Annual GIST Breakfast

Heinrich & Corless LaboratoriesKnight Cancer Institute

Oregon Health & Science University

• An review of projects and the people working on them

• Corless group:• Wild-type GIST project• Heinrich group:• Drug screening team • Combination therapy • Yeast model of SDH mutant GIST• Clinical research program

Topics

Ion Torrent PGM

• Massively parallel (next-gen) sequencing

• Performed on a semi-conductor chip

www.iontorrent.com

Cancer Gene PanelsBased on Semiconductor Sequencing

Panel # Genes

Availability

Non-small cell lung ca panel

23 Now available

GI stromal tumor panel 23 Now availableAML / MDS panel 42 Now availableGeneral solid tumor panel(Melanoma, colorectal ca, many others)

37 Now available

Lymphoma / myeloma panel

TBD Fall 2013

AKT1 KRAS RB1AKT2 MAP2K1 SDHAAKT3 NF1 SDHAF1ATM NRAS SDHAF2BRAF PDGFRA SDHBCDKN2A PIK3CA SDHCHRAS PTEN SDHDKIT PTPN11 TP53

GIST Panel Using Next-Gen DNA Sequencing

Cell Cycle and Wild-Type GIST Projects

• Cell cycle project • Collaboration with Jonathan Fletcher’s group, and others• Analyzing ~85 GISTs to determine the relationship between

malignant behavior and alterations in the following genes: TP53, RB1, CDKN2A

• Goal: be able to predict which GISTs are most likely to come back after initial surgery

• Wild-type GIST project• Collaboration with several groups in Europe• Analyzing ~40 WT GISTs to determine what genes are driving

their growth• Includes the SDH genes, which have recently been implicated in

Carney-Stratakis syndrome

Marina Pukay Dylan Nelson Tanaya Neff Rebecca O’Gara

Carol Beadling, Ph.D.

Nancy Collias, Ph.D.

KIT Exon 1165%

KIT Exon 80.1%

KIT Exon 98%

KIT Exon 132%KIT Exon 17

1%PDGFRA Exon 12

2%

PDGFRA Exon 140.1%

PDGFRA Exon 18 other

3%

PDGFRA D842V

5%

SHDA/B/C/D Mutation (Carney-Stratakis)

6%Loss of SDHB ex-pression6%

BRAF2%

RAS gene mutation0.1%

NF10.2%

Molecular Subtypes of GIST

Imatinib

Crenolanib(?)

SunitinibSorafenib

Regorafenib

Vemurafenib

Moving Forward

Ion Torrent ProtonIon Torrent PGM 4-8 samplesper chip

40-80 samplesper chip

When is a WT GIST not a WT GIST?

Mutations Found using NGS Panel in 32 WT GIST Specimens from a Clinical Study

No mutation (n=19)KIT (n=4)NF1 (n=6)SDH (n=3)

Comparison of Subgroups of KIT/PDGFRA WT GIST

RAS-P MUTANT GIST

NF-1 RAS-BRAF

SDHB+ SDHB+

IGF1R- IGF1R-

Young Adults/ Adults Adults

Equal sex Equal sex

Multifocal No multifocal

Small intestine Gastric/Small intestine

QUADRUPLE Negative GIST

No RAS-P/No SDH

SDHB+

To be defined

Any age?

To be defined

To be defined

Any site?

To be defined

SDH DEFICIENT GIST

SDH mutation NO SDH mutation

SDHB- SDHB-

IGF1R+ IGF1R+

Pediatric/Young Adults Adults

Prevalence of female Prevalence of female

Often Multifocal No multifocal (?)

Gastric Gastric (?)

Lymph nodes metastases

Kinase Inhibitor Screening Team

• Goals• To characterize the activity of novel kinase inhibitors against

GIST-associated KIT mutations (or downstream signaling pathways) using cell lines expressing different KIT mutations

• help select promising agents to move into GIST clinical studies

• To collaborate with other GIST biologists to more rapidly evaluate promising agents and novel targets

• Academic labs (Fletcher, Debiec-Rychter, Druker, etc.)• Pharma companies (Novartis, Ariad, AROG, etc.)

Diana Griffith Arin McKinley Janice Patterson Ajia Town

Kinase Inhibitor Screening Team:Major Accomplishments 2013-2014

• Profiling of regorafenib against GIST-associated mutations• Correlation with outcomes in patients treated as part

of phase 2 study• Study of post-regorafenib surgery or biopsy

specimens to identify mechanisms of regorafenib-resistance

• Profiling of ponatinib against GIST-associated mutations• Lead to phase 2 study that opened June 2013 (OHSU,

Dana Farber, FCCC)• Preliminary results to be presented ASCO 2014

Combination Therapy

Alison Macleod Ph.D.Lilli Klug, future Ph.D.

Imatinib Treatment of Metastatic GIST: How Long?

Le Cesne et al. Lancet Oncology. 2010;11:949.

Persistence

Resistance

PrimaryMutations

Exon 13: 1%K642E

Exon 9 : 12%

Exon 11: 70%

Exon 17: 1%N822H/K, D820Y

ProteinDomain

Ligand binding

JM

ATP binding

Activation Loop

Exon 13 V654A

T670I

D816A/G/H/V

D820A/E/G/YN822H/KY823D

IM SU

A829P

Secondary Mutations

DrugSensitivity

Exon 14

Exon 17

Exon 18

Membrane

ResistantIntermediateSensitive

REG

NR

NR Not reported

GIST Stem Cells

GIST Progenitors

Mature GIST

KIT-dependent, Imatinib-sensitive

KIT-independent, Imatinib-resistant

GIST Stem and Progenitor Cells are Resistant to Imatinib

Adapted from Heinrich et al. Lancet Oncology 2010

Causing a “Traffic Jam”inside a Cancer Cell

Finding Combination Treatments to kill KIT-mutant cells

24,000 different types of marbles = 24,000 different genes50 million marbles to be counted and sorted into 24,000 categoriesProblem: How to identify changes in expression induced by therapy

Analysis using RNASEQ (analogous to counting a mountain made up of 50 million marbles)

DNA RNA Protein

How does combination therapy kill KIT-mutant cells: Analysis using RNASEQ

Blue 3/20Pink 4/20Gray 2/20Green 3/20Yellow 2/20

Blue 3/20Pink 8/20Gray 1/20Green 1/20Yellow 2/20

Combination therapy

Analysis of RNA-Seq Data

KEGG Pathway analysis

4 target pathways

Removed targets not associated with the KIT/NFAT network49 targets

Removed targets with expression 0.5<x <2.0 or p-value >0.002378 targets

Removed uncharacterized and mimimally expressed genes9,164 targets

RNA-Seq Data20,207 targets

B.

Alison Macleod, Ph.D.Carol Beadling, Ph.D.Janice Patterson, Grad studentLilli Klug, future Ph.D.

50 random genes whose expression was not affected by combination therapy

• Identified abundance of JAK-STAT pathway targets

Alison Macleod

JAK-STAT pathway down regulated upon KIT inhibition

p value= 0.00097

KEGG Pathway Target

JAK-STAT MYC, BCL2L1, CCND2, OSM, CISH, SOCS1, IL2RA, IL21R, IL7R, CBLC

“Cancer” FOS, CCND1, VEGFA, MMP2, MYC, BCL2, BCL2L1, CBLC, PDGFRA

Wnt signaling pathway

NFAT1, NFAT2, NFAT3, NFAT4, FOSL, MYC, CCND1, CCND2

MAPK signaling pathway

NFAT1, NFAT3, FOS, TNF, MYC, DDIT3,MYC, PDGFRA, DUSP6

Inhibiting the JAK-STAT Pathway Synergizes with KIT Kinase Inhibitors

Yeast Model of SDH-deficient GIST:

Amber Bannon, future Ph.D.E Smith et al., Human Molecular Genetics 16:3136, 2007

KIT Exon 1165%

KIT Exon 80.1%

KIT Exon 98%

KIT Exon 132%KIT Exon 17

1%PDGFRA Exon 12

2%

PDGFRA Exon 140.1%

PDGFRA Exon 18 other

3%

PDGFRA D842V

5%

SHDA/B/C/D Mutation (Carney-Stratakis)

6%Loss of SDHB ex-pression6%

BRAF2%

RAS gene mutation0.1%

NF10.2%

SDH Deficient GIST

SDH Deficient GIST

Smith et al., Human Molecular Genetics, 2007

SDH Deficient GIST

Yang H et al. Clin Cancer Res 2012;18:5562-5571

Methylated DNA: hypermethylation generally associated with gene silencing, can be inherited during cell division

Methylated histones: can activate or inhibit gene expression depending upon context

SDH Deficient GIST:genome wide DNA hypermethylation

Killian et al., Cancer Discovery 2013

SDH deficient Kinase mutant

Yeast Model of SDH-deficient GIST:Why use Yeast?

• There are no SDH deficient GIST cell lines• In contrast, there are established yeast strains with deficiency of

SDHA, SDHB, etc. • Yeast strains can be easily manipulated to:

• Study effects of gene replacement• Study effects of protein mutation on function including the

evaluation of 15+ novel SDHA/B mutations that we have seen in clinical specimens

NormalSDH deficientSevere mutationNormalSDH deficientModerately severe mutation

NormalSDH deficientNormal variantNormal variantNormal variant

Panizza E et al. Hum. Mol. Genet. 2013;22:804-815

Yeast Model of SDH-deficient GIST:Why use Yeast?

• Yeast can be easily manipulated to study effects of reversible SDH protein loss/replacement on DNA and protein methylation

• Yeast provide a model system to evaluate potential therapies that might reverse the effects of SDH deficiency

• Your experiments will smell good, even if the results stink!

• Saccharomyces cerevisiae = Baker’s yeast = smells like bread

Metabolic Consequences of SDH Deficiency in Yeast

0 50 100 150 200 250 300 350

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Cell Growth Assay

WT pRS416 sc-ura+Dextrose

ΔSDH1 pRS416 sc-ura+Dextrose

ΔSDH2 pRS416 sc-ura+Dextrose

ΔSDH1+ pRS416 SDH1 sc-ura+Dextrose

WT pRS416 sc-ura+Glycerol

ΔSDH1 pRS416 sc-ura+Glycerol

ΔSDH2 pRS416 sc-ura+Glycerol

ΔSDH1+ pRS416 SDH1 sc-ura+Glycerol

Hours

OD

at 6

00nm

Metabolic Consequences of SDH Deficiency in Yeast

WT Δsdh1 Δsdh20

20

40

60

80

100

120

SDH Activity

Perc

ent o

f WT

Clinical Study Team

Lindsay ChandlerResearch Coordinator

Wes WenzelResearch Assistant

Tracy WalkerResearch Nurse

Lindsay OvertonOncology Fellow

Tamara OlenyikResearch Coordinator

Current and Upcoming Clinical Studies• Imatinib/sunitinib/regorafenib resistant GIST: ponatinib

phase 2 will re-open approximately July 1• Imatinib/sunitinib-resistant GIST: phase 1b combination

therapy with imatinib and BYL719 [PI3K inhibitor] is currently open

For more information: Tracy Walker (research nurse) 503-346-1183walkertr@ohsu.edu

THANK YOU FOR YOUR GENEROUS SUPPORT!!