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construction of a computable network Model for DnA
Damage, Autophagy, cell Death, and senescence
Stephan gebel1, rosemarie B. Lichtner1, Brian Frushour3, Walter K. Schlage1, Vy hoang3,
Marja Talikka2, Arnd hengstermann1, carole Mathis2, emilija Veljkovic2, Michael Peck2,
Manuel c. Peitsch2, renee Deehan3, Julia hoeng2 and Jurjen W. Westra3
1
Philip Morris International r&D, Philip Morris research Laboratories gmbh, Koeln, germany. 2Philip Morris International
r&D, Philip Morris Products S.A., neuchâtel, Switzerland. 3Selventa, One Alewife center, cambridge, MA, USA.
corresponding author email: julia.hoeng@pmi.com
Abstract: Towards the development of a systems biology-based risk assessment approach for environmental toxicants, including
tobacco products in a systems toxicology setting such as the “21st Century Toxicology”, we are building a series of computable biological network models specific to non-diseased pulmonary and cardiovascular cells/tissues which capture the molecular events that can be
activated following exposure to environmental toxicants. Here we extend on previous work and report on the construction and evaluation of a mechanistic network model focused on DNA damage response and the four main cellular fates induced by stress: autophagy,
apoptosis, necroptosis, and senescence. In total, the network consists of 34 sub-models containing 1052 unique nodes and 1538 unique
edges which are supported by 1231 PubMed-referenced literature citations. Causal node-edge relationships are described using the
Biological Expression Language (BEL), which allows for the semantic representation of life science relationships in a computable
format. The Network is provided in .XGMML format and can be viewed using freely available network visualization software, such as
Cytoscape.
Keywords: computable, network model, DNA damage, autophagy, apoptosis, necroptosis, senescence, Biological Expression Language
(BEL)
Bioinformatics and Biology Insights 2013:7 97–117
doi: 10.4137/BBI.S11154
This article is available from http://www.la-press.com.
© the author(s), publisher and licensee Libertas Academica Ltd.
This is an open access article. Unrestricted non-commercial use is permitted provided the original work is properly cited.
Bioinformatics and Biology Insights 2013:7
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gebel et al
Introduction
System-wide ‘omics’ data containing measurements of thousands of molecular species in a single
experiment are increasingly being used to unravel
the complex biological mechanisms contributing to
pulmonary and cardiovascular diseases. Detailed
mechanistic network models are needed to place the
differential measurements obtained from molecular
profiling data into the context of known biology. These
mechanistic models can then be used to better understand the impact of biologically active substances/
toxicants and associated disease risks as outlined in
systems toxicology settings such as the “21st Century
Toxicology”.1,2
Previously, we have reported on the construction of network models describing cell proliferation
and cellular stress.3,4 Extending on elements of these
networks (eg, the Cellular Stress Network), which
described the network perturbations occurring during
cellular defense in response to acute exogenous or
endogenous insults, we report here on the construction
and evaluation of a third network model, describing
the mechanisms that can be activated if these cellular
defenses are overwhelmed.
The proper maintenance of homeostatic balance
is essential for cell survival in a constantly changing environment. Human pulmonary tissue forms an
interface between the external and internal microenvironments, and is therefore constantly exposed
to both exogenous stressors including combustion
products (diesel exhaust, carbon monoxide, cigarette smoke (CS)), particulate matter, ozone,5–7 and
endogenous stressors (eg, mitochondrial-derived
reactive oxygen species (ROS), unfolded proteins,
nutrient deprivation), all of which can alter cellular
homeostasis. Pulmonary cells are equipped with a
variety of defense mechanisms to aid in the preservation of cellular homeostasis in the face of such harsh
conditions8–10 as outlined in one of our previous network model describing the main CS-related cellular
stress defense mechanisms in detail.3 However, these
mechanisms can be overwhelmed by chronic stress,
for example, ultimately culminating in the intracellular accrual of free radicals, oxidative damage to
biomolecules including DNA, and the induction of
the DNA damage response as a further protective
mechanism. If all these responses to restore cellular
homeostasis fail, compromised cells may commit to a
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terminal fate for the collective benefit of the surrounding tissue, adopting one of four main fates: apoptosis,
necroptosis, autophagy, or senescence11 to prevent the
nucleation of a potentially deleterious proinflammatory microenvironment.
The DNA damage response activates DNA repair
enzymes and in cycling cells, halts cell division by activating G1/S or G2/M cell cycle checkpoints, allowing
time for DNA repair.12 Apoptosis is initiated through
two main pathways following appropriate extracellular or intracellular signals.13 It fragments a dying cell
into apoptotic bodies, which are subsequently cleared
from tissue by the phagocytic activity of neighboring or immune cells, minimizing local inflammation.
Alternatively, cell clearance can occur through necrosis, a form of death that results in cell lysis and release
of proinflammatory intracellular components into the
surrounding milieu. Accumulating evidence indicates
that at least some forms of necrotic cell death occur in
a regulated manner, termed “necroptosis”.14,15 In contrast to apoptosis and necroptosis, which result in the
removal of damaged cells, the induction of autophagy
or senescence leaves cells surviving, but qualitatively changes their phenotype or function.16,17 During
autophagy, lysosomal enzymes degrade and recycle
damaged intracellular organelles and proteins in an
effort to maintain nutrient and energy homeostasis.18,19
Cellular senescence is characterized by irreversible
growth arrest,20,21 as response to a variety of external
stimuli including DNA damage, oncogene amplification, and telomere dysfunction.
The DNA damage response, apoptosis, necroptosis, autophagy, and senescence are especially
important in the context of CS, as smoke exposure
in human pulmonary experimental systems has been
shown to induce each, depending on the exposure
or experimental context.22–29 Although these cellular fates generally serve in a protective capacity,
emerging research also points to a prominent role for
stress-induced cell fate choices in the pathogenesis of
CS-related diseases, including lung cancer, chronic
obstructive pulmonary disease (COPD), and cardiovascular disease.30–33 Understanding how this protective to pathogenic transition occurs requires both a
thorough mechanistic understanding of the pathways
involved and the appropriate input data.
Here we describe the construction and application of a literature-based network model depicting
Bioinformatics and Biology Insights 2013:7
DnA Damage, Autophagy, cell Death, and Senescence Model
the DNA damage response, apoptosis, necroptosis,
autophagy, and senescence, hereafter referred to by
the acronym DACS (DNA damage, Autophagy, Cell
death (apoptosis and necroptosis), and Senescence).
The DACS Network is modular and computable
with its edges supported by hundreds of scientific
references. We applied the Network to an independent molecular profiling data set, verifying the content and computability of the network in the process.
Together with our previously published network
models, the Network will be an invaluable research
tool to investigate the biological effects of environmental exposures including CS on human systems,
both qualitatively and quantitatively, towards systems
toxicology approaches.
Methods
Biological expression Language (BeL)
The causal relationships in the model are expressed in
the Biological Expression Language (BEL)44, which
allows for the representation of biological processes
in a computable format. BEL is designed to represent
scientific findings by capturing causal and correlative
relationships in context, where context can include
information about the biological and experimental
system in which the relationships were observed, the
supporting publications cited and the curation process used.
Knowledgebase
The nodes and edges comprising the DACS Network
were assembled from the Selventa Knowledgebase, a
comprehensive repository containing over 1.5 million
nodes (biological processes and entities) and over
7.5 million edges (assertions about causal and noncausal relationships between nodes). The assertions
in the Selventa Knowledgebase are derived from
peer-reviewed scientific literature as well as other
public and proprietary databases. Specifically, each
assertion describes an individual experimental observation from an experiment performed in a human,
mouse or rat species context, either in vitro or in vivo.
Assertions in one species (eg, human) are homologized to another species (eg, mouse) in cases where
each element of an assertion has an orthologous counterpart in both species. Assertions also capture information about the referring source (eg, the PubMed
ID (PMID) for journal articles listed in MEDLINE),
Bioinformatics and Biology Insights 2013:7
as well as key contextual information including the
species (human, mouse, or rat) and the tissue or cell
line from which the experimental observation was
derived. An example causal assertion is the increased
transcriptional activity of TP53 (tumor protein p53)
causes an increase in the mRNA expression of
CDKN1A (cyclin-dependent kinase inhibitor 1A)
[fibroblast; Human; PMID 15616590]. The Knowledgebase contains causal relationships derived from
healthy tissues and disease areas such as inflammation, metabolic diseases, cardiovascular injury, liver
injury and cancer.
Analysis of transcriptomic data sets
Four previously published data sets, GSE6206,34
E-MEXP-1968,35 GSE13330,36 and GSE19018 were
used to construct the DACS Network. A fifth data set,
GSE28464,37 was used to evaluate the DACS Network,
with a specific focus on the relevant senescence submodels (Supplementary Table 1). All data sets were
downloaded either from Gene Expression Omnibus
(GEO) (http://www.ncbi.nlm.nih.gov/gds) or from
ArrayExpress (http://www.ebi.ac.uk/arrayexpress).
RNA expression data were analyzed using the “affy”,
“lumiHumanIDMapping”, and “limma” packages of
the Bioconductor suite of microarray analysis tools
available for the R statistical environment.38–42 Robust
Microarray Analysis (RMA) background correction and quantile normalization were used to generate microarray expression values for the Affymetrix
platform (CEL files from GSE6206, E-MEXP-1968,
GSE13330, and GSE19018), while log2 transformation and quantile normalization were used to generate
expression values for the Illumina platform (nonnormalized data file from GSE28464). An overall linear model was fit to the data for all sample groups, and
specific contrasts of interest were evaluated to generate raw P-values for each probe set on the expression
array.43 The Benjamini-Hochberg False Discovery
Rate (FDR) method was then used to correct for multiple testing effects.
For Affymetrix data sets, probe sets were considered to have statistically significant changed expression levels in a specific comparison if they had an
adjusted P-value of less than 0.05, an absolute
fold change greater than 1.3, and average expression intensity greater than 150 in either treatment
group. NetAffx version na32 feature annotation files,
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gebel et al
available from Affymetrix (http://www.Affymetrix.
com), were used to map probe sets to genes. For
the Illumina platform, the criteria used for statistical significance in changed gene expression were if
they had an adjusted P-value of less than 0.05 and an
absolute fold change greater than 1.3. In our analysis,
genes represented by multiple probe sets were considered to have changed if at least one probe set was
observed to change. Gene expression changes that
met these criteria are called ‘State Changes’ and have
the directional qualities of ‘increased’ or ‘decreased’
ie, they were upregulated or downregulated, respectively, in response to the experimental condition. The
number of State Changes for each data set is listed in
Supplementary Table 1.
reverse causal reasoning (rcr):
automated hypothesis generation
Reverse causal reasoning (RCR) analysis of the five
DNA damage and senescence transcriptomic data sets
was used to generate lists of nodes that were predicted
to be increased or decreased, and these lists of nodes
were used to aid in the selection of nodes for inclusion
in the DACS Network, as well as to evaluate the DACS
Network using the data set. RCR interrogates the Selventa Knowledgebase to identify potential upstream
controllers of entities observed to change significantly
in an experiment (see Selventa 201044 and Additional
File 1 for specific detail on RCR). Here we applied
RCR to the mRNA State Changes in the five transcriptomic data sets to predict potential upstream controllers
for the expression changes. These potential upstream
controllers identified by RCR are called HYPs as they
represent statistically significant hypotheses that are
potential explanations for the observed downstream
mRNA State Changes. Specifically, the upstream
HYP is a potential explanation for the subset of State
Changes that are causally downstream of the HYP in
individual assertions in the Selventa Knowledgebase.
Each HYP is scored according to two probabilistic
scoring metrics: richness and concordance. Richness
is the probability that the number of observed mRNA
State Changes connected to a given HYP could have
occurred by chance alone, calculated using the hypergeometric distribution. Concordance is the probability that the number of observed RNA State Changes
that match the direction of the HYP (eg, increased or
decreased activity or abundance of a node) could have
100
occurred by chance alone, calculated using a binomial
distribution. HYPs meeting both richness and concordance P-value cutoffs of 0.1 were considered to be
statistically significant. When performing control analyses, applying these significance cutoffs to randomly
generated data (with similar numbers of RNA State
Changes as the experimental data) generally produces
less than 5% of the number of HYPs meeting both significance criteria than are observed for experimental
data (not shown). For the purposes of network model
construction, top scoring HYPs meeting the minimum statistical cutoffs for richness and concordance
were evaluated and selected for integration based on
their biological plausibility and relevance to the perturbation and biological context (eg, cell type) of the
experiment. For data set interrogation, scored HYPs
meeting these same statistical cutoffs were considered,
with the understanding that as potential explanations
for a subset of State Changes, the connectivity and
consistency of direction of individual HYPs needed to
be considered within context of the models (Selventa
201044 and Additional File 1).
Results
network structure and content
We constructed a network model focused on DNA
damage response and the four main cellular fates
induced by overwhelming stress: autophagy,
apoptosis, necroptosis, and senescence (Fig. 1,
Supplementary Fig. 1). The complete DACS Network is provided in Additional File 2 as an excel file
and in Additional Files 3–7 in .XGMML format. The
.XGMML format can be viewed using freely available
network visualization software, such as Cytoscape.45
The DACS Network was constructed using a highly
modular design, where the larger network is divided
into sub-models. Discrete mechanisms affecting cell
fate (eg, ‘NFKB signaling’ describing the prosurvival
effects of NFKB-mediated transcriptional upregulation of anti-apoptotic genes) in the five DACS Network areas are described by 34 sub-models (Fig. 1).
In total, the DACS Network contains 1052 unique
nodes and 1538 unique edges (959 causal edges and
579 non-causal edges), which are supported by 1231
PubMed-referenced literature citations (Table 1,
Additional Files 2–7). Nodes in the DACS Network
are biological entities such as protein abundances,
mRNA expression levels, and protein activities.
Bioinformatics and Biology Insights 2013:7
DnA Damage, Autophagy, cell Death, and Senescence Model
Caspase cascade
DNA damage response
Autophagy
Apoptosis
ER stress-induced apoptosis
MAPK signaling
NFkB signaling
PKC signaling
Proapoptotic mitochondrial signaling
Prosurvival mitochondrial signaling
TNFR/Fas signaling
TP53 transcriptional signature (TS)
ATG induction of autophagy
mTOR signaling
Nutrient transporter synthesis
Protein synthesis
Components affecting TP53 activity
Components affecting TP63 activity
Components affecting TP73 activity
DNA damage to G1/S checkpoint
DNA damage to G2/M checkpoint
Double-strand break response
Inhibition of DNA repair
NER/XP pathway
Single-strand break response
Senescence
Necroptosis
TP53 TS
Fas activation
Gene signature
Proinflammatory mediators
RIPK/ROS mediated execution
TNFR1 activation
Oncogene induced senescence
Replicative senescence
Stress induced premature senescence
Regulation of CDKN2A expression
Regulation of tumor suppressors
Transcriptional regulation of SASP
Figure 1. Overview of the DAcS Subnetworks.
notes: The DACS Network is comprised of 34 submodels that represent relevant signaling within five areas of biology – apoptosis, autophagy, DNA
damage response, necroptosis, and senescence. each of the 34 submodels describes the molecular signaling mechanisms shown to activate or inhibit
the end process (eg, in the submodel ‘replicative senescence,’ increased cDKn2A and cDKn1A protein abundances lead to the induction of replicative
senescence, while increased abundance of Wrn protein inhibits replicative senescence). The left panel lists the names of the submodels involved in each
area (eg, ‘replicative senescence’ under Senescence), and the right panel shows an agglomerated diagram of all submodels involved in each area, with
different submodels highlighted in unique colors.
In addition, nodes can also represent biological processes (eg, protein biosynthesis). Edges are relationships between the nodes, and are categorized as
either causal or non-causal. Causal edges are directional cause-effect relationships between nodes
Bioinformatics and Biology Insights 2013:7
(eg, NFKB directly increases the gene expression of
BCL2), whereas non-causal edges connect different
forms of a biological entity, such as gene expression
to the related protein abundance. Node-edge relationships in the DACS Network are described using the
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Table 1. DAcS network statistics.
nodes
mrnAs
Proteins
Phosphoproteins
Activities
complexes
Protein families
Biological processes/gO terms
chemicals/small molecules
Other
Total edges
causal
non-casual
Unique PMIDs
submodel name
DnA Damage
Autophagy
Apoptosis
necroptosis
Senescence
1052
138
392
105
224
22
25
48
22
76
1538
959
579
1231
Total nodes (predictable)
272 (72)
161 (49)
280 (112)
94 (30)
365 (186)
notes: Summary of relevant statistics describing the contents of the
DAcS network. For each DAcS network area, the total number of
unique nodes in the agglomerated model is given, along with the number
of those nodes that are capable of prediction by rcr (in parentheses).
Model
boundaries
Scientific
literature
A
catof(B)
Literature model
taof(C)
exp(D)
process
Building
data sets
A
catof(B)
catof(G)
taof(C)
kaof(E)
exp(D)
F
Integrated model
process
Model reviewed
and refined
A
catof(B)
Final network model
catof(G)
kaof(E)
taof(C)
F P@S136
exp(D)
F
process
BEL which allows for the semantic representation
of life science relationships in a computable format
(Selventa 201044 and Additional File 1). Overall, the
DACS Network provides a comprehensive, detailed
representation of the causal pathways involved in
the DNA damage response, apoptosis, necroptosis,
autophagy, and senescence.
Test data
set
A
catof(B)
Evaluation
catof(G)
kaof(E)
taof(C)
F P@S136
exp(D)
F
process
network construction
The DACS Network was constructed using the same
iterative process used to create previously published
network models.3,4 Using this strategy, the network
is populated with nodes and edges from two main
sources: prior knowledge described in the scientific
literature, and results obtained from the computational
analysis of transcriptomic profiling data via RCR
(Selventa 201044 and Additional File 1) (Fig. 2).
In order to build a network model that describes the
DACS-related biological mechanisms in non-diseased
pulmonary and cardiovascular cells/tissues, we first
defined and applied a set of criteria for selecting network content similar to those used in previously published network models.3,4 Starting with a list of nodes
identified by a survey of published literature in the
five DACS Network areas, we searched for causal
102
Figure 2. Workflow used to construct and evaluate the DACS Network.
notes: The DAcS network is a literature based model containing
content derived from two main sources. The literature model was
constructed from causal relationships extracted from relevant scientific
literature following the definition of network boundaries. The literature
model was then augmented with additional nodes derived from reverse
causal reasoning (rcr) analysis of transcriptomic data sets, forming
the integrated model. In this step, rcr analysis was also used to verify
the placement of existing nodes in the literature model. Manual review
and refinement of the integrated model resulted in the final network
model. The final network model was evaluated using RCR analysis of an
independent test transcriptomic data set.
relationships describing the mechanistic relationships
between these nodes with literature support from normal lung and cardiovascular cell types. In cases where
the relevant experiments have not been published in
these contexts, relationships derived from non-lung
contexts using cell types found in normal lung (fibroblasts, epithelial cells, endothelial cells, etc.) were
used. Canonical mechanisms that are well-known in
Bioinformatics and Biology Insights 2013:7
DnA Damage, Autophagy, cell Death, and Senescence Model
the literature were also included in the network model
even if literature support explicitly demonstrating the
presence of the mechanism in normal lung or cardiovascular tissues was not found (eg, the catalytic activity
of the FAS receptor increasing the catalytic activity of
FADD in the activation of TNFR signaling). For direct
and proximal connections such as a kinase phosphorylating a residue on a target or protein-protein interactions, evidence from cell free in vitro systems, which
lack a single specified tissue context, were also used
when normal lung or cardiovascular tissues were not
available. Lastly, relationships derived from human
and rodent (specifically mouse and rat) systems were
included and homologized, with human contexts prioritized (see Methods).
Using these network boundaries, a literature
model was created by compiling causal relationships
extracted from the Selventa Knowledgebase, a unified collection of over 1.5 million elements of biological knowledge captured from public literature and
other resources (see Methods). When critical causal
connections did not exist in the Knowledgebase, they
were identified and manually curated from literature
into the Knowledgebase. During the course of model
building, over 7,500 new causal relationships related
to DNA damage, cell death, and senescence from
685 unique literature references were added to the
Knowledgebase to support the biology reflected in
the DACS Network. Following this effort, the literature model encompassed experimentally proven and
well-established mechanistic signaling within the five
DACS areas.
Next, the literature model was augmented with
additional nodes derived from the computational
analysis of molecular profiling data using RCR.
RCR-derived HYPs were included as new nodes in
the DACS Network model if they had literature support for a mechanistic role in the process of interest.
RCR analysis was done to confirm the relevance
of nodes already present in the literature model,
and to uncover relevant nodes that were not identified during the construction of the literature model.
RCR-based augmentation of the DACS Network was
performed using four transcriptomic data sets (two for
DNA damage and two for senescence), referred to as
‘building’ data sets (Supplementary Table 1). Ideally,
transcriptomic data sets addressing all five DACS
areas would be used in order to maximize network
Bioinformatics and Biology Insights 2013:7
coverage. However, because three of the DACS
Network areas (apoptosis, autophagy and necroptosis) have not been classically described as driven
by or executed through transcriptomic changes,
we focused our efforts on transcriptomic data from
experiments describing DNA damage response and
the induction of senescence. Candidate data sets
for RCR analysis were selected from public gene
expression data repositories GEO and ArrayExpress.
We prioritized data sets according to three main criteria: (1) whether the biological process relevant to
the DACS Network was induced in non-diseased cell
types found in normal lung, (2) whether phenotypic
endpoint data was available to provide additional
verification of the experimental setup/transcriptomic
data, and (3) the statistical rigor of the design of transcriptomic profiling experiments. The four building
data sets (Supplementary Table 1) were all derived
from in vitro experiments done in human or mouse
fibroblasts, and represent the response to DNA
damage, induction of replicative senescence (RS)
and stress-induced premature senescence (SIPS).
Applying RCR to the four network building data sets,
575 HYPs were evaluated for biological plausibility.
From this initial list of 575 HYPs, 63 were considered biologically plausible in the context of previous
literature reports, and were placed into the appropriate sub-model(s) based on their mechanistic connections to the DACS areas (Supplementary Table 2).
The literature model augmented with the data-driven
nodes formed the integrated model. As a final step
in the construction of the DACS Network, the nodes
and edges were manually reviewed and refined (eg,
by additional specific literature curation), producing
the final DACS Network model (Fig. 2).
Application of the DAcS network
to an independent data set
Following network finalization, the DACS Network
was applied to investigate a transcriptomic test data
set, not included in the construction process, from
a well-accepted model of senescence induction ie,
oncogene-induced senescence through tamoxifeninducible HRAS G12V expression in lung fibroblasts
(GSE28464)37 (Supplementary Table 1).46–48 This data
set also met the boundary criteria for data set selection described above. Although the test data set did
not reflect biological activity occurring in all areas
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of the DACS Network, it enabled a detailed proofof-principle evaluation of a specific portion of the
network (ie, relevant senescence sub-models) as a
means to ensure that the nodes and edges placed into
the network through manual curation provided an
accurate reflection of currently known biology. The
four senescence sub-models representing the biology
most closely related to the experimental perturbation (constitutively active HRAS by G12V mutation)
were selected for investigation using this data set:
oncogene-induced senescence (OIS), regulation of
CDKN2A expression, regulation by tumor suppressors, and transcriptional regulation of the senescenceassociated secretory phenotype (SASP). The OIS
sub-model directly reflects the mechanism expected
to be seen given the GSE28464 experimental perturbation. The other three sub-models describe mechanisms that are generally applicable to all modes of
cellular senescence.
In total, the four senescence sub-models used for
evaluation contain 259 unique nodes, 126 (49%) of
which were eligible for prediction (meaning that they
contain four or more downstream gene expression
relationships and thus are capable of prediction as a
hypothesis) by RCR. Eighty three of the 126 RCRcapable nodes (66%) are predicted as HYPs in the
test set, 79 of which (95%) are predicted in directions
consistent with increased oncogene-induced senescence that was experimentally observed.
In particular, the oncogene-induced senescence
sub-model describes the upstream signaling pathways associated with the induction of OIS as well
as the unique SASP proteins produced by cells following OIS.49 When GSE28464 was used to interrogate this sub-model, 30 of the 43 RCR-capable nodes
(70%) comprising this sub-model were predicted as
HYPs, with 28 of the 30 (93%) predicted in directions consistent with increased OIS (Table 2). These
directionally consistent HYP predictions include
increased HRAS mutated at G12V, oncogene-induced senescence, and cell aging, all of which match
the experimental perturbation from the test data set.37
Increased p38 MAPK activity, FOXO1 activity,
RAF1 activity, and HBP1 abundance are all involved
in known pathways leading to OIS (Fig. 3).8,46,50,51
Several SASP proteins were also predicted increased
in abundance, including OSM, MIF, VEGFA, IL1A,
LIF, PPBP and IFNG, consistent with what has been
104
Table 2. nodes from the oncogene-induced senescence
submodel of the DAcS network that are predicted as
hYPs by rcr on the gSe28464 test data set.
Test Data set
Oncogene Induced Senescence HYPs Expected
Direction
GSE28464
Predicted in consistent directions
BNIP3L
cell aging
ETS2
FOXO1
gtpof(Ras family Hs)
HBP1
HRAS
HRAS mutated at G12V
IFNG
IL1A
kaof(MAP2K1)
kaof(MAP2K6)
kaof(MEK Family Hs)
kaof(p38 MAPK family Hs)
kaof(RAF1)
LIF
MAP2K1
MAP2K6
MIF
Oncogene induced senescence
OSM
PPBP
RAF1
RAS Family Hs
SMARCB1
taof(ETS2)
taof(FOXO1)
VEGFA
Predicted in inconsistent directions
KRAS
KRAS mutated at G12V
notes: expected direction is based on internal causality of the oncogeneinduced senescence submodel. Yellow = predicted increase in abundance
or activity; blue = predicted decrease in abundance or activity. Submodel
nodes that are shared with other senescence models are bolded.
Abbreviations: gtpof(X), gTP-bound activity of X; kaof(X), kinase activity
of X; taof(X), transcriptional activity of X.
observed following OIS.49 The two directionally
inconsistent predictions are for KRAS abundance
and KRAS mutated at G12V (which can lead to OIS,
but are predicted to be decreased). These inconsistencies were further clarified by reviewing the underlying State Change support for the KRAS HYPs. First,
we performed a Gene Ontology (GO) biological
process enrichment query on the State Changes supporting both the HRAS mutated at G12V and KRAS
mutated at G12V HYPs using the Database for
Annotation, Visualization and Integrated Discovery
(DAVID). While the State Changes supporting the
HRAS mutated at G12V HYP converged on GO biological processes indicative of cell cycle modulation
known to be affected during cellular senescence, the
State Changes supporting the KRAS at G12V HYP
did not converge on any specific biological process
Bioinformatics and Biology Insights 2013:7
DnA Damage, Autophagy, cell Death, and Senescence Model
Figure 3. graph showing the oncogene-induced senescence submodel as depicted using the BeL framework and colored according to the gSe28464
test data set.
notes: Yellow = predicted increase in abundance or activity; blue = predicted decrease in abundance or activity.
Abbreviations: catof(X), catalytic activity of X; exp(X), mrnA expression of X; gtpof(X), gTP-bound activity of X; kaof(X), kinase activity of X; paof(X),
phosphatase activity of X; sec(X), cell secretion of X; taof(X), transcriptional activity of X.
(data not shown). In addition, underlying evidence
for the KRAS HYP comes, at least in part, from transformed cells that have already bypassed senescence
during the transformation process, thus excluding the
KRAS HYP from further consideration in the OIS
sub-model.
The regulation of CDKN2A expression submodel includes direct transcriptional regulators
of CDKN2A, a cyclin-dependent kinase inhibitor
whose increased expression at the gene and protein
levels are hallmarks of cellular senescence.52 When
interrogated using the test data set, 17 of the 33
RCR-capable nodes (52%) in this sub-model were
predicted as HYPs, all in directions consistent with
Bioinformatics and Biology Insights 2013:7
increased CDKN2A expression (Supplementary
Table 3). Notably, the prediction for increased
CDKN2A protein abundance was also supported
by the observed increase in CDKN2A mRNA levels in the test data set. Both positive (SMARCB1,
HBP1, ETS1, ETS2, SP1, and PPARG) and negative
(HDAC3 and GLI2) regulators of CDKN2A expression were predicted in directions consistent with
their previously reported roles.51,53–57 Finally, members of both the Polycomb Repressive Complexes 1
and 2 (PRC1/2) were predicted decreased (YY1 and
BMI1 for PRC1, EED and EZH2 for PRC2), consistent with their known role as negative regulators of
CDKN2A expression (Fig. 4).58,59
105
gebel et al
Figure 4. graph showing the regulation of cDKn2A expression submodel as depicted using the BeL Framework and colored according to the gSe28464
test data set.
notes: Yellow = predicted increase in abundance or activity; blue = predicted decrease in abundance or activity; red = observed increase in mrnA
expression.
Abbreviations: catof(X), catalytic activity of X; exp(X), mrnA expression of X; taof(X), transcriptional activity of X.
Next, we interrogated the regulation by tumor suppressors sub-model, which describes the cell cycle
exit characteristic of senescence regulated by the
E2F/Rb axis and CDK inhibitors.60 Thirty-eight of the
49 RCR-capable nodes (78%) in this sub-model were
predicted as HYPs in the test data set, all but two
(95%) in directions consistent with cell cycle exit and
increased senescence (Supplementary Table 4). The
consistent HYPs include predictions for decreased
abundance/activity of cell cycle activators (E2F family members and CCND1) and conversely, increased
abundance/activity of cell cycle inhibitors (RB1,
CDKN1A, and CDKN2A). The content of the regulation by tumor suppressors sub-model also included
SASP proteins that are shared between multiple
modes of senescence, and several of these are predicted increased at the HYP level as well, including
106
CCL2, CSF2, CXCL1, HGF, IL1B, IL6ST, IL8, IL13,
and TNFRSF1A (Fig. 5).49 The inconsistent HYPs are
IL6, which is predicted to be decreased, and PTEN,
which is predicted to be increased (Supplementary
Table 4). Upon further exploration of the directionality of the IL6 HYP, we noted that a large fraction
of the supporting State Changes (74 out of 173)
were IL6 targets related to cell proliferation in multiple myeloma, which falls outside of the network
boundaries.61,62 Because many of these proliferative
genes were observed to be downregulated (presumably as a consequence of senescent cells exiting the
cell cycle), this set of genes could account for the
RCR prediction of decreased IL6. When the directionality of the IL6 HYP was re-evaluated excluding
this set of genes, it was predicted increased in abundance (Supplementary Fig. 2), consistent with its role
Bioinformatics and Biology Insights 2013:7
DnA Damage, Autophagy, cell Death, and Senescence Model
Figure 5. graph showing the regulation by tumor suppressors submodel as depicted using the BeL framework and colored according to the gSe28464
test data set.
notes: Yellow = predicted increase in abundance or activity; blue = predicted decrease in abundance or activity; red = observed increase in mrnA
expression. IL6 is shown as predicted increased in this figure, in contrast to the initial prediction by rcr (See Section 3.3 Application of the DAcS network
to an Independent Data Set for additional detail and Supplementary Fig. 2).
Abbreviations: exp(X), mrnA expression of X; kaof(X), kinase activity of X; sec(X), cell secretion of X; taof(X), transcriptional activity of X.
as a proinflammatory mediator. Due to those findings,
the related literature evidence (Supplementary Fig. 2)
will be excluded from future RCR analysis in this
model. PTEN is a multifunctional protein and the prediction for increased abundance may be reflective of
its role in areas outside of senescence.
To further evaluate the mechanisms responsible
for the expression of the SASP proteins, we interrogated the transcriptional regulation of the senescenceassociated secretory phenotype sub-model, which
centers on the transcriptional activities of NFKB and
CEBPB upstream of the mRNA expression of SASP
proteins. Of the 23 RCR-capable nodes in this submodel, 16 (70%) are predicted as HYPs in GSE28464,
and 15 (94%) are predicted in directions consistent
with an increased SASP (Supplementary Table 5). In
addition, the transcriptional activity of both NFKB
and CEBPB are predicted to be increased, consistent
with their roles as central transcriptional mediators of
the SASP.63,64 SASP proteins such as CCL2, CCL5,
CXCL1, IFNG, IL1A, IL13, IL8, IL6, and VEGFA
are all predicted increased. Complementing the RCR
predictions, six of these proteins (CCL2, CXCL1,
IL1A, IL8, IL6 and VEGFA) are also observed
increased at the mRNA level in GSE28464 (Supplementary Fig. 3).
In summary, the evaluation of the transcriptomic
profiling data set from human lung cells expressing oncogenic HRAS (GSE28464) using four relevant sub-networks from the DACS Network reveals
Bioinformatics and Biology Insights 2013:7
molecular processes known to be involved in the
major hallmarks of oncogene-induced senescence,
eg, decreased abundance/activity of cell cycle activators (E2F family members and CCND1), increased
abundance/activity of cell cycle inhibitors (RB1,
CDKN1A, and CDKN2A), and induction of SASP
proteins via activation of the transcription factors
NFKB and CEBPB.
Discussion
comparison with other DAcS-related
computational networks
Several different modeling approaches have been
used to build models of biological systems depending
on the biological complexity being captured, the specific goals of the study, and the experimental details
involved. The DACS Network was constructed using
a prior knowledge of causal relationships from literature, and augmented with nodes derived from RCR, a
data-driven method that infers pathway activity based
on differentially expressed entities and knowledge of
their upstream regulators. Here, we compare and contrast three previously published networks that share
features with the DACS Network.65–67
Behrends et al performed a systematic proteomic
analysis and utilized existing protein interaction databases to construct an autophagy interaction network
(AIN).65 Like the DACS Network, the AIN consists
of functional sub-networks, representing unique
biological areas of autophagy. In contrast to the
107
gebel et al
protein-protein interactions of the Behrends AIN, the
DACS Network shows directionality through mechanistic causal relationships between proteins and other
entities including genes, protein activities, biological
processes, complexes, etc. Additionally, the DACS
Network incorporates transcriptomic data through the
integration of computationally derived nodes to infer
pathway activity.
Caron et al manually constructed a comprehensive,
detailed network of mTOR signaling based on 522
published articles and a protein interaction network
(PIN) using 85 key mTOR proteins and protein-protein
interactions from multiple databases.66 Comparable
to the DACS Network, the mTOR network represents
biochemical modifications, directionality, biological
entities, and annotations (cell lines, cited literature
references). While the integrated mTOR network
provides a highly granular view of mTOR signaling,
the DACS Network covers a wider range in addition
to basic mTOR signaling.
Finally, Passos et al utilized several ‘omics’
approaches to investigate cellular senescence.67 Using
target gene inhibition, in silico interactome analysis
based on the BioGrid database, and statistical inference, they identified a signaling pathway involving
TP53, CDKN1A, GADD45A, MAPK14, GRB2, SRC,
DAB2, TGFRB2, and TGFβ. Overlaying these results
with those from previous gene expression analysis, they
were able to confirm the upregulation of these pathway
genes in senescent MRC5 fibroblasts. Similarly, the
DACS Network uses transcriptomic data, but applies a
computational approach to infer the activity of upstream
controllers that fall in a pathway rather than overlaying the genes onto the network itself. Although the
Passos network depicts the interconnections between
senescence-related entities, it is undirected, as BioGrid
interactions lack inherent directionality.
Thus, although the DACS Network shares many
features with other previously published networks,
we believe the inherent computability conferred upon
it by the BEL Framework and the ability to evaluate biological mechanisms by RCR (as opposed to
direct mapping of differentially expressed genes onto
pathways) differentiates the DACS Network from
previously existing resources. In addition, the broad
scientific coverage of five distinct yet overlapping
biological areas makes the DACS Network a unique
resource for the scientific community.
108
The knowledgebase used to build the network
model contains information curated from published
literature. We concede that the peer review process is
far from perfect and any errors that exist in the public
literature could be translated to the knowledgebase.
However, the prior knowledge encoded in the knowledgebase has been subject to two additional layers of
peer review by PhD level curators. We believe that
any inaccuracies that exist in the knowledgebase
constitute a minor fraction and occur without a systematic bias that would profoundly affect the results
presented here.
While the results shown here indicate that network models have utility in evaluating ‘omics’ data,
there are some elements that could be improved in
the future. The methodology depends on up-to-date
prior knowledge of both the signaling pathways that
are represented by the network models and the genes
that are regulated by network components. As new
discoveries in these areas are made and published, a
process for maintaining the connectivity of the network models will need to be put in place to ensure
the networks constantly reflect the current state of the
field; being dynamic and updatable, any new knowledge can be added to the existing DACS network.
Future application in systems biologybased risk assessment
Understanding how exposure to chemical products
affects biological systems is a key first step in the
development of effective risk assessment programs.
Historically, chemical mechanism-of-action (MOA)
studies used simple in vitro or in vivo models and
measured a relatively limited number of biological entities. Modern toxicological assessment using
system-wide ‘omics’ approaches can now generate
thousands of biological data points for a single experiment, and the field of systems toxicology has evolved
in order to distil discrete MOA information from this
sea of data.68,69 Detailed mechanistic network models are needed to place the differential measurements
obtained from molecular profiling data into the context of known biology. These mechanistic models
can then be used to better understand the impact of
biologically active substances/toxicants and associated disease risks. We are currently developing an
application of these network models to derive quantitative measures of network perturbations to compare
Bioinformatics and Biology Insights 2013:7
DnA Damage, Autophagy, cell Death, and Senescence Model
the impact of biologically active substances, including CS, on human systems in order to assess relative
disease risk.
The biological mechanisms represented in the
DACS Network, combined with its inherent computability, make it an ideal resource in systems toxicology approaches. For example, the DACS Network
could be used in combination with molecular profiling data from human in vitro toxicological studies to
characterize the degree to which a simple chemical
entity induces a DNA damage response or initiates
cell death pathways. In addition, the DACS Network
could be used with molecular profiling data from
rodents exposed to environmental toxicants in vivo
in order to identify the mechanisms whose activation or suppression precedes the development of
known genotoxic markers. In each case, the information obtained by combining systems-level data
with network-level analyses would provide invaluable mechanistic insight into the biological effects of
potentially harmful exposures, and would serve to aid
in the development of risk assessment pipelines.
conclusions
We have presented here a network model that broadly
covers the biology within five distinct yet overlapping
cellular processes: DNA damage and the main cell
fates resulting from cellular stress. The computability
enabled by BEL and the broad coverage of toxicologically relevant biology make the DACS Network an
exceptional, open-source tool for evaluating modern
‘omics’ data.
Acknowledgements
We would like to acknowledge Michael J. Maria for
project management and support with preparation
of this manuscript, Stephanie Boue and Vincenzo
Belcastro for reviewing the manuscript, and Natalie
Catlett for methodological assistance.
Author contributions
Conceived and designed the experiments: SG, RBL,
WKS, MT, AH, CM, EV, MP, MCP, JH. Analysed the
data: SG, RBL, WKS, VH, MT, AH, CM, EV, MP,
JWW. Wrote the first draft of the manuscript: JWW.
Contributed to the writing of the manuscript: SG,
BF, WKS, VH, JWW. Agree with manuscript results
and conclusions: All authors. Jointly developed the
Bioinformatics and Biology Insights 2013:7
structure and arguments for the paper: SG, RD, JWW.
Made critical revisions and approved final version:
All authors. All authors reviewed and approved of the
final manuscript.
Funding
Selventa and PMI authors performed this work under
a joint research collaboration funded by PMI.
competing Interests
Authors disclose no potential conflicts of interest.
Disclosures and ethics
As a requirement of publication author(s) have provided to the publisher signed confirmation of compliance with legal and ethical obligations including but
not limited to the following: authorship and contributorship, conflicts of interest, privacy and confidentiality and (where applicable) protection of human and
animal research subjects. The authors have read and
confirmed their agreement with the ICMJE authorship and conflict of interest criteria. The authors have
also confirmed that this article is unique and not under
consideration or published in any other publication,
and that they have permission from rights holders
to reproduce any copyrighted material. Any disclosures are made in this section. The external blind peer
reviewers report no conflicts of interest.
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supplementary Materials
Table s1. Data sets analyzed by rcr for model augmentation and evaluation.
Model building data sets
Model evaluation data set
process
DnA damage
senescence
Data set ID
PubMed ID
Species
context
cell type
Timepoint(s)
gSe6206
19584263
Mouse
In vitro
embryonic
fibroblasts
cisplatin
(16 μM)
24 hr
control
Untreated
gSe13330
19155301
human
In vitro
Foreskin BJ
fibroblasts
Bleomycin (100 μg/mL)
Long term culture
24 hr
Late passage
early passage (young)
# State
changes
3684
Perturbation
e-MeXP-1968
19363488
Mouse
In vitro
Dermal
fibroblasts
UV irradiation
(4 J/m2)
6 hr after UV
exposure
non-irradiated
(0 J/m2)
472
3355
2799
senescence
gSe19018
Unpublished
human
In vitro
IMr90 lung
fibroblasts
Long term culture
in 20% oxygen
48 population
doublings (old)
30 population
doublings (young)
2257
gSe28464
21512002
human
In vitro
IMR90 lung fibroblasts
Tamoxifen-inducible hrAS
g12V expression
Day 4 post hrAS g12V
induction
Day 0
3691
notes: Four data sets relating to two different DAcS network areas (DnA damage and Senescence) were analyzed by rcr for the data-driven phase of
model construction (Model Building Data Sets). One senescence data set was analyzed by rcr for model evaluation. This table provides a summary of
the experimental details and comparisons used for each data set as well as the number of gene expression State changes observed in each data set.
Senescence
Apoptosis
DNA damage response
Autophagy
Necroptosis
Figure s1. The DAcS network.
notes: Graphical representation of the five central biological areas covered by the DACS Network. An agglomerated view of all subnetwork nodes
and edges relating the mechanisms involved in the cellular DnA damage response (green) and the induction of senescence (teal), necroptosis (red),
autophagy (magenta), and apoptosis (purple), forming an interconnected network of shared and non-overlapping biology.
112
Bioinformatics and Biology Insights 2013:7
DnA Damage, Autophagy, cell Death, and Senescence Model
Table s2. Data-driven nodes that were predicted as hYPs by rcr on the gSe6206, e-MeXP-1968, gSe13330 rS,
gSe13330 SIPS, and gSe19018 building data sets.
Data-Driven Nodes Added to DACS Network
Expected
Direction
ATF2
BACH1
BIRC5
BNIP3L
catof(proteasome complex (sensu Eukarya) Hs)
catof(PTGS2)
CCL5
CCND1
CTNNB1
DDIT3
DNMT3A
ENO1
EP300
ETS2
FHIT
gtpof(RAC1)
gtpof(RHOA)
gtpof(RHOB)
HDAC1
HDAC3
IFNA1
IL1A
IRF1
IRF3
IRF5
kaof(MAP2K1)
kaof(PKC Family Hs)
kaof(RAF1)
MAP2K1
MYC
PKC Family Hs
PPARG
proteasome complex (sensu Eukarya) Hs
PTGS2
RAC1
RAF1
RASSF1
RHOA
RHOB
SMARCB1
SP1
taof(ATF2)
taof(BACH1)
taof(CTNNB1)
taof(EP300)
taof(ETS2)
taof(IRF1)
taof(IRF3)
taof(IRF5)
taof(PPARG)
taof(SP1)
taof(TFDP1)
taof(TP63)
taof(TP73)
taof(XBP1)
taof(YY1)
TFDP1
TP63
TP73
TWIST1
VHL
XBP1
YY1
DNA Damage Data Sets
Senescence Data Sets
GSE6206 E-MEXP-1968 GSE13330 RS GSE13330 SIPS GSE19018
Submode I
DNA Damage—Double—strand break response
Senescence—Stress—induced premature senescence
Apoptosis—NFKB signaling
Senescence—Oncogene—induced senescence
Senescence—Stress—induced premature senescence
Senescence—Stress—induced premature senescence
Senescence—Transcriptional regulation of the SASP
DNA Damage—Double—strand break response
Senescence—Regulation by tumor suppressors
Apoptosis—ER stress—induced apoptosis
Senescence—Replicative senescence
Senescence—Regulation by tumor suppressors
Apoptosis—MAPK signaling
Senescence—Oncogene—induced senescence
DNA Damagee—single—strand break response
Apoptosis—MAPK signaling
DNA Damagee—single—strand break response
DNA Damagee—single—strand break response
Senescence—Regulation by tumor suppressors
Senescence—Regulation of p16INK expression
Senescence—Regulation by tumor suppressors
Senescence—Transcriptional regulation of the SASP
Senescence—Regulation by tumor suppressors
Senescence—Regulation by tumor suppressors
Senescence—Replicative senescence
Senescence—Oncogene—induced senescence
Apoptosis—PKC signaling
Senescence—Oncogene—induced senescence
Senescence—Oncogene—induced senescence
DNA Damage—Double—strand break response
Apoptosis—PKC signaling
Senescence—Regulation of p16INK expression
Senescence—Stress—induced premature senescence
Senescence—Stress—induced premature senescence
Apoptosis—MAPK signaling
Senescence—Oncogene—induced senescence
DNA Damage—Double—strand break response
DNA Damage—Double—strand break response
DNA Damage—Double—strand break response
Senescence—Regulation of p16INK expression
Senescence—Regulation of p16INK expression
DNA Damage—Double—strand break response
Senescence—Stress—induced premature senescence
Senescence—Regulation by tumor suppressors
Apoptosis—MAPK signaling
Senescence—Oncogene—induced senescence
Senescence—Regulation by tumor suppressors
Senescence—Regulation by tumor suppressors
Senescence—Replicative senescence
Senescence—Regulation of p16INK expression
Senescence—Regulation of p16INK expression
Senescence—Regulation by tumor suppressors
DNA Damage—Componenets affecting TP63 activity
DNA Damage—Componenets affecting TP73 activity
Apoptosis—ER stress—induced apoptosis
DNA Damage—Componenets affecting TP53 activity
Senescence—Regulation by tumor suppressors
DNA Damage—Componenets affecting TP63 activity
DNA Damage—Componenets affecting TP73 activity
Senescence—Stress—induced premature senescence
Senescence—Stress—induced premature senescence
Apoptosis—ER stress—induced apoptosis
DNA Damage—Componenets affecting TP53 activity
notes: These data-driven nodes were added to the indicated submodels of the DAcS network based on their mechanistic connections to the processes
reflected by the submodels. Expected direction is based on internal causality of the indicated submodels. Yellow = predicted increase in abundance or
activity, blue = predicted decrease in abundance or activity.
Abbreviations: catof(X), catalytic activity of X; gtpof(X), gTP-bound activity of X; kaof(X), kinase activity of X; taof(X), transcriptional activity of X.
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Table s3. nodes from the regulation of cDKn2A expression submodel of the DAcS network that are predicted as
hYPs by rcr on the gSe28464 test data set.
Expected Test Data set
Direction
GSE28464
Predicted in consistent directions
Regulation of CDKN2A HYPs
CDKN2A
ETS1
ETS2
HBP1
PPARG
SMARCB1
SP1
taof(ETS2)
taof(PPARG)
taof(YY1)
YY1
BMI1
EED
EZH2
GLI2
HDAC3
taof(GLI2)
notes: expected direction is based on internal causality of the regulation
of cDKn2A expression submodel. Yellow = predicted increase in
abundance or activity, blue = predicted decrease in abundance or
activity. Submodel nodes that are shared with other senescence models
are bolded.
Abbreviation: taof(X), transcriptional activity of X.
Table s4. nodes from the regulation by tumor suppressors submodel of the DAcS network that are predicted as
hYPs by rcr on the gSe28464 test data set.
Regulation by Tumor
Suppressors HYPs
Expected
Direction
Test Data set
GSE28464
Predicted in consistent directions
BRCA1
CCL2
CDKN1A
CDKN2A
CDKN2A NP_000068
Cell aging
CSF2
CXCL1
HGF
IFNA1
IL6 *
IL13
IL1B
IL6ST
IL8
ING1
IRF1
IRF3
Kaof(RAF1)
Oncogene induced senescence
RAF1
RB1
RBL2
replicative cell aging
taof(IRF1)
taof(RB1)
TNFRSF1A
CCND1
E2F1
E2F2
E2F3
ENO1
taof(E2F family Hs)
taof(E2F1)
taof(E2F2)
taof(E2F3)
TFDP1
Predicted in inconsistent directions
PTEN
notes: expected direction is based on internal causality of the regulation
by tumor suppressors submodel. Yellow = predicted increase in abundance
or activity, blue = predicted decrease in abundance or activity. Submodel
nodes that are shared with other senescence models are bolded. *IL6
is shown as predicted increased in this table, in contrast to the initial
prediction by rcr (See Section 3.3 Application of the DAcS network to
an Independent Data Set for additional detail and Supplementary Fig. 2).
Abbreviations: kaof(X), kinase activity of X; taof(X), transcriptional
activity of X.
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Subtract proliferative
genes in PMIDs
12791645 and 16188230
IL6
IL6
Score: 55
Contra: 26
Score: 107
Contra: 62
Figure s2. Directionality investigation for the IL6 hYP in the DAcS network Model test data set.
notes: The scored IL6 hYP is shown for the DAcS network evaluation data set gSe28464 before (left) and after (right) removal of proliferation-related
genes in multiple myeloma as reported in two previously published studies. circles represent observed gene expression State changes (red for increased,
green for decreased), and edges connecting the central IL6 hYP node to State changes represent literature-derived causal relationships between IL6 and
its downstream target genes. Solid lines indicate relationships that support the hYP prediction (counted as “Score”) and dotted lines indicate relationships
that are contradictory to the hYP prediction (counted as “contra”). When proliferation-related genes are retained in the IL6 hYP, rcr produces a
prediction for decreased IL6 abundance. excluding this set of proliferation-related genes results in an rcr prediction for increased IL6 abundance.
Table s5. nodes from the transcriptional regulation of the senescence-associated secretory phenotype (SASP) submodel
of the DAcS network that are predicted as hYPs by rcr on the gSe28464 test data set.
Transcriptional Regulation of the SASP HYPs
Expected
Direction
Test Data Set
GSE28464
Predicted in consistent directions
CCL2
CCL5
CEBPB
CXCL1
IFNG
IL1 Family Hs
IL6 *
IL13
IL1A
IL8
kaof(p38 MAPK family Hs)
NFKB Complex Hs
RELA
taof(CEBPB)
taof(NFKB Complex Hs)
VEGFA
notes: expected direction is based on internal causality of the transcriptional regulation of the SASP submodel. Yellow = predicted increase in abundance
or activity, blue = predicted decrease in abundance or activity. Submodel nodes that are shared with other senescence models are bolded. *IL6 is shown
as predicted increased in this table, in contrast to the initial prediction by rcr (See Section 3.3 Application of the DAcS network to an Independent Data
Set for additional detail and Supplementary Fig. 2).
Abbreviations: kaof(X), kinase activity of X; taof(X), transcriptional activity of X.
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Figure s3. graph showing the transcriptional regulation of the senescence-associated secretory phenotype (SASP) submodel as depicted using the BeL
framework and colored according to the gSe28464 test data set.
notes: Yellow = predicted increase in abundance or activity, blue = predicted decrease in abundance or activity, red = observed increase in mrnA
expression. IL6 is shown as predicted increased in this figure, in contrast to the initial prediction by rcr (See Section 3.3 Application of the DAcS network
to an Independent Data Set for additional detail and Supplementary Fig. 2).
Abbreviations: exp(X), mrnA expression of X; kaof(X), kinase activity of X; taof(X), transcriptional activity of X.
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Additional Files
Additional File 1 Reverse Causal Reasoning.pdf
Additional File 2 DACS Network.xlsx
Additional File 3 DNA Damage-Agglomerated.cy.xgmml
Additional File 4_Autophagy-Agglomerated.cy.xgmml
Additional File 5_Apoptosis-Agglomerated.cy.xgmml
Additional File 6_Necroptosis-Agglomerated.cy.xgmml
Additional File 7_Senescence-Agglomerated.cy.xgmml
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