Phosphoproteomic Analysis of Lung Tissue in PAH Reveals Activation of Immune Modulatory, Angiogenic, and Cell Proliferation Pathways

R. Sitapara 1,2, T.T. Lam 3, S. Festin1, L. Zisman1

NIH Support: 5R03HL110821 PHBI Tissue Repository Disclosures

Ravi Sitapara: Current Employee of Pulmokine Inc. stock options in Pulmokine Inc.

Steve Festin: Former Employee of Pulmokine, Inc.

Lawrence Zisman: Founder and Employee of Pulmokine, Inc. Stock in Pulmokine Inc.

2 Background

Signaling Plays an Important Role in PAH

 Preclinical and Clinical Data Suggests Therapeutic potential of kinase inhibition in PAH

 Pathways for Kinase Signaling in PAH are not well understood

3 Hypothesis

An unbiased phosphoproteomic analysis of PAH Lung Tissue will reveal novel kinase regulatory networks implicated in the pathophysiology of PAH

4 Methods iPAH/Control Lung Tissue PHOSPHO PEPTIDE IDENTIFICATION PHBI Repository Progenesis Software MASCOT database

KINASE PREDICTION PhosphoNET and NetworKIN

STRING Database BUILD A KINASE NETWORK INVOLVED IN PAH

5 Clinical Data of Study Subjects from PHBI Repository

Clinical Data RHC Data Medications RA mm Mean PA PCWP Endothelin Receptor Subject Diagnosis Age Gender Hg mm Hg mm Hg CO L/min PDE-V Inhibitor Antagonist Prostanoid 1 IPAH 40 F 7 47 7 6.17 Ambrisentan IV epoprostenol 2 IPAH 41 F 30 55 7 3.86 Sildenafil Bosentan IV epoprostenol 3 IPAH 38 F NA 50 8 2.87 Sildenafil Bosentan IV treprostinil IV epoprostenol/SC 4 IPAH 25 M NA 59 7 4.09 Sildenafil treprostinil 5 IPAH 40 M NA 64 12 3.1 Sildenafil Ambrisentan SC treprostinil 6 IPAH 51 M NA 50 8 4.6 Sildenafil IV epoprostenol 7 APAH SSC 54 F 10 55 10 5.47 Sildenafil Ambrisentan IV epoprostenol 8 APAH SSC 65 F 6 32 6 4.35 Sildenafil Bosentan Inhaled treprostinil 9 APAH SSC 55 F 11 51 11 5.9 Sildenafil Bosentan IV epoprostenol 10 Control 56 F 11 Control 49 F 12 Control 55 F 13 Control 47 M 14 Control 52 M 15 Control 17 M

6 Results

̴1200 ̴2940 phospho- ̴56,000 species phosphoproteins proteins with q<0.05

Volcano Plot 10

8

6 Log(P)

- 4

2

0 -6.000 -4.000 -2.000 0.000 2.000 4.000 6.000 Log2[Female(iPAH/Control)]

7 GO* Pathway Classification

Immune Regulation: 75 proteins

Angiogenesis: 29 proteins

Cell Proliferation: 99 proteins

*GO= Ontology

8 Examples of the Most regulated phosphoproteins (iPAH vs Control)

Female Protein Function Phospho-site Male iPAH/control iPAH/control Lymphocyte IKZF3 Ser-378 #DIV/0! #DIV/0! Differentiation HMHA1 Rho-GTPase Activator Ser-73 #DIV/0! 6.49 BCAS3 Angiogenesis Ser-709 119.26 10.70 RHG25 Rho-GTPase Thr-442 18.95 2.75 NUMA1 Mitotic Spindle Ser-2047 15.32 #DIV/0! Thr-180, Tyr- MK14 MAP Kinase 14 8.74 3.18 182 HDGF DNA binding Ser-107 8.09 37.15 Protein Tyrosine PTPRB Ser-119 0.00 0.00 Phosphatase HDAC2 Epigenetic Regulation Ser-442 0.29 0.21

9 Validation Studies: Example of pIKZF3 in iPAH Lung

iPAH Control

N=6 N=5 ** p<0.01

10 Perivascular Co-localization of pIKZF3 to B and T Cells

11 Kinase Prediction: NetworKin

Kinase N Kinase N Kinase N ADRBK1 1 HIPK2 8 PRKAA2 2 AKT1 5 HYRC 5 PRKACB 1 ATM 1 MAP2K1 6 PRKACG 1 AURKA 5 MAP2K2 3 PRKCA 3 BCKDK 1 MAP2K3 2 PRKCG 3

CAMK2B 3 MAP2K4 2 PRKCI 7 CDK1 20 MAP2K5 1 PRKD1 2 CDK2 4 MAP2K6 3 PRKD2 2 CDK4 4 MAPK1 5 ROCK2 4 CDK5 8 MAPK11 1 RPS6KA1 1 CLK1 1 MAPK12 1 RPS6KB1 4 CLK4 1 MAPK3 9 RPS6KB2 2

CSNK1A1 2 PAK2 2 SLK 1

CSNK2A1 39 PAK3 2 STK11 1

CSNK2A2 3 PAK4 2 TGFBR2 2 DMPK 1 PDK1 5 TTK 2 GSK3A 3 PDK2 2 GSK3B 4 PRKAA1 3 12 Kinase Prediction: Phosphonet

Biomarker Phosphosite Top Predicted Kinase(s) Candidate IKZF3 S378 GSK3alpha/beta* HMHA1 S73 CAMK4 RHG25 T442 PIM1 NUMA1 S2047 A ASPP1 S710 CAMK4 HDGF S107 PLK3, GSK3alpha/beta HDAC2 S422 CK2* PTPRB S119 RSK2, MK14, PRKG1

*Confirmed with Kinexus in vitro assays

13 String Kinase Network CSNK2A1 + CDK1 Substrates

14 String Network for GSK3A and GSK3B

15 Conclusions

 This first reported phosphoproteomic analysis in PAH lung tissue revealed several novel kinase targets of potential interest for therapeutic and biomarker development.

 Initial validation confirmed that pIKZF3 was highly regulated in iPAH Lung.

 String Network Analysis Demonstrated a high degree of interaction between multiple both directly and indirectly including interactions between CDK1 and .

16 Future Directions

Functional significance of phosphoproteins Multiplex Phosphoprotein Biomarker Assay Computer simulations of Network Perturbations with physiologic readouts

17 Acknowledgments

Pulmokine Inc. University of Colorado Steve Festin Todd Bull Ravi Sitapara Rubin Tuder Larry Zisman Yale University Tukiet Lam RCTR Ravi Sitapara PHBI Tissue Repository

18 Back Up Slides: String Network Analyses

MAPK PRKG and ROCK1 GSK3 all CAMK

19 String Network MAP Kinases

20 String Network PRKG and ROCK1 Kinase

21 String Network Analysis GSK3 (Networkin>3)

String Analysis GSK3 Networkin All

22 String Network Analysis CAMK (NetworKin>3)

23