Researchers give update on GIST Resistance Project

By Rick Ware
Member of LRG Science Team

One of the highlights of Life Fest 2006 was a presentation of research updates by several members of the LRG GIST Resistance Research Team. Our GIST Resistance Research Team, led by Jonathan Fletcher, M.D., is made up of world-renowned clinical/research medical experts who share a common focus and ultimate goal – The Defeat of GIST! Dr. Fletcher and this remarkable team of top international GIST Specialists have committed to helping to successfully lead us into the future of GIST cancer treatment. Development of research methodologies that promote international collaboration along with transparent sharing of individual/ collective research excellence and seamless communication between project investigators/laboratories are the guideposts of their novel research paradigm. Everyone works together for success of the project goal – A CURE for GIST!

Presentations from the GIST Resistance Research Team included:
1) Dr. Peter Besmer (Memorial Sloan-Kettering Cancer Center) – “Oncogenic Signaling Mechanisms”
2) Dr. Matt van de Rijn (Stanford University) – “KIT/PDGFRA WT GIST”
3) Dr. Christopher Corless and Dr. Michael Heinrich (Oregon Health and Science University) – “Primary Resistance and Secondary Resistance Mechanisms”
4) Dr. Maria Debiec-Rychter (Catholic University of Leuven, Belgium) – “Stable Disease”
5) Dr. Jonathan Fletcher (GIST Resistance Research Team Leader – Harvard/ Dana-Farber/Brigham and Women’s Hospital) – “KIT Degradation”
6) Dr. Cristina Antonescu (Memorial Sloan-Kettering Cancer Center) – “Gene Profiles in Pediatric GIST”
7) Dr. Brian Rubin (University of Washington and The Cleveland Clinic) – “Murine (mouse) Imatinib Sensitive and Resistant Models”

Dr. Jonathan Fletcher, with his commanding presence and distinguished flowing wispy locks, immediately reminded me of “Doc” Emmett Brown from the movie “Back to the Future.” Dr. Fletcher’s compelling and enthusiastic desire to take us all into the future of GIST treatment was mesmerizing. In his opening remarks, “Doc” as I now affectionately think of him, shared that the Research Team Goal is two fold: Elucidation of the mechanisms of GIST treatment resistance and development of novel therapeutic agents to successfully overcome these resistance mechanisms. Dr. Fletcher sincerely believes that through the concerted efforts of our Research Team, GIST as a disease process will be increasingly medically controllable, if not curable, within 4 to 5 years. What a fantastic thought – “Hope for the Future and a Future with Hope.” Way to go Research Team!

Dr. Peter Besmer (Dr. Fletcher presenting) is the group leader of the Oncogenic Signaling Mechanisms Project. Dr. Fletcher and Dr. Besmer, co-discover of the KIT protein and a pioneer in the study of KIT and its oncogenic signaling mechanisms, shared insights into KIT activation and the concept of resistance heterogeneity. Primary KIT mutations in the exon 9 (dimerization domain) and exon 11 (juxtamembrane domain) with secondary imatinib resistance mutations in the ATP binding site and catalytic site were highlighted. Secondary imatinib resistant mutations may activate downstream pathways via abnormal KIT signaling with subsequent effects on GIST cell survival and proliferation. Potential downstream cascades include the JAK/ STATs, RAS/RAF/MAPK and AKT/ mTOR pathways. Preliminary data from Dr. Fletcher’s laboratory (Dr. Sebastian Bauer, in press) of incubation of GIST882 (imatinib sensitive) and GIST48 (imatinib resistant) cell culture lines with 17-AAG (an HSP90 inhibitor) showed marked reduction/absence of phosphoKIT (activated KIT), phosphoAKT and phospho-S6 activity in both cell culture lines at concentrations above 0.5 uM.

Dr. Maria Debiec-Rychter, group leader of the Stable Disease Project, has done extensive GIST research into the molecular pathogenesis of imatinib response/ resistance mechanisms. Dr. Debiec- Rychter presented research on a GIST xenograft model developed for pre-clinical testing of novel agents. Using this GIST xenograft model (nude mice without hair or immunity that do not reject human tumors), Dr. Debiec- Rychter studied the growth-inhibitory effect of an experimental histone deacetylase (HDAC) inhibitor. HDACs are thought to act in a number of ways, one of which may include degrading KIT, and showed promising results against the murine xenograft GIST tumors. Pathology studies indicated significant effects of HDAC inhibition, as shown by increased apoptotic index (HDAC inhibitor > Imatinib > Control) and decreased mitotic index (Imatinib > HDAC inhibitor > Control). Also, review of the GIST tumor specimens from HDAC treated xenografts showed that HDAC inhibition induced regression with evidence of ischemia, necrosis, arrest of cell growth and induction of cell death. These findings are very interesting but still in the preliminary phase of study.

Dr. Christopher Corless and Dr. Michael Heinrich are group leaders of the Primary and Secondary Resistance Projects. Drs. Corless and Heinrich specialize in development and application of molecular diagnostics for use in the classification of solid tumors. Dr. Corless presented research on an allele specific polymerase chain reaction (AS-PCR) diagnostic assay for the detection of resistance mutations in KIT. This novel diagnostic assay is highly sensitive (1 in 1,000 cells) and will be of use in the detection of low-level resistance mutations in tumor biopsies, and possibly in the analysis of blood samples from patients on imatinib or other inhibitor therapies. Dr. Heinrich is developing a new “library” of cell culture lines that demonstrate mutational resistance to kinase inhibitors. After genotyping and determination of resistance point mutation(s), this new library of resistant cell lines will be cultured for sensitivity testing of combination therapies and new kinase inhibitors.

Dr. Matt van de Rijn, group leader of the KIT/PDGFRalpha Wild-Type GIST Research Project and host of the Adult GIST Tissue Bank has expertise in gene expression profiling, genomic hybridization and tissue microarray analysis. Dr. van de Rijn and his laboratory have developed a new high-through-put gene expression profiling technique that permits the use of paraffin-embedded tumor specimens. Up until now, conventional gene expression profiling methods have required freshly frozen tissue samples for analysis. With Dr. van de Rijn’s new high-through-put gene expression profiling technique, as little as 8 sections of 20 um thick FFPE (formaldehyde-fixed-paraffin embedded) tissue would be needed for analysis. The ability to use FFPE tumor tissue from slides or blocks would allow for almost unlimited availability of GIST research specimens. The key to success of this new high-through-put methodology is use of HEEBO (Human Exonic Evidence based Oligonucleotide DNA Microarray) platform arrays. HEEBO gene arrays are spotted oligonucleotides, which allow for analysis of smaller isolated RNA fragments. Dr. van de Rijn has used this new technique to validate Gleevec-sensitive and Gleevec-resistant GIST cell lines as representative models for drug testing. His laboratory demonstrated that immortal GIST 48 and GIST 882 cell lines cluster with known GIST patient samples. This new technology needs further validation, but the early results look very promising.

Dr. Cristina Antonescu (Dr. Fletcher presenting), group leader of the Pediatric GIST Project and host of the Pediatric GIST Tissue Bank, is investigating pediatric GIST and its imatinib resistance mechanism(s). Dr. Antonescu and her laboratory are developing strategies to successfully treat Pediatric GIST. Dr. Antonescu has shown that most pediatric GIST patients lack KIT or PDGFRA mutations (wild-type) and, according to gene expression profiling, group together (are biologically related) with adult wild-type GIST. Also, KIT is strongly activated in pediatric GIST and demonstrates both activity levels and signaling mechanisms very similar to those observed in adult GIST. Genes that are uniquely upregulated in pediatric GIST include FGF4 (fibroblast growth factor 4), PLAG1 (pleomorphic adenoma gene1) and IGF1R (insulin growth factor 1 receptor). An observed 10- to 20-fold increase in expression of these genes may have direct effects on cellular proliferation. Dr. Antonescu and her Research Team are also developing lab models for testing and evaluating novel KIT inhibitors for activity against wildtype KIT, some of which may inhibit wildtype KIT better than imatinib.

Dr. Brian Rubin, group leader of the Mouse Imatinib and Resistant Models Project has expertise in genomics and proteomics of sarcomas and in the development of mouse sarcoma models. Dr. Rubin working in conjunction with Dr. Besmer is constructing murine (mouse) models that mimic the development and biology of human GISTs as closely as possible. These genetically engineered mouse models will be used to evaluate novel GIST therapies and assist in prioritizing the most promising new GIST drugs for clinical trials in human patients. GIST mouse models would have corresponding KIT and PDGFRA activating mutations similar to those observed in human GIST. In addition, Drs. Rubin and Besmer are also constructing GIST mouse models that harbor secondary resistance mutations. These new GIST mouse lines with secondary resistance will be used to expedite development and testing of novel drugs or combination therapies for imatinib-resistant GIST. “Stuart Little,” Dr. Rubin’s ablebodied lab assistant, served as the model for the newlydeveloped mouse PET Scanner. This new PET Scan methodology will allow for “mini-clinical GIST mouse trials” that permit both timedependent evaluation of drug therapies and harvesting of tissue specimens for additional gene expression profiling and pathologic studies. Stuart was quite the Mouseketeer!

On behalf of our Life Fest 2006 participants, as well as all the members of our extended GIST Family who were unable to join us in Dallas, we extend our personal gratitude and a “very special thank you” to Dr. Daniel Vasella and Novartis AG for their generous financial support and to each member of the GIST Resistance Research Team for helping make this gathering a complete success. Your altruism and compassion, your selfless commitment to each individual patient and your unwavering dedication to research excellence have built a strong foundation upon which “Our Hope for the Future” now stands. Let us all continue to link arms together and pledge to complete our noble task – A Cure for GIST.