9034 Background: Many GIST pts respond to KIT inhibition with imatinib (IM), yet eventually develop resistance. IM resistance mechanisms are heterogeneous, and little is known about KIT functional roles in IM-resistant GIST. Methods: GIST cell lines were established from biopsy specimens. Biological consequences of KIT, PI3-K, PLCgamma, MEK/MAPK and mTOR inhibition were determined by immunoblotting for protein activation, and by cell proliferation and apoptosis assays. Results: Novel cell lines, GIST430 and GIST48, were developed from GISTs progressing clinically on IM. These lines contain primary IM-sensitive KIT juxtamembrane mutations and secondary IM-resistance mutations in the ATP-binding (GIST430) and catalytic (GIST48) portions of the KIT kinase domain. Kinase activation was compared with GIST882 cells, which contain only a primary IM-sensitive mutation. KIT expression was equally strong in the 3 GIST cell lines, whereas KIT activation was 3-to-6 fold higher in GIST430 and GIST48 than in GIST882. In addition to the higher set point for KIT activation, GIST430 and GIST48 had intrinsic IM-resistance: IM IC50s for KIT activation were 1.0, .2 and .1 μM in GIST430, GIST48 and GIST882, respectively. After treatment with 1 μM IM, residual KIT activation was 6 and 2.8-fold higher in GIST430 and GIST48, respectively, compared to GIST882. Both GIST430 and GIST48 were sensitive to the alternate KIT inhibitor, PKC412. PKC412 IC50s for KIT activation were .1, .1, and >1.0 μM, and for proliferation were .4, .25, and 1.0 μM, in GIST430, GIST48 and GIST882, respectively. In all GIST lines, cell growth arrest resulted from PI3-K inhibition, and - to a lesser extent - from MEK/MAPK and mTOR inhibition (Table). Conclusions: GIST secondary KIT mutations can be associated with KIT hyperactivation and IM resistance. IM resistance may be overcome with alternate KIT inhibitors, or by targeting critical downstream signaling proteins. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis Novartis Novartis