GALK2 -2.825, MAP2K7 -2.816, PTK7 - E- 2.791, MINK -2.576, COASY -2.548, CDKL3 SUM44 Not AMP POS POS Luminal Cadherin, -2.532, CSNK1D -2.338, CDK10 -2.289, TP53 ADRBK1 -2.249, PIK3C2A -2.182, LOC390226 -2.114, PTK6 -2.046, P101- PI3K -2.045, CAMK2B -2.043, MARK3 - 2.014, MAP3K7 -2.003, MAPK3 -2.001

EPHA6 -4.464, CALM2 -3.498, PLK1 -3.292, SRMS -3.29, EEF2K -3.139, PIK3CA -3.104, KSR -3.001, AURKA -2.986, MUSK -2.952, ADRBK1 -2.915, SYK -2.869, EXOSC10 - 2.74, COASY -2.631, CDKL2 -2.619, PIK3CA, PRKAB1 -2.613, DAPK3 -2.597, PTK9L - T47D Not AMP POS POS Luminal TP53 2.522, MAP2K3 -2.458, PRKCH -2.41, RPS6KA5 -2.321, NEK3 -2.314, TLK1 - 2.312, CSNK2B -2.23, DLG4 -2.198, FN3KRP -2.162, MAPK4 -2.157, GUCY2D - 2.043, PRKAR1B -2.016, ERN1 -2.014, NEK8 -2.009

CDK4 -5.985, PLK1 -5.595, DCK -5.292, JAK2 -4.915, GUCY2D -4.396, PRPF4B - 4.133, PCTK3 -3.516, TTBK1 -3.383, PRKAG3 -3.31, PRKAR2B -3.208, FLJ23074 -3.066, CDC2L2 -2.969, FRAP1 -2.947, AURKA -2.833, MAPK1 -2.816, CIT -2.661, VP229 AMP NEG NEG CALM3 -2.561, LAK -2.508, PANK4 -2.503, CALM2 -2.486, PIK3CA -2.428, MAP3K9 - 2.426, PRKCN -2.409, RPS6KA5 -2.405, CSNK2A2 -2.398, RELA -2.39, PACE-1 - 2.362, CIB2 -2.32, PRKAB1 -2.306, NRBP2 -2.292

Functional Breast Cancer Profiles Sup.Info.12

Supplementary Table 4: Integration of mRNAexpression, aCGH and z score data for amplified (AMP) genes. (i) amplified genes in the breast tumour cell line panel as definced by aCGH; (ii) genes where the amplification event is also characterized by elevated mRNA expression; (iii) genes where siRNA targeting causes cell inhibition in breast tumour cell lines with amplification and overexpression of the gene; (iv) genes where siRNA targeting causes cell inhibition in breast tumour cell lines with overexpression of the gene.

iii. Hit iv. Z score ii. AMP and when AMP correlates i. AMP Kinases Over- and with expressed Overexpr- Expression essed ADK MPP2 TBK1 CDK4 AURKA ADCK2 ADRBK1 MPP3 TESK1 CRKRS ERBB2 AK5 AURKA NEK7 TEX14 EGFR ADRBK1 BMX BRD4 NEK8 TLK2 ERBB2 CDK4 CDADC1 CAMK1G NLK TNIK FGFR1 GUCY2E CHEK1 CDC2 NME1 TP53RK GUCY2E MAP3K3 DYRK2 CDK4 NME2 TRIB1 PAK1 PCTK3 ERBB2 CDKN1A NPR2 TRIO PDK2 RPS6KB1 GOLGA5 CHKA NRBP1 TRPM7 PHKG1 GUCY2E CKS1B NUAK2 WNK3 PIP4K2B IGF1R CLK2 NUCKS1 XRCC6BP1 PIP4K2C ITK CPNE3 OXSR1 PTK2 ITPKA CRKRS PAK1 RPS6KB1 KALRN DCLK2 PCK1 RPS6KB2 LYN DGKQ PCTK1 STK4 MAP4K5 DSTYK PCTK3 XRCC6BP1 MAPK15 DYRK2 PDK2 MAPKAPK5 DYRK3 PFKFB2 MAST1 EFNA3 PHKB MINK1 EFNA4 PHKG1 MKNK1 EGFR PIK3C2B MST4 ERBB2 PIK3CA MUSK ERN1 PIM1 NAGK ETNK2 PIP4K2B PACSIN1 FGFR1 PIP4K2C PANK1 FGFRL1 PKIA PCK2 FYN PKLR PFKP GAK PMVK PHKG1 GNE PRKAA1 PI4KB GTF2H1 PRKAB2 PIK3CD GUCY2E PRKAR1A PIK3R5 HIPK1 PRKCA PIM1 HIPK3 PRKCI PKM2 IKBKB PSKH2 PRKAA1 IKBKE PTK2 PRKAR1B IKBKG RBKS RPS6KB1 IRAK3 RIPK2 STK16 KHK RPS6KB1 KSR1 RPS6KB2 MAGI3 SCYL3 MAP2K6 SGK196 MAP3K3 SGK196 MAPK13 SGK494 MAPK14 SKAP1 MAPK6 SRC MAPKAPK2 SRPK1 MELK STK38 MET STK4 MPP1 STRADA Functional Breast Cancer Profiles Sup.Info.13

Supplementary Table 5: PIK3CA status in panel of cell lines. The PIK3CA mutation, copy number, expression (as assessed by microarray and western blot, Supplementary Fig 2d) and cell line dependency for PIK3CA (as measured by Z score) is summarized here. Highlighted are seven PIK3CA mutant lines. MUT = mutant, WT = wild-type, NA = not applicable.

Relative PIK3CA PIK3CA PIK3CA PIK3CA copy mRNA Domain affected siRNA mutation number Expression (arbitarty units) Assessed z score sequencing sequencing aCGH microarray by: BT20 -2.51 P539R,H1047R Kinase gain 7.98 Neither kinase nor BT474 -5.48 K111N no change 7.24 helical CAL120 -0.12 WT NA no change 6.75 CAL51 -0.50 E542K Helical no change 7.15 CAMA1 -1.08 WT NA no change 6.86 HCC1143 -1.62 WT NA gain 7.60 HCC202 -6.83 H1047R Helical gain 7.65 HS578T 0.09 WT NA gain 7.40 JIMT1 -1.34 WT NA gain 7.50 MCF7 -1.96 E545K Helical gain 7.58 MDAMB134 -0.52 WT NA no change 7.11 MDAMB157 0.38 WT NA no change 7.25 MDAMB231 0.58 WT NA no change 7.32 MDAMB453 -10.46 H1047R Kinase gain 7.49 MDAMB468 -0.68 WT NA no change 7.12 SKBR3 -3.19 WT NA gain 7.43 SUM149 -0.19 WT NA no change 7.18 SUM44 -0.83 WT NA loss 6.96 T47D -3.09 H1047R Kinase gain 7.81 VP229 -2.42 WT NA AMP 7.42

Functional Breast Cancer Profiles Sup.Info.14

Supplementary Table 6: ERBB2 status in panel of cell lines. The ERBB2 copy number, expression (as assessed by microarray and western blot, Supplementary Fig. 1g) and cell line dependency for ERBB2 (as defined by Z score and lapatinib sensitivity) are summarized here. Highlighted are six ERBB2 amplified lines. AMP = amplified; not AMP = not amplified; ND = not determined. SF50 = concentration of lapatinib at which 50% of cells are inhibited.

Relative ERBB2 ERBB2 ERBB2 copy Lapatinib Expression siRNA number sensitivity (arbitary units) Assessed z score aCGH microarray SF50 (uM) by: BT20 0.64 not AMP 6.92 ND BT474 -6.96 AMP 9.48 0.073 CAL120 0.62 not AMP 7.01 ND CAL51 1.39 not AMP 7.08 ND CAMA1 0.69 not AMP 7.15 ND HCC1143 0.73 not AMP 6.82 ND HCC202 -2.00 AMP 9.06 0.581 HS578T 1.15 not AMP 8.28 ND JIMT1 0.67 AMP 7.66 5.01 MCF7 1.12 not AMP 6.85 >10 MDAMB134 0.43 not AMP 6.98 ND MDAMB157 1.96 not AMP 6.82 ND MDAMB231 0.31 not AMP 6.99 ND MDAMB453 -0.95 AMP 7.68 2.365 MDAMB468 -0.44 not AMP 6.86 ND SKBR3 -6.40 AMP 9.63 0.095 SUM149 0.06 not AMP 6.89 ND SUM44 1.02 not AMP 6.72 ND T47D 0.88 not AMP 7.22 ND VP229 -0.24 AMP 7.82 1.078

Functional Breast Cancer Profiles Sup.Info.15

Supplementary Table 7: PTEN and TTK status in cell line panel. The PTEN mutation, copy number, expression (as assessed by microarray and western blot, Supplementary Fig. 2d) and cell line dependency for TTK (as defined by Z score and TTK inhibitor) is summarized here. In addition, the expression and genomic copy number of TTK is listed as assessed by microarray and aCGH, respectively. Highlighted are four PTEN mutant lines and CAL51 cells, which have a PTEN deletion. MUT = mutant, WT = wild-type, ND = not determined.

Relative TTK Relative PTEN mRNA TTK PTEN PTEN copy mRNA TTK copy TTK siRNA Expression inhibitor mutation number Expression number (arbitary sensitivity (arbitary units) units) Assessed z score Sequencing aCGH microarray microarray aCGH SF50 uM by: BT20 -0.82 WT Loss 8.18 10.11 no change 11.81 BT474 -0.51 WT no change 10.10 9.87 no change 50.00 CAL120 0.90 WT Loss 8.25 8.87 no change 17.36 CAL51 -3.10 DELETION no change 7.96 9.68 no change 9.96 D92H, CAMA1 -7.83 T277fs*13 no change 8.03 8.42 loss 27.00 HCC1143 -0.70 WT no change 9.84 10.55 no change 12.79 HCC202 -0.63 WT no change 9.55 8.00 loss 12.55 HS578T -0.61 WT Loss 9.06 9.69 no change 50.00 JIMT1 -2.00 WT no change 8.69 7.11 no change 50.00 MCF7 -2.29 WT no change 8.90 10.01 no change 1.59 MDAMB134 0.49 WT no change 8.85 9.07 no change 50.00 MDAMB157 -0.59 WT Loss 8.56 10.78 gain ND MDAMB231 -0.80 WT no change 9.63 9.77 no change 15.01 MDAMB453 -6.26 E307K no change 9.41 9.72 no change 12.55 MDAMB468 0.97 A72fsX5 no change 9.22 10.21 gain ND SKBR3 ND WT Loss 8.04 8.97 no change 12.57 Gross SUM149 -1.97 Mutations no change 6.65 9.79 no change 14.96 SUM44 1.27 WT no change 9.33 9.41 no change 50.00 T47D -0.50 WT Loss 10.01 9.81 no change 7.35 VP229 -0.09 WT no change 8.47 9.63 no change 16.79

Functional Breast Cancer Profiles Sup.Info.16

Supplementary Table 8: ADCK2 and estrogen receptor (ESR1) information in the breast tumour cell line panel. The ADCK2 and ESR1 copy number, expression (as assessed by microarray, immunohistochemistry (IHC; for ESR1 only) and western blot, Supplementary Fig. 7f) and cell line dependency for ADCK2 and ESR1 (as defined by Z score) is summarized here. Highlighted are six ER positive lines. ND = not determined. NPI = normalized percent inhibition, which is an estimation of survival where 1 = no cell inhibition and 0 = complete cell inhibition. Expression by western blot analysis was graded none, low, medium and high as determined by the intensity of the ADCK2 and ESR1 bands shown in Supplementary Fig. 6f and 2e, respectively. IHC positive refers to the presence of ESR1 staining and negative refers to the absence of staining.

Relative ADCK2 Relative ESR1 ADCK2 ADCK2 copy mRNAExpression mRNAExpression ER Status ESR1 siRNA ESR1 siRNA ESR1 siRNA siRNA number (arbitary units) (arbitary units) Assessed z score aCGH microarray microarray IHC NPI oligo 1 NPI oligo 2 NPI oligo 3 by: BT20 -1.08 no change 9.95 7.38 Negative ND ND ND BT474 -1.95 no change 10.02 9.33 Positive 0.566272925 0.77298535 0.74081153 CAL120 0.30 no change 9.74 6.66 Negative ND ND ND CAL51 1.09 no change 9.61 6.71 Negative ND ND ND CAMA1 -6.87 gain 11.08 8.64 Positive 0.58720165 0.19285192 0.29054295 HCC1143 -0.42 no change 9.41 7.42 Negative ND ND ND HCC202 -0.51 gain 10.75 6.66 Negative ND ND ND HS578T -0.77 no change 9.19 8.17 Negative ND ND ND JIMT1 -1.48 no change 10.14 7.66 Negative ND ND ND MCF7 -3.50 no change 10.69 9.78 Positive 0.201682325 0.09995058 0.28418073 MDAMB134 -2.85 no change 10.46 11.20 Positive 0.398024025 0.44175705 0.42471533 MDAMB157 0.15 gain 10.38 6.64 Negative ND ND ND MDAMB231 -1.95 no change 9.66 6.68 Negative ND ND ND MDAMB453 -0.41 no change 9.84 6.68 Negative ND ND ND MDAMB468 -1.35 no change 10.38 6.95 Negative ND ND ND SKBR3 -0.35 no change 9.88 6.85 Negative ND ND ND SUM149 0.41 no change 9.96 7.06 Negative ND ND ND SUM44 0.69 loss 9.52 11.45 Positive T47D -1.24 loss 10.33 10.62 Positive 0.272133467 0.1307487 0.1292707 VP229 -0.60 no change 9.79 7.77 Negative ND ND ND

Functional Breast Cancer Profiles Sup.Info.17

Supplementary Table 9. Optimised transfection conditions for the 20 breast cancer cell lines screened in the functional viability profiling.

Volume Doubling lipid per Cell Time Cell line Direction Reagent well (ul) no (days) CAL51 Reverse Lipo2000 0.25ul 2500 1.3 CAL120 Reverse RNAiMax 0.1ul 2000 1.6 CAMA1 Forward oligofectamine 0.5ul 2000 1.8 HCC1143 Forward RNAiMax 0.2ul 2000 1 MDAMB134 Reverse RNAiMAX 0.1ul 5000 2.8 SUM44 Forward RNAiMax 0.1ul 2000 4 VP229 Reverse RNAiMax 0.1ul 2000 2.5 MCF7 Reverse RNAiMAX 0.2ul 2000 0.9 JIMT1 Reverse RNAiMax 0.1ul 2000 1.4 SKBR3 Reverse RNAiMax 0.1ul 2000 1.9 BT474 Reverse RNAiMax 0.2ul 5000 1.9 HCC202 Reverse RNAiMax 0.1ul 4000 3.1 MDAMB453 Reverse RNAiMax 0.05ul 3000 2.2 SUM149 Forward RNAiMax 0.1ul 2000 0.9 MDAMB468 Reverse RNAiMax 0.1ul 3000 1.6 MDAMB231 Forward DF4 0.2ul 2000 1.5 HS578T Forward DF4 0.2ul 2500 2 MDAMB157 Reverse RNAiMax 0.1ul 2500 1.9 T47D Reverse RNAiMax 0.1ul 2000 2.6 BT20 Reverse RNAiMax 0.1ul 4000 2.5