Targeting Gain of Function and Resistance Mutations in Abl and KIT by Hybrid Compound Design

Mutations in the catalytic domain at the gatekeeper position represent the most prominent drug-resistant variants of kinases and significantly impair the efficacy of targeted cancer therapies. Understanding the mechanisms of drug resistance at the molecular and atomic levels will aid in the design and development of inhibitors that have the potential to overcome these resistance mutations. Herein, by introducing adaptive elements into the inhibitor core structure, we undertake the structure-based development of type II hybrid inhibitors to overcome gatekeeper drug-resistant mutations in cSrc-T338M, as well as clinically relevant tyrosine kinase KIT-T670I and Abl-T315I variants, as essential targets in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). Using protein X-ray crystallography, we confirm the anticipated binding mode in cSrc, which proved to be essential for overcoming the respective resistances. More importantly, the novel compounds effectively inhibit clinically relevant gatekeeper mutants of KIT and Abl in biochemical and cellular studies

[¹⁸F]FLT PET indicates response to therapy after 2 days of erlotinib treatment.

<p>In (A) a representative [¹⁸F]FLT PET image of a mouse bearing the sensitive PC9, HCC827 and the resistant H1975 xenografts before beginning of treatment, 48h and 96h after daily erlotinib treatment (Tarceva, 50mg/kg). (B) Quantitative analysis of changes in [¹⁸F]FLT and [¹⁸F]FDG uptake ratios after 48h and 96h daily erlotinib treatment vs. vehicle only as control (PC9: n = 8; vehicle, n = 2; HCC827: n = 7; vehicle, n = 2; H1975: n = 8; vehicle, n = 2).</p

Immunohistochemistry of tumor tissue for Ki-67 expression and TUNEL, relation of [¹⁸F]FLT and [¹⁸F]FDG uptake to Ki-67 expression, and measurement of tumor volume for the assessment of treatment response.

<p>(A) Frozen tissue was stained for Ki-67 and TUNEL (magnification 10×). Columns, average number of TUNEL positive cells (green cells) were counted in three randomly selected field (area 0.625mm²) in two tumor samples for each cell line. The Ki-67 labeling index as assessed by the percentage of nuclei stained with MIB-1 per total number of nuclei was compared to uptake ratios of [¹⁸F]FLT and [¹⁸F]FDG (B). Effects of daily Erlotinib treatment on the tumor size of the xenografts for the assessment of tumor response (C).</p

Erlotinib treatment induces down-regulation of EGFR/EGFR-coupled signaling pathways and cell cycle arrest with subsequent induction of apoptosis in EGFR sensitive tumor cells.

<p>The erlotinib sensitive cell lines HCC827 and PC9 and the erlotinib-resistant cell line H1975 were treated with the indicated doses of erlotinib for 24 hours. Whole-cell lysates were subjected to immunoblotting with the indicated antibodies (A). PC9, HCC827 and H1975 cells were treated with erlotinib (0.5 µM) for 24h, 48h and 72h; nuclei were prepared, stained with propidium iodide and analyzed by flow cytometry. Results are shown for the G1 and S phases of the cell cycle (A). Apoptotic effects of erlotinib on EGFR-sensitive cell lines in comparison to the T790M mutant H1975 (B). Annexin V FACS was performed 12h, 24h, 36h, 48h, 72h and 96h after 0.5 µM erlotinib treatment. Images show Annexin V-positive cells after 48h in the different cell lines.</p