Background: TRK inhibition is the standard of care for patients with TRK fusion-positive solid tumors. TRK kinase domain mutations that impair drug binding are common mechanisms of resistance to 1st-generation TRK inhibitors. While 2nd-generation TRK inhibitors were designed to maintain kinase inhibition in this setting, the resistance to these agents is still poorly characterized.

Methods and Results: We sequenced paired tumor biopsies and serial cell-free DNA (cfDNA) collected before therapy and at progression from patients treated with 2nd-generation TRK inhibitors (selitrectinib or repotrectinib). We identified 5 cases in which the acquisition of xDFG (G667) TRKA mutations was associated with resistance. Two patients whose tumors carried these substitutions pre-selitrectinib never responded to therapy, while three additional cases acquired these mutations upon progression to either selitrectinib or repotrectinib.

In-silico molecular modeling combined with molecular dynamic simulations predicted that TRKA xDFG substitutions can confer resistance to 2nd-generation TRK inhibitors by generating steric hindrance that compromises drug binding. Accordingly, in vitro kinase assays showed that the IC50 for selitrectinib of TRKA xDFG mutants was >12 to >8000 fold higher compared to the IC50 of either TRKA wild type or the selitrectinib-sensitive TRKA G595R solvent front mutant.

Interestingly, our data also suggest that TRKA xDFG substitutions induce conformational changes that stabilize the inactive (xDFG-out) conformation of the kinase, thus sensitizing it to type II inhibition. In vitro microscale thermophoresis revealed that the binding affinity of type II TRK inhibitors (cabozantinib or foretinib) to the TRKA G667C-mutant kinase was 8-10-fold higher compared to the type I inhibitor selitrectinib. We then tested the efficacy of type II TRK inhibitors against TRKA xDFG mutants in different cell models. A Bcan-Ntrk1-driven mouse model knocked in by CRISPR Cas9 to express the xDFG mutations was sensitive to type II but not to type I TRK inhibitors. Similar results were obtained using an LMNA-NTRK1-positive colorectal cell line that acquired the G667C substitution upon chronic selitrectinib treatment.

Type II TRK inhibitor therapy achieved complete and durable responses also in patient-derived models with TRKA xDFG-mediated resistance to type I 2nd-generation agents.

Conclusions: Our study uncovers a molecular switch induced by xDFG mutations that limits the sensitivity to type I kinase inhibitors by conformational changes that favor the inactive xDFG-out kinase state. This same switch in turn sensitizes these mutant kinases to type II inhibitors that effectively engage this inactive conformation. These results provide a paradigm for the rational development of 3rd-generation TKIs that address the problem of conformational resistance in tumors that are driven by oncogenic kinases.

Citation Format: Emiliano Cocco, Ji Eun Lee, Srinivasaraghavan Kannan, Alison M. Schram, Helen H. Won, Sophie Shifman, Amanda Kulick, Laura Baldino, Eneda Toska, Sabrina Arena, Benedetta Mussolin, Ram Kannan, Neil Vasan, Alexander N. Gorelick, Michael F. Berger, Yi Liao, Uwe Rix, Alberto Bardelli, Jacklyn Hechtman, Elisa de Stanchina, David M. Hyman, Chandra Verma, Andrea Ventura, Alexander Drilon, Maurizio Scaltriti. TRK xDFG mutations trigger a sensitivity switch from type I to II kinase inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5680.