Annual GIST Breakfast

1. Annual GIST Breakfast Heinrich & Corless Laboratories Knight Cancer Institute Oregon Health & Science University

2. Topics • 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

3. Ion Torrent PGM • Massively parallel (next-gen) sequencing • Performed on a semi-conductor chip www.iontorrent.com

4. Cancer Gene PanelsBased on Semiconductor Sequencing

5. GIST Panel Using Next-Gen DNA Sequencing

6. Cell Cycle and Wild-Type GIST Projects Carol Beadling, Ph.D. Nancy Collias, Ph.D. • 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

7. Molecular Subtypes of GIST Imatinib Crenolanib(?) Sunitinib Sorafenib Regorafenib Vemurafenib

8. Moving Forward Ion Torrent PGM Ion Torrent Proton 40-80 samples per chip 4-8 samples per chip

9. When is a WT GIST not a WT GIST?

10. 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 Comparison of Subgroups of KIT/PDGFRA WT GIST 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

11. 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.) Ajia Town Janice Patterson Diana Griffith Arin McKinley

12. 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 ponatinibagainst GIST-associated mutations • Lead to phase 2 study that opened June 2013 (OHSU, Dana Farber, FCCC) • Preliminary results to be presented ASCO 2014

13. Combination Therapy Lilli Klug, future Ph.D. Alison MacleodPh.D.

14. Imatinib Treatment of Metastatic GIST: How Long? Resistance Persistence Le Cesne et al. Lancet Oncology. 2010;11:949.

15. Secondary Mutations Drug Sensitivity Primary Mutations Protein Domain IM SU REG Exon 13 V654A Ligand binding Exon 14 T670I Exon 9 : 12% Membrane Exon 11: 70% JM D816A/G/H/V Exon 13: 1% K642E ATP binding D820A/E/G/Y Exon 17 N822H/K Y823D NR Activation Loop Exon 17: 1% N822H/K, D820Y A829P Exon 18 Resistant Intermediate Sensitive NR Not reported

16. GIST Stem and Progenitor Cells are Resistant to Imatinib Mature GIST GIST Progenitors GIST Stem Cells KIT-dependent, Imatinib-sensitive KIT-independent, Imatinib-resistant Adapted from Heinrich et al. Lancet Oncology 2010

17. Causing a “Traffic Jam”inside a Cancer Cell

18. Finding Combination Treatments to kill KIT-mutant cells DNA  RNA  Protein Analysis using RNASEQ (analogous to counting a mountain made up of 50 million marbles) 24,000 different types of marbles = 24,000 different genes 50 million marbles to be counted and sorted into 24,000 categories Problem: How to identify changes in expression induced by therapy

19. How does combination therapy kill KIT-mutant cells: Analysis using RNASEQ Combination therapy Blue 3/20 Pink 4/20 Gray 2/20 Green 3/20 Yellow 2/20 Blue 3/20 Pink 8/20 Gray 1/20 Green 1/20 Yellow 2/20

20. Analysis of RNA-Seq Data B. Alison Macleod, Ph.D. Carol Beadling, Ph.D. Janice Patterson, Grad student Lilli Klug, future Ph.D.

21. 50 random genes whose expression was not affected by combination therapy

22. JAK-STAT pathway down regulated upon KIT inhibition • Identified abundance of JAK-STAT pathway targets p value= 0.00097 Alison Macleod

23. Inhibiting the JAK-STAT Pathway Synergizes with KIT Kinase Inhibitors

24. Yeast Model of SDH-deficient GIST: Amber Bannon, future Ph.D. E Smith et al., Human Molecular Genetics 16:3136, 2007

25. SDH Deficient GIST

26. SDH Deficient GIST Smith et al., Human Molecular Genetics, 2007

27. SDH Deficient GIST 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 Yang H et al. Clin Cancer Res 2012;18:5562-5571

28. SDH Deficient GIST:genome wide DNA hypermethylation SDH deficient Kinase mutant Killian et al., Cancer Discovery 2013

29. 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 Normal SDH deficient Severe mutation Normal SDH deficient Moderately severe mutation Normal SDH deficient Normal variant Normal variant Normal variant Panizza E et al. Hum. Mol. Genet. 2013;22:804-815

30. 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

31. Metabolic Consequences of SDH Deficiency in Yeast

32. Metabolic Consequences of SDH Deficiency in Yeast

33. Clinical Study Team Lindsay Chandler Research Coordinator Tracy Walker Research Nurse Tamara Olenyik Research Coordinator Lindsay Overton Oncology Fellow Wes Wenzel Research Assistant

34. 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) • 503-346-1183 • walkertr@ohsu.edu

35. THANK YOU FOR YOUR GENEROUS SUPPORT!!