Aurora Kinase A (AURKA) is often overexpressed in neuroblastoma (NB) with poor outcome. The causes of AURKA overexpression in NB are unknown. Here, we describe a gene regulatory network consisting of core regulators of AURKA protein expression and activation during mitosis to identify potential causes. This network was transformed to a dynamic Boolean model. Simulated activation of the serine/threonine protein kinase Greatwall (GWL, encoded by MASTL) that attenuates the pivotal AURKA inhibitor PP2A, predicted stabilization of AURKA. Consistent with this notion, gene set enrichment analysis showed enrichment of mitotic spindle assembly genes and MYCN target genes in NB with high GWL/MASTL expression. In line with the prediction of GWL/MASTL enhancing AURKA, elevated expression of GWL/MASTL was associated with NB risk factors and poor survival of patients. These results establish Boolean network modeling of oncogenic pathways in NB as a useful means for guided discovery in this enigmatic cancer. Crowther Audrey audrey.crowther@huskers.unl.edu University of Nebraska-Lincoln 2017-04-20T16:52:20Z 2018-04-23T08:16:31Z

The concentration of STMN required to regulate PLK1.

Protein: Stathmin

UniProt ID: P16949

Gene: STMN1

Gene ID: 3925

 2018-03-21T16:20:27Z

Glycogen synthase kinase-3 beta

Uniprot ID: P49841

Gene Name: GSK3B

Gene ID: 2932

 2018-03-21T16:20:27Z

The possible activation of PLK1 by microtubule (MT)

 2018-03-21T16:20:27Z

LIM domain-containing protein ajuba/ sprindle pole protein

Uniprot ID: Q96IF1

Gene Name: AJUBA

Gene ID: 84962

 2018-03-21T16:20:27Z

CDK1 bound to CCNB

Gene Name: CDK1

Gene Name: CCNB1

Protein: G2/mitotic-specific cyclin-B1

Uniprot ID: P14635

Protein: Cyclin-dependent kinase 1

Uniprot ID: P06493

Gene ID: 983

Gene ID: 891

 2018-03-05T19:42:00Z

M-phase inducer phosphatase 2

Uniprot ID: P30305

Gene Name: CDC25B

Gene ID: 994

 2017-07-14T17:19:30Z

Microtubule

 2017-04-21T13:56:57Z

mitotic spindle assembly

 2017-04-20T17:31:02Z

Alpha-Endosulfine

Uniprot ID: O43768

Gene Name: ENSA

Gene ID: 2029

 2017-04-21T14:00:13Z

Aurora Kinase A Active Form

Uniprot ID: O14965

Gene Name: AURKA

Gene ID: 6790

 2018-03-21T10:54:05Z

Targeting protein for Xklp2 / Spindle Protein

Uniprot ID: Q9ULW0

Gene Name: TPX2

 2017-07-14T17:19:30Z

Protein aurora borealis/ spindle pole protein

Uniprot ID: Q6PGQ7

Gene Name: BORA

Gene ID: 79866

 2018-03-21T14:05:21Z

Aurora Kinase A presence

Gene Name: AURKA

Uniprot ID: O14965

Gene ID: 6790

 2018-03-21T10:54:05Z

Serine/threonine-protein kinase greatwall

Uniprot ID: Q96GX5

Gene Name: MASTL

Gene ID: 84930

 2017-05-19T13:16:00Z

Wee1-like protein kinase

Uniprot ID: P30291

Gene Name: WEE1

Gene ID: 7465

 2017-07-14T17:19:30Z

Enhancer of filamentation 1/ Spindle Protein

Uniprot ID: Q14511

Gene Name: NEDD9

Gene ID: 4739

 2018-03-21T10:54:05Z

Stathmin

Uniprot ID: P16949

Gene Name: STMN1

Gene ID: 3925

 2017-04-21T14:00:59Z

Serine/threonine-protein kinase PLK1 / polo-like kinase

Uniprot ID: P53350

Gene Name: PLK1

Gene ID: 5499

 2018-03-21T14:05:21Z

Last process required for cell cycle division

The physical division into two daughter cells

 2018-03-21T14:05:21Z

Serine/threonine-protein phosphatase PP1-alpha catalytic subunit

Uniprot ID: P62136

Gene Name: PPP1CA

Gene ID: 5499

 2018-03-22T16:25:44Z

Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform

Uniprot ID: P67775

Gene Name: PPP2CA

Gene ID: 5515

 2017-04-21T14:00:13Z

Cytoplasmic polyadenylation element-binding protein 1

Uniprot ID: Q9BZB8

Gene Name: CPEB1

Gene ID: 64506

 2017-07-14T17:19:30Z

Centrosome maturation

 2017-04-20T17:31:02Z

CDC25B and hCPEB activate CDK1CCNBComplex.

Cytokinesis inhibits CDK1CCNBComplex and is dominant to CDC25B and hCPEB.

CDK1CCNBComplex is active when all positive and negative regulators are inactive.

WEE1 inhibits CDK1CCNBComplex but is not dominant to CDC25B and hCPEB.

WEE1 inhibits the Cdk1-cyclin B complex (CDK1CCNBComplex) by phosphorylating Tyr15 on Cdk1. This inhibition is relieved by the dephosphorylation of Cdk1 via CDC25B.

CDC25B is required for cyclin B accumulation.

hCPEB stimulates elongation of the poly(A) tails of cyclin B1 and Cdk1.

βTrCP-dependent degradation of CDC25B begins after the first stages of mitosis. Thus, Cytokinesis inhibits the activation of CDK1CCNBComplex by CDC25B inhibition.

After mitotic exist (completion of Cytokinesis), Cdk1-cyclin B complex (CDK1CCNBComplex) activity decreases.

 S_22 1 S_18 1 S_2 1 S_18 1 S_14 1 S_2 1 S_22 1 S_18 1

AURKAActive and PLK1 activate CDC25B.

Cytokinesis inhibit CDC25B and is dominant to AURKAActive and PLK1.

Aurora A (AURKAActive) phosphorylates CDC25B on Ser353.

PLK1 promotes nuclear localization and subequent activation of CDC25B.

βTrCP-dependent degradation of CDC25B begins after the first stages of mitosis. This is required for mitotic exit.

 S_17 1 S_18 1 S_7 1 S_18 1

STMN inhibits MT.

Stathmin (STMN) is a microtubule (MT) destabilizer.

 S_16 1

CentrosomeMat activates SpindleAssembly.

Cytokinesis inhibits SpindleAssembly and is dominant to CentrosomeMat.

Centrosome maturation (CentrosomeMat) occurs prior to mitotic spindle assembly (SpindleAssembly).

Cytokinesis occurs after mitotic spindle assembly (SpindleAssembly). Thus, Cytokinesis and SpindleAssembly are mutually exclusive events.

 S_23 1 S_18 1

GWL_MASTL activates ENSA.

GWL_MASTL phosphorylates and activates ENSA.

 S_13 1

PP1 inhibits AURKAActive when is AURKAActive is active and is dominant to AJUBA and PP1.

AJUBA activates AURAActive when either BORA and AURKAPresent are active or when NEDD9 and AURKAPresent are active. TPX2 activates AURKAActive when AURKAPresent is active.

PP1 inhibits hyperphosphorylated active Aurora A kinase (AURKAActive) via dephosphorylation of Thr288. Thus AURKAActive is required for inhibition of AURKAActive via PP1.

PP1 inhibits hyperphosphorylated active Aurora A kinase (AURKAActive) via dephosphorylation of Thr288. Thus AURKAActive is required for inhibition of AURKAActive via PP1.

The N-terminal of TPX2 interacts with Aurora A kinase (AURKPresent), which induces a conformational change to induce autophosphorylation of Aurora A kinase.

NEDD9 binding to Aurora A kinase (AURKAPresent) is required for protein stabilization and decreases the efficacy of Aurora A kinase inhibitors.

Binding of AJUBA to Aurora A kinase (AURKAPresent) induces autophosphorylation and activation of Aurora A kinase.

BORA binds to and activates Aurora kinase A (AURKAPresent). Activation of Aurora A kinase by BORA requires autophosphorylation of Aurora A kinase.

Aurora A kinase needs to be present in the system (AURKAPresent) in order to be activated.

 S_8 1 S_10 1 S_19 1 S_7 1 S_20 1 S_15 1 S_10 1 S_9 1 S_10 1 S_19 1 S_7 1

PLK1 activates TPX2.

PLK1 phosphorylates TPX2 at Ser204.

 S_17 1

GSK3B activates BORA.

PLK1 inhibits BORA when cytokinesis is active and is dominant to GSK3B.

GSK3B phosphorylates Ser274 and Ser278 of BORA.

PLK1 binds to BORA and initiates β-TrCP-mediated degradation.

While PLK1 is activated during the G2/M transition, BORA is protected from PLK1-induced degratation during early mitosis. This protection is lost later during mitosis.

 S_11 1 S_17 1 S_18 1

PP2A inhibits AURKAPresent.

PP2A inhibits AURKAPresent via dephosphorylation of Ser51, leading to proteosomal degradation.

 S_21 1

CDK1CCNBComplex activates GWL_MASTL.

PP2A inhibits GWL_MASTL and is dominant to CDK1CCNBComplex.

PP2A dephosphorylates CDK1 substrates in addition to inhibiting Cdc25, an activator for CADK1CCNBComplex.

GWL_MASTL is activated downstream of CDK1CCNBComplex.

 S_1 1 S_21 1

PLK1 inhibits WEE1.

PLK1 promotes degradation of WEE1 through phosphorylation of Ser53.

 S_17 1

AURKAActive activates NEDD9.

Activated Aurora A kinase (AURKAActive) phosphorylates NEDD9.

 S_7 1

AURKAActive inhibit STMN.

The active form of Aurora A kinase (AURKAActive) phosphorylates Stathmin (STMN).

 S_7 1

AURKAActive activates PLK1. MT activates PLK1 when MTCanAct is active. STMNCanAct activates PLK1 when STMN is inactive.

AURKAActive activates PLK1 by phosphorylation of Thr210.

Microtubules (MT) restores PLK1 activation in stathmin-depleted cells.

MTCanAct models the possible activation of PLK1 by MT. Microtubules are required for the activation of PLK1.

Stathmin (STMN) overexpression decreases PLK1 activity.

STMNCanAct represents the required concentration of STMN to activate PLK1.

 S_7 1 S_4 1 S_16 1 S_3 1 S_12 1

SpindleAssembly activates Cytokinesis.

CentrosomeMat inhibits Cytokinesis and is dominant to SpindleAssembly.

Spindle assembly (SpindleAssembly) occurs before Cytokinesis during mitotic progression. Successful completion of SpindleAssembly activates Cytokinesis.

Centrosome maturation (CentrosomeMat) occurs prior to spindle assembly (SpindleAssembly) during mitotic progression. Thus, SpindleAssembly cannot activate Cytokinesis until CentrosomeMat is inactive.

 S_5 1 S_23 1

AURKAActive, CDK1CCNBComplex, and Cytokinesis inhibit PP1.

Hyperphosphorylation Aurora A kinase (AURKAActive) phosphorylates and inhibits PP1.

CDK1CCNBComplex phosphorylates and inhibits PP1

PP1 is important in progession through mitosis, and thus PP1 activity increase during G2. Successful completion of mitosis leads to a decrease in PP1 activity and the decrease in nuclear concentrations of PP1.

 S_18 1 S_1 1 S_7 1

PP1 activates PP2A.

ENSA inhibit PP2A and is dominant to PP1.

Recruitment of PP1 to the regulatory subunits of PP2A activates PP2A.

When ENSA is phosphorylated on Ser67 by Greatwall, it inhibits PP2A.

 S_19 1 S_6 1

AURKAActive activates hCPEB.

Aurora A kinase (AURKAActive) binds to and phosphorylates hCPEB.

 S_7 1

CDK1CCNBComplex activates CentrosomeMat.

SpindleAssembly inhibits CentrosomeMat and is dominant to CDK1CCNBComplex.

Mitotic spindle assembly (SpindleAssembly) occurs after Centrosome maturation (CentrosomeMat). Thus, SpindleAssembly and CentrosomeMat are mutually exclusive events.

Activation of CDK1CCNBComplex is required for mitotic entry (represented as CentrosomeMat).

 S_1 1 S_5 1