The blood cancer T cell large granular lymphocyte (T-LGL) leukemia is a chronic disease characterized by a clonal proliferation of cytotoxic T cells. As no curative therapy is yet known for this disease, identification of potential therapeutic targets is of immense importance. In this paper, we perform a comprehensive dynamical and structural analysis of a network model of this disease. By employing a network reduction technique, we identify the stationary states (fixed points) of the system, representing normal and diseased (T-LGL) behavior, and analyze their precursor states (basins of attraction) using an asynchronous Boolean dynamic framework. This analysis identifies the T-LGL states of 54 components of the network, out of which 36 (67%) are corroborated by previous experimental evidence and the rest are novel predictions. We further test and validate one of these newly identified states experimentally. Specifically, we verify the prediction that the node SMAD is over-active in leukemic T-LGL by demonstrating the predominant phosphorylation of the SMAD family members Smad2 and Smad3. Our systematic perturbation analysis using dynamical and structural methods leads to the identification of 19 potential therapeutic targets, 68% of which are corroborated by experimental evidence. The novel therapeutic targets provide valuable guidance for wet-bench experiments. In addition, we successfully identify two new candidates for engineering long-lived T cells necessary for the delivery of virus and cancer vaccines. Overall, this study provides a bird's-eye-view of the avenues available for identification of therapeutic targets for similar diseases through perturbation of the underlying signal transduction network. Crowther Audrey audrey.crowther@huskers.unl.edu University of Nebraska-Lincoln 2017-07-20T11:02:32Z 2017-07-20T16:59:45Z

G protein coupled receptors (mouse)

UniProt AccessionID: P35413

 2017-07-20T12:11:33Z

small mothers against decapentaplegic 2

UniProt AccessionID: Q15796

small mothers against decapentaplegic 3

UniProt AccessionID: P84022

(MAPK Network [1])

SMAD family member 4 small mothers against decapentaplegic 4 9

UniProt AccessionID: Q13485

Gene Name: SMAD4 Gene ID: 408

 2017-07-20T12:11:33Z

Zeta-chain (TCR) associated protein kinase 70kDa [Homo sapiens(human)] UniProt ID: Q6ISU1

Gene Name: ZAP70

NCBI Gene ID: 7535

T Cell Receptor complex coreceptors in CD8 cells

For more info: http://www.ncbi.nlm.nih.gov/mesh/68019672

 2017-07-20T11:24:37Z

myeloid cell leukemia sequence 1 (BCL2-related)

UniProt AccessionID: Q07820

For more info: http://www.ncbi.nlm.nih.gov/mesh/67579095

 2017-07-20T12:56:07Z

<font size="2" style="font-style: normal;" face="Georgia">baculoviral IAP repeat-containing 2 (BIRC2)</font><div style="font-style: normal;"><font face="Georgia"><font size="2">UniProt Accession ID: Q13490</font><font size="2"><br/></font></font><div><font size="2" face="Georgia"><br/></font></div><div><font face="Georgia" size="2">baculoviral IAP repeat-containing 3 (BIRC3)</font></div><div><font face="Georgia" size="2">UniProt Accession ID: Q13489</font></div><div><font face="Georgia" size="2"><br/></font></div><div><font face="Georgia" size="2">baculoviral IAP repeat-containing 4 (BIRC4)</font></div></div><div style="font-style: normal;"><font face="Georgia" size="2">UniProt Accession ID: P98170</font></div><div style="font-style: normal;"><font face="Georgia" size="2"><br/></font></div><div><font face="Georgia" size="2">In the <em>Apoptosis Network</em>, IAP represents inhibitors of apoptosis [1].</font></div><div><font face="Georgia" size="2"><br/></font></div><div><font face="Georgia" size="2">In Survival Signaling 2008 and 2011, IAP represents inhibitors of apoptosis.</font></div>

 2017-07-20T12:51:45Z

Cell death

 2017-07-20T12:57:22Z

Death inducing signaling complex

UniProt AcessionID: O14763

For more info: http://www.ncbi.nlm.nih.gov/mesh/?term=death+inducing+signaling+complex

 2017-07-20T12:56:07Z

FLICE inhibitory protein/ CASP8 and FADD-like apoptosis regulator

For more info: http://www.ncbi.nlm.nih.gov/mesh/68053446

 2017-07-20T12:49:19Z

Sphingosine-1-phosphate

PubChem ID: 5283560

Molecular Formula: C18H38NO5P

Molecular Weight: 379.471782

 2017-07-20T12:56:07Z

T cell activation signal stimulation

 2017-07-20T12:07:09Z

Fas (TNF receptor superfamily, member 6)

 2017-07-20T12:15:09Z

Interferon, gamma [Homo sapiens(human)]

UniProt Accession ID: P01579

Gene Name: IFNG

NCBI Gene ID: 3458

 2017-07-20T12:07:09Z

cytotoxic T-lymphocyte-associated protein 4

UniProt AccessionID: P16410

Gene Name: CTLA4

Gene ID: 1493

For more info: http://www.ncbi.nlm.nih.gov/mesh/67556706

 2017-07-20T11:24:37Z

Fas (TNF receptor superfamily, member 6)

Uniprot ID: P25445

 2017-07-20T12:31:45Z

Ceramide, a small molecule

for more info: http://www.ncbi.nlm.nih.gov/mesh/68002518

 2017-07-20T12:56:07Z

BH3 interacting domain death agonist [Homo sapiens (human)]

UniProt Accession ID: P55957

Gene Name: BID

Gene ID: 637

 2017-07-20T12:51:45Z

<font face="Arial, Verdana" size="2">Caspase3, Apoptosis cysteine related peptidase</font><div style="font-family: Arial, Verdana; font-size: 10pt; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;">UniProt Accession ID:&nbsp;<a href="http://www.uniprot.org/uniprot/P42574" style="font-size: 10pt; text-decoration: none; color: rgb(64, 148, 180); border-bottom-width: 0px; cursor: pointer; font-family: verdana, arial; line-height: 20.0819721221924px; background-color: rgb(255, 255, 255);">P42574</a></div><div style="font-family: Arial, Verdana; font-size: 10pt; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;">Caspase8, Apoptosis cysteine related peptidase</div><div style="font-family: Arial, Verdana; font-size: 10pt; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;">UniProt Accession ID:&nbsp;<a href="http://www.uniprot.org/uniprot/Q14790" style="font-size: 10pt; text-decoration: none; color: rgb(64, 148, 180); border-bottom-width: 0px; cursor: pointer; font-family: verdana, arial; line-height: 20.0819721221924px; background-color: rgb(222, 239, 245);">Q14790</a></div><div style="font-family: Arial, Verdana; font-size: 10pt; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"><br/></div><div style="font-family: Arial, Verdana; font-size: 10pt; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;">For more info on Caspase3:</div><div><font face="Arial, Verdana" size="2">http://www.ncbi.nlm.nih.gov/mesh/68053148</font></div><div><font face="Arial, Verdana" size="2"><br/></font></div><div style="font-family: Arial, Verdana; font-size: 10pt; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;">For more info on Caspase8:</div><div style="font-family: Arial, Verdana; font-size: 10pt; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;">http://www.ncbi.nlm.nih.gov/mesh/68053181</div><div style="font-family: Arial, Verdana; font-size: 10pt; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"><br/></div>

 2017-07-20T12:57:22Z

cAMP responsive element binding protein 1

Cyclic AMP-responsive element-binding protein 1

UniProt ID: P16220

Gene Name: CREB1

Gene ID: 1385

 2017-07-20T11:24:37Z

Sip activates GPCR. Apoptosis inhibits GPCR and is dominant to SiP.

S1P binds to a number of GPCRs.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

 S_9 1 S_6 1

GPCR activates SMAD. Apoptosis inhibits SMAD and is dominant to GPCR.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

GPCR indirectly activates SMAD.

 S_1 1 S_6 1

Apoptosis and CTLA4 inhibit TCR.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

CTLA is an important negative regulator of T cells.

 S_6 1 S_13 1

Apoptosis and DISC inhibit MCL1.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

DISC inhibits MCL1.

 S_6 1 S_7 1

Apoptosis and BID inhibit IAP.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

BID indirectly inhibits IAP through the release of Omi/HtrA2 and Smac/DIABLO into the cytosol.

 S_16 1 S_6 1

Apoptosis and Caspase activate Apoptosis.

Apoptosis is considered an irreversible cell fate. A positive feedback loop on apoptosis models this irreversibility.

Activation of pro-apoptotic caspases, such as caspase-3, induces apoptosis.

 S_6 1 S_17 1

Fas and Ceramide activate DISC. Apoptosis inhibits DISC and is dominant to both Fas and Ceramide. FLIP inhibits DISC and is dominant to Fas.'

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

Binding of Fas to its ligand leads to the formation of DISC.

FLIP inhibits Fas-mediated apoptosis by inhibiting caspase-8 processing at the DISC.

Ceramide generation induces Fas clustering, which triggers the formation of DISC.

 S_15 1 S_6 1 S_14 1 S_6 1 S_8 1

Apoptosis and DISC inhibit FLIP.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

DISC inhibits FLIP.

 S_6 1 S_7 1

Apoptosis and Ceramide inhibit S1P.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

Increase in Ceramide levels triggers apoptosis and inhbits the pro-survival signals mediated by S1P.

 S_6 1 S_15 1

P2 and IFNG activate P2. Apoptosis inhibits P2 and is dominant to P2 and IFNG.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

IFNG activates P2.

P2 activates P2.

 S_12 1 S_6 1 S_10 1 S_6 1

S1P activates sFas. Apoptosis inhibits sFas and is dominant to S1P.

S1P activates sFas.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

 S_9 1 S_6 1

Apoptosis, SMAD, and P2 inhibit IFNG.

Apoptosis considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

SMAD inhibits IFNG.

Ps inhibits IFNG.

 S_6 1 S_10 1 S_2 1

TCR activates CTLA4. Apoptosis inhibits CTLA4 and is dominant to TCR.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

CTLA4 is activated after TCR stimulation. CTLA4 is important in eliminating potentially autoreactive T cells right after naive T cell activation.

 S_3 1 S_6 1

Apoptosis and sFas inhibit Fas.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

sFas inhibits Fas-induced apoptosis by binding to Fas ligand, thereby preventing the interaction between Fas ligand and membrane-bound Fas.

 S_11 1 S_6 1

Fas activates Ceramide. Apoptosis and S1p inhibit Ceramide and are dominant to Fas.

S1P promotes cell survival, which inhibits Ceramide-mediated apoptosis.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

The connection between Fas and Ceramide is controversial, with some studies finding that Fas induces the generation of Ceramide.

 S_14 1 S_9 1 S_6 1

Apoptosis and MCL1 inhibits BID.

Mcl-1 binds to truncated BID and inhibits its induction of cytochrome c.

Apoptosis considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

 S_6 1 S_4 1

DISC and BID activate Caspase. Apoptosis inhibits Caspase and is dominant to DISC and BID. IAP inhibits Caspase and is dominant to BID.

IAPs are a family of proteins that inhibit caspase activity.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

BID indirectly activates a caspase cascade through the release of cytochrome c.

Caspases are activated in the DISC. Caspase 8 and 10 are activated this way via induced proximity dimerization.

 S_7 1 S_6 1 S_16 1 S_6 1 S_5 1

IFNG activates CREB. Apoptosis inhibits CREB and is dominant to IFNG.

Apoptosis is considered an irreversible cell fate. All cellular processes stop after the cell has successfully undergone apoptosis.

IFNG stimulates CREB phosphorylation and CREB DNA binding.

 S_12 1 S_6 1