The effect of crenolanib (CP-868596) on phosphorylation of the imatinib-resistant D842V PDGFRA activating mutation associated with advanced gastrointestinal stromal tumors.

Abstract

10012 Background: 8-10% of gastrointestinal stromal tumors (GIST) have activating mutations of the platelet-derived growth factor receptor alpha (PDGFRA) gene. The most common PDGFRA mutation is the D842V mutation encoded by exon 18. This gain-of-function mutation results in constitutive tyrosine kinase activity. Currently approved tyrosine kinase inhibitors (TKIs), such as imatinib (IM) or sunitinib, have little to no in vitro activity against this mutation. Clinically, these drugs are ineffective for the treatment of patients whose GIST has a D842V mutation. In addition, the D842V mutation can develop as a secondary IM resistance mutation during treatment of patients with primary IM-sensitive GIST or hypereosinophillic syndrome. Crenolanib (formerly CP-868596) is an orally bioavailable, highly potent and selective PDGFR TKI. Phase I trials of crenolanib (CR) have shown a favorable toxicity profile, and achievable serum concentrations as high as 2,000 nM. Methods: Mutant PDGFRα isoforms were expressed by transient transfection of Chinese hamster ovary cells. The transfected cells were treated with various concentrations of CR or IM. Protein lysates were immunoprecipitated with an anti-PDGFRA antibody, followed by sequential immunoblotting for activated- and total-PDGFRA. IC50 (50% inhibitory concentration) was measured by densitometry of the phosphoPDGFRA bands and normalization using total PDGFRA expression. Results: CR was effective in blocking the activity of single or compound PDGFRA D842V mutant kinases. In contrast, IM had no significant activity against these same mutant kinases. Conclusions: GISTs with PDGFRA D842V mutations are clinically resistant to currently approved TKIs. CR blocks the kinase activity of PDGFRA D842V mutants (single and compound) at clinically achievable concentrations, providing a potential new therapeutic modality for GIST patients. A phase II clinical study of CR for treatment of GIST patients with primary or secondary PDGFRA D842V mutation is currently being initiated. Mutation CR IC50(nM) IM IC50(nM) PDGFRA WT 13 30 PDGFRA D842V 20 >1,000 PDGFRA V561D + D842V 35 >1,000 PDGFRA T674I + D842V 26 >1,000