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Annual GIST Breakfast Heinrich & Corless Laboratories Knight Cancer Institute Oregon Health & Science University 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 Ion Torrent PGM • Massively parallel (next-gen) sequencing • Performed on a semiconductor chip www.iontorrent.com Cancer Gene Panels Based on Semiconductor Sequencing Panel Non-small cell lung ca panel GI stromal tumor panel AML / MDS panel General solid tumor panel (Melanoma, colorectal ca, many others) Lymphoma / myeloma panel # Genes Availability 23 Now available 23 Now available 42 Now available 37 TBD Now available Fall 2013 GIST Panel Using Next-Gen DNA Sequencing AKT1 AKT2 AKT3 ATM BRAF CDKN2A HRAS KIT KRAS MAP2K1 NF1 NRAS PDGFRA PIK3CA PTEN PTPN11 RB1 SDHA SDHAF1 SDHAF2 SDHB SDHC SDHD TP53 Cell Cycle and Wild-Type GIST Projects Carol Beadling, Ph.D. Nancy Collias, Ph.D. Marina Pukay • Cell Dylan Nelson Tanaya Neff Rebecca O’Gara 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 CarneyStratakis syndrome Molecular Subtypes of GIST KIT Exon 8 0.1% KIT Exon 17 1% KIT Exon 13 2% KIT Exon 9 8% PDGFRA Exon 12 2% PDGFRA Exon 14 0.1% PDGFRA Exon 18 other 3% Crenolanib(?) KIT Exon 11 65% SHDA/B/C/D Mutation (Carney-Stratakis) 6% Sunitinib Sorafenib Regorafenib Vemurafenib BRAF 2% RAS gene mutation NF1 0.1% 0.2% Moving Forward Ion Torrent PGM 4-8 samples per chip Ion Torrent Proton 40-80 samples per 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 QUADRUPLE Negative GIST SDH DEFICIENT GIST SDH mutation NO SDH mutation No RAS-P/No SDH NF-1 RAS-BRAF SDHB+ SDHB+ SDHB- SDHB- SDHB+ IGF1R- IGF1R- IGF1R+ IGF1R+ To be defined Young Adults/ Adults Adults Pediatric/Young Adults Equal sex Equal sex Prevalence of female Multifocal Small intestine No multifocal Gastric/Small intestine Often Multifocal (?) Gastric Lymph nodes metastases Adults Prevalence of female No multifocal Gastric (?) Any age? To be defined To be defined Any site? To be defined Kinase Inhibitor Screening Team Diana Griffith Arin McKinley Janice Patterson Ajia Town • Goals • To characterize the activity of novel kinase inhibitors against GISTassociated 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.) 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 Lilli Klug, future Ph.D. Alison Macleod Ph.D. Imatinib Treatment of Metastatic GIST: How Long? Resistance Persistence Le Cesne et al. Lancet Oncology. 2010;11:949. Primary Mutations Secondary Mutations Protein Domain Drug Sensitivity IM SU REG Ligand binding Exon 13 V654A Exon 14 T670I Exon 9 : 12% Exon 11: 70% JM Membrane D816A/G/H/V Exon 13: 1% ATP binding K642E Exon 17: 1% N822H/K, D820Y D820A/E/G/Y Exon 17 N822H/K Y823D Activation Loop Exon 18 NR A829P Resistant Intermediate Sensitive NR Not reported GIST Stem and Progenitor Cells are Resistant to Imatinib Mature GIST GIST Progenitors GIST Stem Cells KIT-dependent, Imatinib-sensitive Adapted from Heinrich et al. Lancet Oncology 2010 KIT-independent, Imatinib-resistant Causing a “Traffic Jam” inside a Cancer Cell 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 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 Analysis of RNA-Seq Data RNA-Seq Data 20,207 targets Removed uncharacterized and mimimally expressed genes 9,164 targets B. Removed targets with expression 0.5<x <2.0 or p-value >0.002 378 targets Removed targets not associated with the KIT/NFAT network 49 targets KEGG Pathway analysis 4 target pathways Alison Macleod, Ph.D. Carol Beadling, Ph.D. Janice Patterson, Grad student Lilli Klug, future Ph.D. 50 random genes whose expression was not affected by combination therapy JAK-STAT pathway down regulated upon KIT inhibition • Identified abundance of JAKSTAT pathway targets 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 p value= 0.00097 Alison Macleod 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 SDH Deficient GIST KIT Exon 8 0.1% KIT Exon 17 1% KIT Exon 13 2% KIT Exon 9 8% PDGFRA Exon 12 2% PDGFRA Exon 14 0.1% PDGFRA Exon 18 other 3% KIT Exon 11 65% SHDA/B/C/D Mutation (Carney-Stratakis) 6% BRAF 2% RAS gene mutation NF1 0.1% 0.2% SDH Deficient GIST Smith et al., Human Molecular Genetics, 2007 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 SDH Deficient GIST: genome wide DNA hypermethylation SDH deficient Killian et al., Cancer Discovery 2013 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 • 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 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 Cell Growth Assay 4.5 WT pRS416 sc-ura+Dextrose 4 ΔSDH1 pRS416 sc-ura+Dextrose 3.5 OD at 600nm 3 ΔSDH2 pRS416 sc-ura+Dextrose 2.5 ΔSDH1+ pRS416 SDH1 sc-ura+Dextrose 2 WT pRS416 sc-ura+Glycerol 1.5 1 ΔSDH1 pRS416 sc-ura+Glycerol 0.5 ΔSDH2 pRS416 sc-ura+Glycerol 0 0 -0.5 50 100 150 200 Hours 250 300 350 ΔSDH1+ pRS416 SDH1 sc-ura+Glycerol Metabolic Consequences of SDH Deficiency in Yeast SDH Activity Percent of WT 120 100 80 60 40 20 0 WT Δsdh1 Δsdh2 Clinical Study Team Tracy Walker Research Nurse Lindsay Chandler Research Coordinator Tamara Olenyik Research Coordinator Wes Wenzel Research Assistant Lindsay Overton Oncology Fellow 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 [email protected] THANK YOU FOR YOUR GENEROUS SUPPORT!!