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Abstract
The emergence of drug resistance is a primary concern in any cancer treatment, including with targeted kinase inhibitors as exemplified by the appearance of Bcr-Abl point mutations in chronic myeloid leukemia (CML) patients treated with imatinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations. To predict resistance mutations in the receptor tyrosine kinase MET that could emerge during inhibitor treatment in patients, we conducted a resistance screen in BaF3 TPR-MET cells using the novel selective MET inhibitor NVP-BVU972. The observed spectrum of mutations in resistant cells was dominated by substitutions of tyrosine 1230 but also included other missense mutations and partially overlapped with activating MET mutations that were previously described in cancer patients. Cocrystallization of the MET kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972, as previously reported for multiple other selective MET inhibitors. A second resistance screen in the same format with the MET inhibitor AMG 458 yielded a distinct spectrum of mutations rich in F1200 alterations, which is consistent with a different predicted binding mode. Our findings suggest that amino acid substitutions in the MET kinase domain of cancer patients need to be carefully monitored before and during treatment with MET inhibitors, as resistance may preexist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a condition that may be addressed by MET inhibitors with alternative binding modes.
Highlights
Inhibition of oncogenic tyrosine kinases with small molecules is a commonly pursued approach for cancer therapy.Authors' Affiliations: 1Disease Area Oncology, 2Global Discovery Chemistry, and 3Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland; 4Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, California; 5Novartis Oncology, Translational Medicine; and 6Disease Area Oncology, Novartis Institutes for BioMedical Research, Cambridge, MassachusettsNote: Supplementary data for this article are available at Cancer Research Online.Current address for E
NVP-BVU972 is a selective MET kinase inhibitor NVP-BVU972 (Fig. 1A) was synthesized in the context of a medicinal chemistry program that was aimed at identifying selective MET inhibitors
In an attempt to prospectively identify resistance mutations affecting the novel MET inhibitor NVP-BVU972, we carried out a cellular drug resistance screen in TPR-MET–transformed BaF3 cells and detected a large proportion of Y1230 mutations
Summary
Inhibition of oncogenic tyrosine kinases with small molecules is a commonly pursued approach for cancer therapy. Mutated Bcr-Abl constructs were expressed in BaF3 cells to select inhibitor-resistant clones One advantage of this method is that one can estimate the complexity of the library of mutants and the coverage of the respective kinase. The strategy we used was based on the previously described Bcr-Abl ENU mutagenesis screens in BaF3 cells [9, 10], in this case using BaF3 cells driven by the oncogenic TPR-MET fusion gene, in which the MET kinase domain is constitutively activated These cells are strictly dependent on MET kinase activity [14] and highly sensitive to MET inhibitors like NVP-BVU972. The observed mutation spectra largely correlated with the results obtained by traditional sequencing with variants detected at frequencies as low as 0.1%
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