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PDF file, 289KB, Western immunoblots and their densitometric analyses showing the pharmacodynamic response of GIST430 xenograft to 22 days of AT13387, imatinib or combination treatment.

PDF file, 56KB, Graphs showing the typical dose response of GIST882, GIST430 and GIST48 cell lines to AT13387 and imatinib.

The majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that HSP90 inhibition might be a valuable treatment option for GIST, which would be equally effective on imatinib-sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently being evaluated in clinical trials, in both in vitro and in vivo GIST models. AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to the geldanamycin analogue HSP90 inhibitor, 17-AAG. Treatment with AT13387 resulted in depletion of HSP90 client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines, irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW, xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70, depletion of phospho-KIT and inhibition of KIT signaling were seen in tumors from both models after treatment with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model significantly enhanced tumor growth inhibition over either of the monotherapies. Importantly, the combination of AT13387 and imatinib was well tolerated. These results suggest AT13387 is an excellent candidate for clinical testing in GIST in combination with imatinib. Mol Cancer Ther; 11(8); 1799–808. ©2012 AACR.

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 >1000 300 GIST430: KIT Ex 11 + V654A (ABP) 20 300 >1000 Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B184.