Mechanisms of sunitinib malate (SU) resistance in gastrointestinal stromal tumors (GISTs)

10006 Background: SU, an oral, multitargeted inhibitor of KIT, PDGFRs, VEGFRs, FLT3, and RET receptor tyrosine kinases, is now the standard therapy for treatment of imatinib mesylate (IM)-resistant GIST tumors or IM-intolerant GIST patients. However, it is unclear if the clinical efficacy of SU in this setting is due to inhibition of KIT and/or PDGFRA in tumor cells, inhibition of VEGFRs and PDGFRs in endothelial cells and pericytes, respectively, or a combination of antitumor and antiangiogenic effects. In order to address this question, we studied the molecular mechanisms of SU resistance in vitro and in patient-derived tumors. Methods: We studied the in-vitro effects of IM or SU on cells with KIT exon 11 mutations, either alone or in combination with known IM-resistant secondary mutations. In addition, we analyzed tumor samples obtained from patients undergoing salvage surgery after SU treatment failure. Tumor specimens were genotyped for primary and secondary mutations. Results: SU was approximately 2–5-fold more potent than IM against typical GIST-associated KIT exon 11 mutations. In addition, the presence of secondary mutations involving the ATP/drug binding pocket (V654A or T670I) did not substantially alter SU potency. In contrast, these ATP/drug-binding-pocket mutations conferred high-level resistance to IM. However, SU had no significant inhibitory effect on the kinase activity of secondary IM-resistant mutations involving the KIT activation loop (exon 17, codons 816, 820, 822, and 823). Surgical resection was performed on two GIST patients who had clinical progression on SU. Among nine progressing lesions, substitutions were detected in codons 816, 820, and 822; a novel mutation, L783V, was also identified. In contrast, two non-progressing tumors both contained a V654A secondary mutation. Conclusions: Consistent with in-vitro studies, acquired IM-resistant mutations of the KIT activation loop in GIST are associated with clinical resistance to SU and IM. In addition, novel kinase mutations not previously associated with IM resistance may contribute to clinical SU resistance. These findings suggest that the antiangiogenic effects of SU may be insufficient to inhibit GIST progression when the targeted oncogenic kinase remains active. [Table: see text] </jats:p

Abstract B184: HSP90 inhibitor STA-9090 potently suppresses heterogeneous KIT kinase-domain mutations responsible for gastrointestinal stromal tumor progression during imatinib therapy

Abstract Background: Most GISTs express mutant KIT or PDGFRA oncoproteins, which are targets of tyrosine kinase inhibitors (TKIs), such as front-line imatinib (IM) or second-line sunitinib (SU). GIST clinical resistance to IM or SU is commonly associated with the acquisition of heterogeneous secondary mutations in the KIT/PDGFRA ATP-binding pocket (ABP) or activation loop (AL), which maintain the constitutively activated state of these kinases. We therefore asked whether the heterogeneous IM-resistant KIT oncoproteins in GIST are uniformly HSP90 clients, and whether they can be inhibited by STA-9090, a synthetic small molecule HSP90 inhibitor that is structurally unrelated to the first-generation natural product-derived ansamycin HSP90 inhibitor 17-AAG. Methods: KIT and PDGFRA were genotyped in up to 15 metastases from each of 10 patients whose metastatic GIST had progressed after IM therapy. Kinase mutants were biochemically profiled for IM and STA-9090 sensitivity in: 1. Ba/F3 cells transformed by mutant KIT constructs; 2. GIST cell lines (IM-sensitive and IM-resistant); and 3. a novel assay measuring inhibition of kinase phosphorylation after drug treatment in GIST48B (KIT-negative) GIST cells transfected with mutant KIT constructs. Drug effects on proliferation, apoptosis and cell cycle were evaluated in five GIST cell lines, including a KIT-dependent GIST subline (GIST882B) that is resistant to 17-AAG. Results: As many as 8 different secondary KIT IM-resistance mutations (both ABP and AL) were detected in individual patients whose GISTs progressed after IM therapy. All mutations were sensitive to STA-9090. STA-9090 was 5-15 fold more potent than 17-AAG against these IM-resistant KIT secondary mutations, and was at least as effective against the primary + secondary (IM-resistant) mutations in combination, as compared to the primary IM-sensitive mutation alone (Table). STA-9090 also potently inhibited the 17-AAG resistant GIST882B cell line. Conclusions: STA-9090 was more potent than 17-AAG against a panel of KIT mutations found in TKI-resistant GISTs. STA-9090 potency was undiminished against combination activating KIT mutations (including secondary kinase-domain resistance mutations), as typically occur in IM-resistant GIST clones. Based on these results, we hypothesize that STA-9090 might have broad clinical activity against IM-resistant GIST. Table: IC50 (nM) for GIST viability: HSP90-inhibition vs. imatinib GIST Line STA-9090 17-AAG Imatinib GIST882: KIT Ex 13 40 200 300 GIST882B 35 &amp;gt;1000 300 GIST430: KIT Ex 11 + V654A (ABP) 20 300 &amp;gt;1000 Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B184.</jats:p