Pathway studiosymposium lorenzi

Census of the Apoptosis Pathway (and Other Uses of Pathway Analysis)

Phil Lorenzi, Ph.D.

Co-Director, The Proteomics and Metabolomics Core Facility Dept. of Bioinformatics & Computational Biology

The University of Texas MD Anderson Cancer Center

Disclosures

• My attendance at this symposium was supported by Elsevier

The Drug Discovery & Development Process

Hypothesis Generation

Target Identification

Target Validation Lead Optimization

Pharmacodynamics

Pharmacokinetics

Clinical Trials

In Vivo Animal Studies

IND Application NDA

Chemical Profiling Data

Database Construction

Exploration

In Vitro Absorption

and Metabolism

Discovery Preclinical

Toxicology

Old View:

Pharmacological Profiling Data Phase I: Safety in Healthy Subjects

Phase II: Efficacy in Patients

Phase III: Large-scale Trial in Patients

“omics”

Mechanism of Action Delivery

Biological Profiling Data

Drug Combinations

Small Molecule Screens

Technology and Information

New View:

Systems Pharmacology of L-Asparaginase (2005)

L-asparaginase (drug)

N

Q

ASNS

Ø (total [D])

E

N

Q + D + ATP

?

Ø

SP2

Ara-C

ATF-2

ATF-4

ATF-3

NSRE-2 NSRE-1

CHOP

AARE

RA

? ?

ASNS

ATF-6

5-aza-2’-deoxycytidine

diazo-4-oxo-L-norvaline

6-Diazo-5-oxo-L-norleucine &/or 8-N3-ATP

DMT P

ATF-5

CHOP

•

AARE C/EBP-β

C/EBP-β

GC-III GC-II GC-I

SP1

SP3

P

CD95 = FAS

FAS-L

Caspase-8

Caspase-3

↓Cell Volume

FADD

APOPTOSIS

D

Ø (< 0.4 mM)

GADD45 GRP78

ASK1 JNK

QRS

p53

p38

Ca2+

CAM kinase 2 PKC

NOS

NFκB

MAPK

NO

ROS

Calpain

Bid

Q (Ø)

GSH Bcl-2 cFLIP

HSP70

Ø

Ø

cPLA2

ERK

p21

p27

H2O

PLC

RAF

MEK

Proliferation

GCN2

P

EIF2A

↑Metabolism

The Autophagy Pathway (2014)

Lorenzi et al., Autophagy, 2014

Outline

1. Comparison of pathway analysis strategies

• introduction

• the autophagy pathway

• the apoptosis pathway

2. Pathway analysis for target validation (pharmacovigilance)

• COX2 inhibitors (post-market failure)

• AKT inhibitors (many years of failed clinical trials)

• EGFR inhibitors (already approved)

• MEK inhibitors (already approved)

• RANKL inhibitor (already approved)

• PARP inhibitors (already approved)

• GLS inhibitors (in clinical trials)

Pathway Analysis

• Growing demand in biological sciences to perform pathway analysis on “big data”

• Common Goals: – build pathway maps

– obtain insight not provided by specific gene/protein/metabolite-level analyses

– drug development: because cancer cells acquire resistance to targeting single nodes, targeting pathways may improve outcome

Popular Pathway Analysis Options

• Ingenuity Pathway Analysis (Ingenuity Systems) • MetaCore (Thomson Reuters GeneGo)

– MetaDrug

• GeneSpring (Agilent) – MPP – NGS

• DAVID (open source) • KEGG (http://www.kegg.jp)

• Roche Pathways (http://biochemical-pathways.com/#/map/1)

• Biocyc (http://biocyc.org)

• Pathway Studio (Elsevier) – ChemEffect – DiseaseFx

http://www.kegg.jp/

http://biochemical-pathways.com/#/map/1



http://biocyc.org/

Pathway Studio - Introduction

• MedScan • Mammalian cartridge

• ChemEffect cartridge

• DiseaseFx cartridge

Database

The Autophagy Pathway (2014)


Construction of the Autophagy Pathway

Integrated Analysis of the Autophagy Pathway

1: siRNA Screen 1

2: siRNA Screen 2 3: siRNA Screen 3

4: siRNA Screen 4

5: IPA

6: MetaCore

7: Pathway Studio (raw)

8: Pathway Studio (curated)

No single method enables comprehensive pathway interrogation


Integration of Proteomic Data

No single method enables comprehensive pathway interrogation

Behrends et al. Nature 466:68 (2010)

Quality Assessment – How Accurate is PS?

• False positives – manual curation of all extracted sentences indicated 1181/6215 (19%) of

the relations were incorrect (false positives)

• reduced to ~0% by manual curation

– Example: “The ability of the excess Atg4B mutant to inhibit autophagy...”

• Change direction of regulation from NEGATIVE to POSITIVE

• False negatives – Example: “We show that ATF4 and LC3B play a critical role in activating

autophagy and protecting cells from Bortezomib-induced cell death.”

• Change direction of regulation from UNKNOWN to POSITIVE

Integrated Analysis of the Autophagy Pathway

1: siRNA Screen 1

2: siRNA Screen 2 3: siRNA Screen 3

4: siRNA Screen 4

5: IPA

6: MetaCore


8: Pathway Studio (curated)


Key Question

• Are these conclusions generalizable?

– Examine additional biological pathways to assess whether:

• siRNA screening limitations persist • pathway analysis outperforms experimental approaches

Integrated Analysis of the Apoptosis Pathway

1: siRNA screen

2: siRNA screen 3: ORFeome screen

4: siRNA/miRNA screen

5: shRNA screen

6: siRNA screen

7: shRNA screen

The previous conclusions are generalizable

8: siRNA screen

9: siRNA screen 10: siRNA screen

11: siRNA screen

12: siRNA screen

13: IPA


2894

3487

484

Lorenzi et al., unpublished

Deriving Novel Therapeutic Strategies from Pathway Analysis

• Clinical insight – Temsirolimus (MTOR inhibitor/autophagy stimulator) – why is there not a

clear clinical response? • Answer: induces mitophagy, which degrades mitochondrial proteins

(e.g., RIPK1, RIPK3) that promote cell death

• Our proposal – Combination of an autophagy inhibitor + autophagy stimulator

• e.g., hydroxychloroquin (HCQ) + temsirolimus (TEM; rapamycin analog) • Hypothesized outcome: inhibition of mitophagy (by HCQ) would

preserve death-promoting mitochondrial proteins that are activated in response to the autophagy stimulator (TEM)

Deriving Novel Therapeutic Strategies from Pathway Analysis

Outline

1. Comparison of pathway analysis strategies

• introduction

• the autophagy pathway

• the apoptosis pathway

2. Pathway analysis for target validation (pharmacovigilance)

• COX2 inhibitors (post-market failure)

• AKT inhibitors (many years of failed clinical trials)

• EGFR inhibitors (already approved)

• MEK inhibitors (already approved)

• RANKL inhibitor (already approved)

• PARP inhibitors (already approved)

• GLS inhibitors (in clinical trials)

Vignette: COX2 Inhibitors, Vioxx

• Vioxx/rofecoxib was a COX2 inhibitor and NSAID developed by Merck that received FDA approval in 1999 but was removed in 2004 due to concerns about heart attack and stroke.

• Could text mining / literature searching have predicted the disaster?

• Detective work using Pathway Studio:

Vignette: Vioxx

1999: “The question whether the suppression of COX2 gene expression is fully or partially responsible for ADR-induced selective cardiovascular toxicity is still open.”

Vignette: AKT Inhibitors

• AKT activation is among the most frequently observed genetic alterations in cancer, yet no AKT inhibitors have been approved for cancer treatment. Why?

• Hypothesis generation using Pathway Studio:

Vignette: AKT Inhibitors

!

Vignette: EGFR Inhibitors

• Approvals:

– Iressa/gefitinib (AstraZeneca, Teva); non-small cell lung cancer (NSCLC)

– Erbitux/cetuximab (Bristol-Myers Squibb); KRAS-WT colon cancer

– Tarceva/erlotinib (Astellas, Genentech, OSI, Roche); metastatic, EGFR-positive non-small cell lung cancer (NSCLC)

– Gilotrif/afatinib (Boehringer-Ingelheim); metastatic, non-small cell lung cancer (NSCLC)

– Vectibix/panitumumab (Abgenix, Amgen); EGFR-positive colon cancer

• Pathway Studio: – Are text mining results consistent with approval of EGFR inhibitors for

clinical use?

Vignette: EGFR Inhibitors

No serious concerns

Vignette: MEK Inhibitors

• Approval: – 2013: Mekinist/trametinib (GSK); MEK1 and MEK2 co-inhibitor; metastatic

melanoma with BRAF V600 mutations (V600E or V600K)

• Pathway Studio: – Are text mining results consistent with approval of MEK inhibitors for

clinical use?

Vignette: MEK Inhibitors

No serious concerns

Vignette: RANKL Inhibitors

• Approvals:

– 2013: Xgeva/denosumab (Amgen); mAb; giant cell tumor of bone

• Pathway Studio: – Are text mining results consistent with approval of RANKL inhibitors for

clinical use?

Vignette: RANKL Inhibitors

No serious concerns

Vignette: PARP1 Inhibitors

• First agent approved (2014); many others in development • Pathway Studio:

– Are text mining results consistent with approval of PARP1 inhibitors for clinical use?

Vignette: PARP Inhibitors

! !

!

! !

! ! !

!

! ! ! ! !

Vignette: GLS Inhibitors

• In clinical development now • Pathway Studio:

– Are text mining results consistent with approval of GLS inhibitors for clinical use?

Vignette: GLS Inhibitors

No serious concerns

Doing Your Part for the Future of Text Mining

• Facilitate accurate text-mining of your own publications

– Remember: regulation has direction

• not good writing: “Protein X regulates Protein Y.” • good writing: “Protein X positively regulates Protein Y.”

Our Team

David Hawke

Wai Kin Chan Phil Lorenzi

John Weinstein Thomas Horvath

Michael Pontikos

Bih-Fang Pan Ron Bouchard

Daniel Du Yulun Chiu

Bo Peng

Lin Tan

Acknowledgements and Collaborators

John Weinstein Gordon Mills David Hawke

Wai Kin Chan Tom Horvath

Lin Tan Leona Martin

Michael Pontikos Bih-Fang Pan

Yiling Lu Tracy Guo

Jennifer Dennison Pradeep Raghavan

Susan Rempe David Rogers Juan Vanegas

Sergei Sukharev Andriy Anishkin

Yann Godfrin Fabien Gay

Willy Berlier

Bo Peng Daniel Du Yulun Chiu Jing Wang

Bradley Broom Jiexin Zhang

Rehan Akbani Shiyun Ling

Keith Baggerly

Marina Konopleva Gautam Borthakur

Steve Kornblau Ken Aldape

The Chapman Foundation The Michael and Susan Dell Foundation NCI grant CA14883 (TCGA GDAC) NCI grant CA083639 (Ovarian SPORE) CPRIT grant RP130397 MD Anderson Cancer Center

Cliff Stephan David Brunell Peter Davies

+ _

+ _

Bill Reinhold Natasha Caplen Yves Pommier Sudhir Varma

Chris Beecher Felice de Jong

MDACC

MDACC

MDACC

MDACC

Sandia/U Maryland

Texas A&M NCI

Erytech Pharma

Elsevier

IROA Technologies

Virginia Heatwole Travis Vaught Chris Cheadle

Jaqui Hodgkinson Dave Arndt

ThermoFisher

Ken Miller Bob Swaim

Jamie Humphries Bernard Delanghe

Health & Medicine

Pathway studiosymposium lorenzi