Abstract 5886: High-throughput mutagenesis reveals novel mechanisms of drug resistance in the proto-oncogene Src kinase
Authors: Ahler Ethan , Chakraborty Sujata , Fan Linglan , Maly Dustin J. , Fowler Douglas M. 
Published: July 2017

Kinase inhibitors are integral for the treatment of certain cancers, but the emergence of drug resistance limits their long-term effectiveness. Resistance can arise through multiple routes, including via mutations in the kinase that render it insensitive to drug. Although some resistance mutations for a few inhibitors are well characterized, most remain uncharacterized or even unidentified, particularly in new or emerging kinase targets. Ideally, we could characterize every possible resistance mutation that could arise during treatment with each inhibitor. The resulting comprehensive data could improve clinical decision-making for patients with drug-resistant tumors. However, current experimental methods for discovering resistance mutations cannot achieve the required scale, preventing us from realizing this goal.

We developed a method for parallel assessment of the degree of inhibitor resistance of nearly all possible single mutants of the promising drug target Src kinase. We generated a comprehensive single mutant library of Src and subjected it to selection for Src activity either alone or in the presence of the inhibitor dasatinib. Then, we measured the change in frequency of each mutant during selection by deep sequencing.

Using this data, we classified the impact of each Src mutant, both in terms of activity and resistance to dasatinib. Our method identified dozens of previously uncharacterized dasatinib-resistance mutations in Src. As expected, some of these novel resistance mutations cluster near the ATP-binding cleft of the catalytic domain. However, a surprisingly large number occur at residues distal to dasatinib’s binding site. Biochemical validations of a subset of these newly identified resistance mutants are ongoing. Although dasatinib is not currently approved for Src inhibition, these results could be useful to direct treatment for those patients currently enrolled in dasatinib clinical trials.

Our results demonstrate the power of our method for the characterization of resistance mutations in drug targets. In contrast with previous low-throughput and retrospective methods, our approach enables the comprehensive and prospective identification of resistance mutations. The approach can reveal resistance mechanisms, even before a drug is clinically approved. Importantly, our method allows for the rapid profiling of other Src inhibitors and potentially the discovery of inhibitor-specific resistance mutations. Finally, we are using our data to explore hypotheses regarding the structure, function, or regulation of Src kinase.

Note: This abstract was not presented at the meeting.

Citation Format: Ethan Ahler, Sujata Chakraborty, Linglan Fan, Dustin J. Maly, Douglas M. Fowler. High-throughput mutagenesis reveals novel mechanisms of drug resistance in the proto-oncogene Src kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5886. doi:10.1158/1538-7445.AM2017-5886