Article

KDC YJMBI-65651; No. of pages: 12; 4C:

Rationalizing Drug Response in

Cancer Cell Lines

Teresa Juan-Blanco 1 , Miquel Duran-Frigola 1 and Patrick Aloy 1, 2

1 - Joint IRB-BSC-CRG Program in Computational Biology, Institute for Research in Biomedicine (IRB Barcelona),
The Barcelona Institute of Science and Technology, Barcelona, Catalonia, Spain
2 - Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, Spain

Correspondence to Patrick Aloy: IRB-BSC-CRG Program in Computational Biology, Institute for Research in
Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Catalonia, Spain.
patrick.aloy@irbbarcelona.org
https://doi.org/10.1016/j.jmb.2018.03.021
Edited by Juan Fuxman

Abstract
Cancer cell lines (CCLs) play an important role in the initial stages of drug discovery allowing, among others,
for the screening of drug candidates. As CCL panels continue to grow in size and diversity, many
polymorphisms in genes encoding drug-metabolizing enzymes, transporters and drug targets, as well as
disease-related genes have been linked to altered drug sensitivity. However, identifying the correlation
between this variability and pharmacological responses remains challenging due to the heterogeneity of
cancer biology and the intricate interplay between cell lines and drug molecules. Here, we propose a network-
based strategy that exploits information on gene expression and somatic mutations of CCLs to group cells
according to their molecular similarity. We then identify genes that are characteristic of each cluster and
correlate their status with drug response. We find that CCLs with similar characteristic active network regions
present specific responses to certain drugs, and identify a limited set of genes that might be directly involved in
drug sensitivity or resistance.
© 2018 Elsevier Ltd. All rights reserved.

Introduction To overcome these limitations, CCL panels

Cancer cell lines (CCLs) are one of the most widely
used experimental models to understand cancer
biology, as well as to test the efficacy of novel
anticancer therapies [1]. A large number and variety
of CCLs exist, and their cost-effectiveness and
unlimited auto-replicative nature facilitate the fast
acquisition of results [2]. However, the clinical
relevance of CCLs remains controversial. First,
CCLs present technical restrains such as cross-
contamination with other cell lines or genomic
instability produced by culture conditions [3]. Second,
and more important, CCLs often do not represent
primary tumors [4,5], which may lead to misleading
conclusions and difficulties to translate the outcomes
to in vivo models. For example, two-dimensional
monolayer cultures are more sensitive to cytotoxic
agents, and therefore, sensitivity results are over-
optimistic [6–8].
continue to improve in size and in-depth character-
ization. In 1990, the NCI-60 was launched, contain-
ing only 59 cell lines representing 9 cancer types [9].
Modern panels such as the pioneering Cancer Cell
Line Encyclopedia (CCLE) offer a comprehensive
collection of gene expression, chromosome copy
number and sequencing data for about one thousand
CCLs [10]. Currently, efforts are put into increasing the
number of drugs that are screened against these vast
collections, and to map clinical data on their molecular
profiles [11].
The large volume of molecular data accumulated
allows for the systematic study of drug response
in CCLs. Many polymorphisms in genes encoding
drug-metabolizing enzymes, transporters and drug
targets, as well as disease-related genes have been
proposed as determinants of drug sensitivity [12].
However, true mechanistic understanding of drug
action remains challenging. One of the main difficulties

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org/10.1016/j.jmb.2018.03.021
2 Rationalizing Drug Response in Cancer Cell Lines

is that differential pharmacological profiles are caused Results and Discussion

by the interplay of multiple genes [13], and other factors
such as the tissue of origin may confound the analysis
and translation to the clinics [3,14]. Often, there is Stratifying CCLs according to their molecular
no correlation between the expression of the intended profiles
pharmacological targets and the observed drug
activity [14,15]. All the above suggest that systems To describe molecular variability in CCLs, we
biology approaches may help elucidating the exploited the mutational and transcriptional land-
complexity of drug response [16]. So far, and scapes provided by the CCLE. This panel contains
given the significant amount of data collected, the somatic mutations and indels for 1651 genes, and
most successful strategies to predict drug activity are expression data for 18,987 genes. We kept for
based on supervised machine learning approaches analysis the 448 CCLs, representing 23 tissues, for
[17,18]. However, although they yield promising which somatic mutations, transcriptional data and
results in terms of accuracy of the predictions, these pharmacological profiles are available. Differential
methods are largely unable to pinpoint the biological gene expression analysis was done by comparing
players (i.e., over/under-expression of certain genes, basal expression of CCLs to the rest of the panel
mutations, etc.) responsible for the observed drug (see Methods).
effects. Figure 1 summarizes the strategy implemented and
Here, we describe a systems biology, cell-centered content of our data set. The most represented tissues
approach to characterize drug response in CCL in the data set are lung and hematopoietic and
panels. Instead of grouping CCLs according to their lymphoid cells. The number of somatic mutations per
drug sensitivity, we exploited molecular data provided CCL is, on average, 10 times smaller than the number
by the CCLE, together with a protein interaction of differentially expressed genes.
network, to bring together CCLs with similar molecular To integrate the CCL molecular profiles and to
profiles. Only then we correlate these molecular determine their area of influence, we applied the
profiles to differential drug response and propose network-based stratification (NBS) method [19]. In
potential gene expression signatures that are brief, NBS combines network propagation with clus-
beneficial for predicting drug sensitivity/resistance tering analysis to identify groups of CCLs whose
in CCLs. somatic mutations and other molecular signals affect

(a) Network Propagation (b)

1500
# Dif exp genes

Somatic Mutations 1000

1651 genes
Interaction network
12486 proteins 500
Gene expression
18987 genes 62387 interactions

1400
448 CCLs
# Mutated genes

1200
1000
800

CCL Clusters 600

400
●
200
**
0
●

**
●

**
●
●

*
80
●
● ● ●
●
● ●

● ●

*
●

60
# CCLs

40
20
Map drug sensitivity Pharmacological profiles 0
Skin

Thyroid
Lung
Liver
Endometrium
Bone

UAT
Pleura
H&L

Ovary

Prostate
Kidney

Stomach
Breast
CNS

Soft tissue

Urinary tract
Bilary tract
Autonomic ganglia

Large intestine

Pancreas

on clusters
Salivary gland
Oesophagus

24 antineoplastic drugs

Tumor type

Fig. 1. Summary of the methodology and data set. (a) Somatic mutations and gene expression data from the CCLE was
integrated with PPI data through the network propagation algorithm. Then, CCLs were clustered and drug sensitivity to 24
antineoplastic drugs was mapped onto them. (b) Distribution of the number of differentially expressed and mutated genes
among the 448 CCLs grouped by tumor type and number of CCls per tumor type. CNS stands for central nervous system;
H&L, hematopoietic and lymphoid; and UAT, urinary aerodigestive tract.

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Rationalizing Drug Response in Cancer Cell Lines 3

similar areas of the interactome. We used an in-house
binary protein–protein interaction (PPI) network gen-
erated by merging major public PPI repositories [20]. In
total, the PPI network used in this study contains
12,486 proteins and 62,387 interactions.
The NBS protocol requires that both mutation and
expression are expressed as binary features (see
Methods). The network propagation step smoothens
these profiles so that they are compliant with
the network architecture, leading to a molecular
signature that is no longer binary but captures the
proximity to the altered genes. Then, we clustered
the resulting network-smoothed profiles into an
optimal number of groups and used the tissue of
origin as a proxy to assess the biological relevance
of the clusters [21]. The results are displayed in the
co-clustering networks in Fig. 2a and b. As evident,
gene expression and somatic mutation data do not
cluster cell lines in the same way. Differential
expression clusters are able to recapitulate the
tissue of origin of the cell lines (e.g., hematopoietic
and lymphoid cells form a well-distinguished group),

(a) Differential expression co-clustering network (b) Mutations co-clustering network

Color legend
(c) Merged co-clustering network Skin

Thyroid
Lung
Liver
Endometrium
Bone

UAT
Pleura
H&L

Ovary

Prostate
Kidney

Stomach
Breast
CNS

Soft tissue

Urinary tract
Bilary tract
Autonomic ganglia

Large intestine

Pancreas

Salivary gland
Oesophagus

(d) 1.0
% co-clustering

0.8

0.6

0.4

Dif. exp Merged Mutations

(e) 1.0
0.8
Dif. exp vs. merged
NMI

0.6 Dif. exp vs. mutation

0.4 Mutation vs. merged

0.2
0.0
20 40 60 80 100
# Clusters

Fig. 2. Co-clustering network using differential expression (a), somatic mutations (b) and the merged data set (c) after
applying NR-NMF onto the network-smoothed matrix for a number of clusters from 3 to 100. Nodes symbolize the CCLs.
Edges connect two CCLs if they cluster together at least in 30% of the clustering results. Node colors represent the tissue
of origin. Edge transparency is proportional to the co-clustering score. (d) Co-clustering scores distribution for differential
expression, mutation and merged data set. (e) Normalized mutual information between the clusters results between the
three data sets. CNS stands for central nervous system; H&L, hematopoietic and lymphoid; and UAT, urinary
aerodigestive tract.

Please cite this article as: T. Juan-Blanco, et al., Rationalizing Drug Response in Cancer Cell Lines, J. Mol. Biol. (2018), https://doi.
org/10.1016/j.jmb.2018.03.021
4 Rationalizing Drug Response in Cancer Cell Lines

17.AAG F = 3.46 p−value = 2.25e−05 AEW541 F = 6.71 p−value = 2.03e−12 AZD0530 F = 4.03 p−value = 1.44e−06 AZD6244 F = 13.32 p−value = 2.76e−26

* ** **
6 4 6
** 3 **
* 3
**
4
* 4 *
* * * * *
ActA

ActA

ActA

ActA
2
2
*
2 **
2 1
1

0 0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cluster Cluster Cluster Cluster
Erlotinib F = 5.5 p−value = 9.15e−10 Irinotecan F = 9.84 p−value = 1.34e−17 L.685458 F = 13.53 p−value = 1.43e−26 Lapatinib F = 6.69 p−value = 2.17e−12
6
** ** **
4 4
* 3 * *
3
* ** 3
4
* ** **
* 2 *
ActA

ActA

ActA

ActA
2 2 *
*
* * 2 1 ** **
1 1 *
0 0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cluster Cluster Cluster Cluster
Nilotinib F = 14.18 p−value = 6.61e−27 Nutlin.3 F = 4.08 p−value = 1.09e−06 PD.0325901 F = 16.03 p−value = 1.46e−31 PD.0332991 F = 10.76 p−value = 1.52e−20
** **
** **
3
6 ** 3
6 ** ** **
* ** * ** * *
2 *
** ** *
2
4 4
ActA

ActA

ActA

ActA
**
* ** ** *
2
1
2
1 *

0 0 0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cluster Cluster Cluster Cluster
PF2341066 F = 10.11 p−value = 9.88e−20 PHA.665752 F = 4.79 p−value = 3.14e−08 PLX4720 F = 10.6 p−value = 1.03e−20 Paclitaxel F = 7.6 p−value = 2.25e−14
8
* ** ** **
****
3
**
5
4
** ** * *
** 4 **
3 ** 2 **
6

3
**
ActA

ActA

ActA

ActA

2 * 4
1
2 *
1 * 1
2

0 0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cluster Cluster Cluster Cluster
Panobinostat F = 20.92 p−value = 2.32e−40 RAF265 F = 5.53 p−value = 9.27e−10 Sorafenib F = 9.68 p−value = 7.98e−19 TAE684 F = 8.34 p−value = 5.6e−16
7
** 6
6
** ** 4 **
** * * * * *
** **
6
* * * * * *
* 3 4 4 *
5
*
** * * *
ActA

ActA

ActA

ActA

*
**
2
4
2 2
*
3 1
*
2 0 0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cluster Cluster Cluster Cluster
TKI258 F = 8.18 p−value = 1.26e−15 Topotecan F = 9.58 p−value = 1.26e−18 ZD.6474 F = 4.78 p−value = 3.37e−08

** 4
* * ** *
6
** * *
4 * 3 *
* * * * *
** 4 * *
ActA
ActA

ActA

2
*
2
* 2
1

0 0 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Cluster Cluster Cluster

Fig. 3 (legend on next page)

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Rationalizing Drug Response in Cancer Cell Lines 5

Table 1. Summary of the functional enrichment analysis of genes altered in each cluster
Cluster Function annotation (GO terms, KEGG and Reactome pathways)
1 Neuron differentiation and development, signaling by GPCR, synaptic transmission
2 Pathways in cancer, signaling by NGF, ErbB signaling pathway, protein kinase activity
3 Apoptosis, ectoderm and endodermis development, cytoskeleton, calcium ion binding, cell–cell junction
5 Extracellular matrix structural constituent, ECM–receptor interaction, growth factor binding, focal adhesion, axon guidance,
hemostasis, integrin cell surface interactions
6 Primary immunodeficiency, intestinal immune network for IgA production, signaling in Immune system, homeostasis, cytokine–cytokine
interaction, B-cell receptor signaling pathway
7 Glutathione metabolism
8 Complement and coagulation cascades, hemostasis, metabolism of lipids and lipoproteins
11 MHC class II receptor activity, ECM–receptor interaction
12 B-cell homeostasis, cytokine binding
13 Signaling in immune system, hemostasis, natural killer cell-mediated cytotoxicity, hematopoietic cell lineage, T-cell receptor signaling
pathway, chemokine signaling pathway, integral cell surface interactions. Cell adhesion molecules, Fc gamma R-mediated
phagocytosis, Jak–STAT signaling pathway, leukocyte transendothelial migration, regulation of actin cytoskeleton
14 Apical part of cell, sensory perception of sound and mechanical stimulus
15 Epidermis and ectoderm development, axon guidance, signaling by PDGF, collagen type IV

and the result of the clustering is robust (Fig. 2d).
However, and despite their recognized importance in
tumor development [22] and drug response [23], we
found that CCL clusters obtained from somatic
mutations alone are less robust (Fig. 2d), rather
scattered, and do not correlate with the tissues of
origin (Fig. 2b). In order to retain mutation data in our
clusters, we combined the network-smoothed
expression and mutation profiles into a single, merged
profile (see Methods). The resulting co-clustering
network of this merged data set is depicted in Fig. 2c.
Here, clusters resemble those obtained by gene
expression analysis alone (Fig. 2e), thereby keeping
their biological relevance and robustness (Fig. 2d),
while still incorporating the valuable mutation data. We
chose this merged data set for further analyses.
To optimize the clustering parameters, we required
the number of CCLs per cluster to be comparable
among clusters and be large enough to enable
statistical analysis. We chose to carry on our analyses
with 15 clusters, as it ensured a minimal number of
groupings with less than 10 CCLs and a small standard
deviation of the number of cells per group (Fig. S1).
Figure S2 shows that the number of CCLs in each
cluster varies from 16 to 45, and illustrates tissue
distribution along the 15 clusters. Some clusters are
clearly dominated by CCLs belonging to a single tissue
(i.e., clusters 6, 12 and 13 are composed only by
hematopoietic and lymphoid cells), while others are
more diverse. Finally, we re-ran our pipeline using two
functional networks gathered from STRING [24] and
inBioMap [25] to measure the robustness of the results
and to check whether we could increase the protein
coverage (Figs. S3–S5). The clusters obtained from
functional networks were considerably similar to the
previous results with physical protein interaction in
terms of cluster robustness and protein coverage
(normalized mutual information of 0.66 and 0.68 for
STRING and inBioMap, respectively).
Cluster correlation with drug response
We then assessed whether CCLs in the same cluster
respond similarly to drug treatment. Following recom-
mendation by the CCLE authors, we used the area
above the drug-response curve (ActA) as the drug
activity measure—the higher the ActA, the more
effective the drug is. Remarkably, for 23 of the 24
drugs, we could find at least one cluster whose CCLs
had a differential drug sensitivity (ANOVA p value b
0.05; Fig. 3). Some of the drugs, like Panobinostat, had
specific drug activity ranges in most of the clusters,
while others, like PLX4720, were mostly related to a few
of them. Some clusters contained CCLs that were in
general more sensitive, as it is the case for two out of
the three hematopoietic and lymphoid clusters (12
and 13). Also, several tissue-diverse clusters were
specific to some of the drugs (e.g., cluster 3 and
Erlotinib), supporting the notion that the key molecular
features may drive drug response across tumor types
[14,15,26].
Interestingly, CCL clusters respond similarly to drugs
with the same mechanism of action. For instance,
clusters 11, 13 and 14 are more sensitive to the
two MEK inhibitors AZD6244 and PD-0325901. Their
drug targets, MEK1 and MEK2, are down- and over-
expressed, respectively, in these sensitive clusters
(Wilcoxon's p value b 0.005). Another example relates
to cluster 3, whose CCLs are tissue heterogeneous and
respond to EGFR inhibition by Erlotinib and Lapatinib.

Fig. 3. Drug activity distribution along CCL clusters for the 23 drugs with a response significantly associated with
at least one cluster. “F” and “p value” are the ANOVA F value and p value, respectively. Red asterisks indicate that the
drug response of the cluster is significantly different to more than 5 (*) or 10 (**) clusters (Benjamini–Hochberg adjusted
p value b 0.05).

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6 Rationalizing Drug Response in Cancer Cell Lines
Molecular signatures and drug response
Given the observed relevance of our CCL clusters to
drug response, we sought to identify the representative
genes of each cluster, that is, those with a similar
behavior in all the CCLs belonging to it. For this, we
applied the Significant Analysis Of Microarray (SAM)
algorithm [27] and selected genes with a significantly
high SAM score (FDR b 0.01). We then compared
every CCL cluster with the remaining cohort. The figure
shows the number of significant genes identified per
cluster. Finally, we exploited the DAVID database [28]
to functionally annotate the relevant genes identified.
Table 1 summarizes the biological terms associated
with the clusters.
The number of detected genes is highly variable
across clusters (Fig. 4). The clusters with a higher
number of genes are clusters 6 and 13, which contain
hematopoietic and lymphoid cells, as well as cluster 11,
composed by skin cells. As expected, we found a
lesser number of associated genes when CCLs in the
cluster were more diverse, as is the case of clusters 9
and 10, containing cell lines from all of the tissues.
A noteworthy observation is that, thanks to the
network-propagation algorithm, we could detect 724
gene–cluster associations (i.e., 21% of the total) that
would have been otherwise lost. Seventy-one out of
these 724 involve genes whose expression was not
measured by the CCLE, 214 comprise genes that
do not significantly change their expression in any
CCL, and the remaining 439 are not differentially
expressed nor mutated in the cluster compared to the
other clusters. Some interesting examples among
these associations are cancer-related genes such as
the transcription factor FOXO1, the kinase FYN, the
proto-oncogene VAV1 and PIDD1, which promotes
apoptosis downstream of TP53 and mediates NF-ΚΒ
activation in response to DNA damage [29,30].
To check whether cluster-specific molecular
signatures can help discovering drug response
500
400
# Genes
300 321
200
203
171
100 132
1 2 3 4
Fig. 4. Number of significant genes identified by SAM in each cluster. FDR b 0.01.
Please cite this article as: T. Juan-Blanco, et al., Rationalizing Drug Response in Cancer Cell Lines, J. Mol. Biol. (2018), https://doi.
org/10.1016/j.jmb.2018.03.021
biomarkers, we compared the expression/mutation
status of the previous cluster-associated genes be-
tween sensitive and resistant CCLs. That is, for each
drug–gene pair, we tested whether the expression/
mutation status of the gene was significantly different in
sensitive/resistant CCLs. Figure 5a has examples of
the three potential drug sensitivity associations, namely
down-, up-regulated gene expression, and mutated
genes. Figure 5b–e quantifies the potential biomarkers
that we could find (Bonferroni-corrected Fisher's
p value b 0.05). With gene expression data, we
identified 1733 gene–drug correlations, involving 563
genes and 23 drugs. A portion of these associations is
listed in Table 2, and the rest are available in Table S1
(please note that genes associated with clusters only
through the network-propagation step had no chance to
enter this differential expression analysis). Regarding
mutations, we could only find six significant mutation–
drug associations, all of them involving BRAF and
KRAS (Table 3). Reassuringly, all of them are well-
documented drug response biomarkers [31,32]; unfor-
tunately, due to the low mutation rate and to the smaller
number of mutated genes, we lacked statistical power
to find novel biomarker mutations.
Benefit of the system biology approach
As we showed above, the network-propagation
algorithm was helpful to detect cluster-associated
genes that would have remained otherwise unper-
ceived. To further evaluate this feature, we re-run our
clustering analysis, this time skipping the network
propagation step. By considering the basal gene
expression of 16,077 genes, we now grouped the
CCLs into 7 clusters (Fig. S6A). Similar to the results
obtained with NBS, 23 of the 24 drugs could be
associated with one or more of these clusters (Fig. S7).
However, we found a rather different profile of genes in
the clusters, detecting more than 2000 genes per
cluster and, overall, associating 88% of the genes to at
514
466
440
306
212
194
124 118
109
87
25
5 6 7 8 9 10 11 12 13 14 15
Cluster
Rationalizing Drug Response in Cancer Cell Lines 7

(a) (c)
FLI1 — L-685458 BTBD3 — L-685458 BRAF — AZD6244 20
Up-regulated Down-regualted Mutated

# Unique drugs
Yes No Yes No Yes No 15
Sens.

Sens.

Sens.
Response

Response

Response
21 30 14 34 33 48
10
Res.

Res.

Res.
7 337 14 330 23 300 5

Fisher p-value = 2e-15 Fisher p-value = 6e-7 Fisher p-value = 2e-12 0

9 10 4 7 8 15 5 1 14 2 11 3 12 6 13
(b) (d) Cluster
1%
500 51%
2% 300
80% 1% > 100
44%
400 250 ≤ 100

# Unique genes
# Unique genes

≤ 10
Expression

1% No expression 200
2% data in CCLE
300 98%
97%
48% 55%
No significant 150
7% 1% Significant
200 1% 89% biomarkers 100
2% 42%
89%
89%
1% 11% 50
98% 96% 57% 6%
100 2% 89% 18% 90%
93% 0

PLX4720
Nilotinib
PF2341066
PD.0332991
Sorafenib
Paclitaxel
Topotecan
Panobinostat
L.685458
RAF265
AZD0530
X17.AAG
ZD.6474
Nutlin.3
Lapatinib
Erlotinib
PHA.665752
PD.0325901
AEW541
TKI258
AZD6244
Irinotecan
TAE684
4% 10%
4% 9% 2% 1%
0 2% 5% 3% 96% 5%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cluster

83%
500
92% Drugs
87%
(e)
400
Mutations

# Unique genes

No mutation
93% 91% data in CCLE 150
300 No significant
Significant
# Genes

64% 93% biomarkers 100

200 89%
94%
94% 97% 90%
91%
100 17% 92% 50
36%
13%
7% 9% 7% 88% 8% 11% 8%
7%
0
6% 6% 1% 3% 10% 12% 1%
0
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
# Drugs
Cluster

Fig. 5. Statistics on drug response associations. (a) Examples of the contingency tables to find associations between
differential expression or mutations and drug response. (b) Proportion of genes significantly, no significantly associated
with drug response and without molecular data available in the CCLE for both differential expression and mutation.
(c) Number of unique drugs per cluster. (d) Number of unique genes per drug. (e) Specificity of the drug–gene associations, that
is, number of genes associated with a certain number of drugs.

least one cluster (Fig. S6B). By integrating network based on Bayesian Regression Trees (BART) [33].
information and starting from a comparable number of Like most Bayesian methods, BART works wells with
genes, we decreased this percentage to 25%. Hence, relatively few samples and, thanks to its tree structure,
our NBS approach has permitted to drastically reduce can natively deal with highly correlated variables.
the number of genes associated with clusters, allowing We applied the BART algorithm on the Network-
a much simpler interpretation. smoothed profiles to classify CCLs into sensitive or
resistant, according to their ActA (Fig. 6a). Further-
Drug response prediction more, we included the cluster identity and the
cluster-specific genes identified to assess whether
The described methodology has grouped CCLs the predictive power 11of our model would improve
according to their basal molecular profiles, and we with this information (Fig. 6).
have shown that these cell clusters, in many cases, can We used the area under the ROC curve (AUROC) to
be associated with the response to specific drugs. We evaluate the performance of the leave-one-out cross-
have also identified characteristic molecular features of validated classifier (Fig. 6b). In total, by using cluster
each cluster, and it remains to be seen whether these information and cluster-specific genes, we could rank
are sufficient to predict drug sensitivity or resistance sensitive CCLs with an AUROC higher than 0.7 for 16
of CCLs. To address this question, and given the drugs. Remarkably, the overall performance of the
relatively small number of samples and the recognized classifier does not drop if we only use the set of cluster-
coexpression between genes, we used a classifier associated genes (i.e., four times fewer genes), and the

Please cite this article as: T. Juan-Blanco, et al., Rationalizing Drug Response in Cancer Cell Lines, J. Mol. Biol. (2018), https://doi.
org/10.1016/j.jmb.2018.03.021
8 Rationalizing Drug Response in Cancer Cell Lines

Table 2. Drug response associations to differentially expressed genes

Drug Gene Expression Cluster % Total % Sensitive % Resistant Bonferroni p value
Nilotinib ASAP2 Down 6, 12, 13 12 65.5 7.9 1.34E−08
TKI258 BIN2 Up 12, 13 6 57.9 4.0 1.75E−06
Sorafenib ARHGAP15 Up 6, 13 10 55.6 7.1 8.16E−06
Sorafenib SNX7 Down 6, 13 11 55.6 8.6 6.37E−05
Sorafenib TJP1 Down 6, 12, 13 11 55.6 8.1 1.47E−05
Nilotinib SH3BP4 Down 6, 12, 13 12 55.2 8.5 1.74E−05
L.685458 ASAP2 Down 6, 12, 13 11 53.7 4.7 8.11E−15
L.685458 SNX7 Down 6, 13 12 53.7 5.8 4.86E−13
TKI258 CD53 Up 12, 13 10 52.6 7.9 6.78E−03
TKI258 DOCK8 Up 6, 13 8 52.6 6.5 3.31E−03
TKI258 LCP2 Up 13 7 52.6 4.7 2.46E−04
“Expression” indicates whether the gene is up- or down-regulated. “% Total” is the total percentage of CCLs with the expression biomarker.
“% Sensitive” and “% Resistant” are the percentages of sensitive or resistant CCLs with the expression biomarkers. “Bonferroni p value” is
the corrected Fisher's test p value.

number of drugs with an AUROC of N 0.7 is higher.
Under these settings, the best performing drugs are
L-685458 and Nilotinib, with a cross-validated AUROC
above 0.8.
In addition, we sought to confirm the predictive power
of our approach with an external data set. We chose the
GDSC CCL panel described by Iorio et al. [11] in 2016,
where they characterized molecular alterations in 1001
human CCLs and correlated them with sensitivity to
265 drugs. The CCLE and the GDSC data sets share
343 CCLs and 15 drugs. However, the drug activities
reported by the two studies are poorly correlated
(Fig. S9), which effectively limits the validity of the
comparison. We nevertheless carried out the analysis
by using the BART models obtained from the CCLE
panel to predict drug sensitivity in the GDSC data set.
Figure 6c shows the performance of the BART
classification for all methods. As it can be observed,
the performance of the classification decreases in the
external data set compared to the cross-validation
results.
We repeated the analyses considering only those
drugs that, at least, had the same type of qualitative
response in the two data sets (i.e., sensitive or
resistant; Fig. S10), obtaining better results for 8 out
of 10 drugs (SAM & cluster approach). Finally, we
cleaned up the sets further by including only those
CCLs whose drug response falls in the percentile 33
and 66 in both cases (i.e., CCLs that are in the top
sensitive or the top resistant in both data sets; Figs. 6d
and S11). We can appreciate that the median AUROC
raises for all the approaches and, although the increase
is not significant, the performance improved for 9 out of
15 drugs. This increase, however, has the drawback of
a lower applicability since the number of sensitive CCLs
is very limited for some drugs.
Conclusions
In this report, we have described an approach to
group CCLs according to their molecular profiles and
to capture how specific profile alterations are spread
across their protein–protein wiring. Combining gene
expression and mutational data, we obtained robust
clusters that recapitulate the CCL tissue of origin,
indicating that gene expression profiles are highly
influenced by the histology. Moreover, we observed
that, starting from the molecular variability described in
the CCLE alone, our CCL clusters showed significant
specificity for most of the drugs. Moreover, we identified
those genes that were altered in each cluster and
looked for associations between their expression status
and the observed drug response. In total, we identified
1712 potential gene–drug associations and found, in
addition, a strong, potentially confounding influence of
the CCL tissue of origin on drug response, especially in
hematopoietic and lymphoid cells, which tend to be
more sensitive.

Table 3. Drug response associations for mutations

Drug Gene Cluster % Total % Sensitive % Resistant Bonferroni p value
PLX4720 BRAF 11 16 66 10 3.21E− 13
AZD6244 BRAF 11 16 42 9 2.69E− 08
PD.0325901 BRAF 11 16 27 6 6.39E− 06
PD.0325901 KRAS 7, 15 22 32 13 3.87E− 03
PLX4720 KRAS 7, 15 21 0 24 1.56E− 02
RAF265 BRAF 11 16 26 9 4.04E− 02
“% Total” is the total percentage of CCLs with the mutated gene. “% Sensitive” and “% Resistant” are the percentages of sensitive or
resistant CCLs with the mutation biomarker. “Bonferroni p value” is the corrected Fisher test p value.

Please cite this article as: T. Juan-Blanco, et al., Rationalizing Drug Response in Cancer Cell Lines, J. Mol. Biol. (2018), https://doi.
org/10.1016/j.jmb.2018.03.021
Rationalizing Drug Response in Cancer Cell Lines 9

(a)
100
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Response
50 46%
40% 41% 39% Resistant
35% 34%
29% Sensitive
25 20% 17% 16% 15% 14%
12% 7% 12% 12% 8% 9% 11% 11%
6% 4% 9%
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(b)
NS matrix SAM genes NS matrix & Clusters SAM & Clusters
Median AUC = 0.709 Median AUC = 0.715 Median AUC = 0.722 Median AUC = 0.72
1.00

0.75
TPR

0.50

0.25

0.00
0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00
FPR FPR FPR FPR
(c)
NS matrix SAM genes NS matrix & Clusters SAM & Clusters
Median AUC = 0.535 Median AUC = 0.497 Median AUC = 0.514 Median AUC = 0.519
1.00

0.75
TPR

0.50

0.25

0.00
0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00
FPR FPR FPR FPR
(d) SAM genes NS matrix & Clusters SAM & Clusters
NS matrix
Median AUC = 0.551 Median AUC = 0.545 Median AUC = 0.534 Median AUC = 0.522
1.00

0.75
TPR

0.50

0.25

0.00
0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00
FPR FPR FPR FPR

Fig. 6. Drug response prediction. (a) Percentage of sensitive CCLs per drug. (b) ROC curves after applying a leave-one-out
cross-validation. Lines represent the 23 drugs. “NS matrix” is the complete network-smoothed matrix—448 CCLs and 12,486
genes; “SAM genes” only includes genes identified as relevant in the clusters—448 CCLs and 3119 genes; “NS matrix &
Clusters” is the complete network-smoothed matrix plus the cluster information; and “SAM & cluster” refers to both the relevant
genes and the cluster information. (c) ROC curves for the prediction of the external data set and (d) including only the top resistant/
sensitive CCLs in both data sets.

Please cite this article as: T. Juan-Blanco, et al., Rationalizing Drug Response in Cancer Cell Lines, J. Mol. Biol. (2018), https://doi.
org/10.1016/j.jmb.2018.03.021
10
Thanks to the systems biology approach to propa-
gate the effect of over/under-expression and mutations
throughout the network, we detected 724 genes that
were not characterized in the CCLE and might, we
believe, guide mechanistic interpretation due to their
proximity to altered genes.
Finally, we checked whether the network-propagated
profiles could be used to predict drug response. We
predicted drug sensitivity with an AUROC higher than
0.7 for 13 of the 24 drugs. However, the poor correlation
between drug responses in different CCL panels
makes the external validation of the predictions tough.
This has been a classical concern of machine-learning
based prediction of CCL drug response [34–36]. We
hope that approaches like the one presented here,
more focused on the identification of key, robust genetic
determinants of drug response, will lead to more
informed predictions and finally shed light onto the
molecular bases of drug action.
Methods
CCL data
We retrieved CCL data from the CCLE portal (https://
portals.broadinstitute.org/ccle). We downloaded the
gene expression data (version 2012-09-29), the Hybrid
capture sequence (version 2012-10-18) and the drug
profiling (version 2015-02-24). We included in our
analysis the 448 CCLs that are present in the three
data sets. The final data set contains somatic mutations
and indels for 1651 genes, expression data for 18,987
genes and pharmacological profiles for 24 antineo-
plastic drugs.
PPI data
We used an in-house PPI network, generated by
merging data available from major public PPI data-
bases (version 2016_06) [20]. We only considered
physical binary interactions. To incorporate the molec-
ular data onto the network, we mapped ENTREZ Gene
IDs to UniProtACs using the UniProtID mapping tool.
We also gathered two functional interaction networks
from STRING [24] and inBioMap [25] databases. We
applied confidence cutoffs of 0.7 and 0.2, respectively,
regarded as “high confidence” by the databases'
authors. The STRING network contains 14,725 pro-
teins and 300,686 interactions and the inBioMap
network, 10,100 proteins and 168,970 interactions.
NBS of CCLs
We applied the NBS method [19] to integrate gene
expression and somatic mutations data with the PPI
network. We used the MATLAB code provided by the
authors. Expression and mutation data were binarized
Please cite this article as: T. Juan-Blanco, et al., Rationalizing Drug Response in Cancer Cell Lines, J. Mol. Biol. (2018), https://doi.
org/10.1016/j.jmb.2018.03.021
Rationalizing Drug Response in Cancer Cell Lines
to map them onto the PPI network. For somatic
mutations, we considered 1 if a given gene is mutated
in a CCL and 0 otherwise. The following variants were
filtered out: common polymorphisms, allelic fraction
b 10%, putative neutral variants (missenses present in
less than 2 warm-blooded vertebrates) and located
outside of the CDS for all transcripts. Regarding
transcriptomic data, 1 refers to differentially expressed
genes, either up- or down-regulated. To detect
differentially expressed genes, we normalized the
basal expression of each gene in a cell line to the
expression distribution of the gene in all CCLs, yielding
a differential gene-expression Z-score. We defined
as up- or down-regulated those genes with Z ≥ 2 or
Z ≤ −2, respectively. To merge expression and muta-
tion data, we summed up the network-smoothed
matrices, and afterward, we scaled the merged matrix
between 0 and 1. To optimize the number of clusters,
we minimized the groups with less than 10 CCLs and
the standard deviation of the number of cells per group
(Fig. S1). To compare the clustering results, we
calculated the normalized mutual information.
We applied the SAM [27] method to identify those
genes with a significantly high score within clusters
(FDR b 0.01). We compared every cluster with the
remained cohort. We run the functional enrichment
analysis on the DAVID database Web Services. We
considered biological processes and molecular func-
tions from the Gene Ontology, as well as KEGG and
Reactome pathways.
Cluster correlation with drug response
To assess whether CCLs within clusters present
similar drug response, we performed the ANOVA test
on the drug response distribution along clusters. We
used the activity area (ActA) as drug response measure
since it provides a comprehensive representation of
drug activity according to the CCLE. Clusters were also
compared pairwise to identify significant differences
in drug response (Wilcoxon's rank test, Benjamini–
Hochberg p value b 0.05).
Gene–drug response associations
To analyze whether the genes identified previously
are associated with differential drug response, we
compared their expression/mutation status between
sensitive and resistant CCLs. For each drug–gene pair,
we built a contingency table to see whether the
expression/mutation status of the gene is significantly
different between sensitive and resistant CCLs. We
applied the Bonferroni correction to adjust for multiple
Fisher's exact testing. We used the waterfall plot to
classify CCLs as resistant or sensitive as described in
Haibe-Kains et al. [34]. In brief, for each compound, the
shape of the rank-ordered plot of the responses values
was inspected to classify CCLs into sensitive or
resistant. If the drug response distribution was linear,
Rationalizing Drug Response in Cancer Cell Lines
we used as inflection point the median. If it was not
linear, we took the point on the curve with the maximal
distance to a line drawn between the start and end
points of the distribution.
Drug response prediction
To predict drug sensitivity in each CCL, we applied
the BART classifier [33] on the network-smoothed
matrix, using the implementation provided in the R
package bartMachine [37]. We applied both a leave-
one-out and fivefold cross-validation (Fig. S8). In the
fivefold cross-validation, we applied the SMOTE R
package [38] to balance the number of resistant/
sensitive CCLs. SMOTE blends under-sampling of
the majority class with a special form of over-
sampling the minority class. We split the training
and test data set keeping the proportion of resistant/
sensitive CCLs and then we computed SMOTE only
in the training set.
We chose the GDSC CCL panel described in
Ref. [11] as external data set to validate the classifier.
GDSC contains 15 drugs in common with CCLE. We
included only CCLs analyzed in both data set (343
CCLs). We mapped the binarized expression gene
expression values onto the network and we applied
the network-propagation algorithm as described
previously. We finally used these data to test the
BART classifier trained with the results from the CCLE
panel.
Supplementary data to this article can be found
online at https://doi.org/10.1016/j.jmb.2018.03.021.
Acknowledgments
T.J.-B. is a recipient of and FPI-SO fellowship. P.A.
acknowledges the support of the Spanish Ministerio
de Economía y Competitividad (BIO2013-48222-R;
BIO2016-77038-R) and the European Research
Council (SysPharmAD: 614944).
Received 31 January 2018;
Received in revised form 19 March 2018;
Accepted 22 March 2018
Available online xxxx
Keywords:
cancer cell lines;
drug response;
molecular signatures;
antineoplastic drugs;
network-based stratification
Abbreviations used:
CCLs, cancer cell lines; NBS, network-based stratifica-
tion; PPI, protein–protein interaction; CCLE, Cancer Cell
Please cite this article as: T. Juan-Blanco, et al., Rationalizing Drug Response in Cancer Cell Lines, J. Mol. Biol. (2018), https://doi.
org/10.1016/j.jmb.2018.03.021
11
Line Encyclopedia; SAM, Significant Analysis Of
Microarray; BART, Bayesian Regression Trees; AUROC,
area under the ROC curve.
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