Abstract P247: Evaluating TRKB activity of novel preclinical brain-penetrant ROS1 and ALK inhibitors
Authors: Tangpeerachaikul Anupong , Horan Joshua C. , Pelish Henry E. 
Published: December 2021

Oncogenic ROS1 and ALK rearrangements are detected in up to 3% and up to 5% of advanced non-small cell lung cancer (NSCLC), respectively, with up to 40% of patients presenting with brain metastases at diagnosis. Although there are FDA-approved ROS1 and ALK inhibitors, there is a need for next-generation brain-penetrant inhibitors that have activity against drug-resistance mutations while sparing tropomyosin kinase B (TRKB) function in the central nervous system (CNS). TRKB inhibition in the CNS has been implicated in adverse events such as cognitive impairment, mood disorders, sleep effects or sleep disturbances, dizziness, ataxia or gait/motor disturbances, and weight gain, which have been observed with FDA-approved dual TRK/ROS1 inhibitor entrectinib and FDA-approved ALK inhibitor lorlatinib. TRKB-related CNS adverse events present a key challenge for the development of next-generation ROS1 and ALK therapies. We previously reported the discovery of NVL-520 and NVL-655, preclinical inhibitors of ROS1 and ALK, respectively, which have demonstrated activity against common drug-resistance mutations, selectivity versus TRKB, and brain penetrance in preclinical experiments. An important part of the discovery process was generating reliable and relevant TRKB potency measurements to enable structure-activity relationship (SAR) efforts. Here we describe four in vitro TRKB potency assays and explain their utility in guiding selectivity optimization for new inhibitors. These assays include (1) biochemical, (2) cell viability, (3) cellular coupled-enzyme, and (4) direct cellular phosphorylation readouts. Although biological contexts vary, each assay exhibits good cross-correlations, and we recommend multi-assay testing to improve confidence in evaluating TRKB inhibition. Using these assays, we present a multi-assay analysis of the preclinical TRKB potency and selectivity of NVL-520 and NVL-655 alongside FDA-approved or experimental inhibitors of ROS1 (crizotinib, entrectinib, repotrectinib, and taletrectinib) and ALK (crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, and ensartinib). In these preclinical assays, we observed entrectinib and lorlatinib to exhibit poor ROS1-vs-TRKB and ALK G1202R-vs-TRKB selectivity, respectively, which we believe is consistent with their CNS adverse events. In the same experiment, we observed NVL-520 and NVL-655 to exhibit selectivity across all assays, including for ROS1 G2032R and ALK G1202R/L1196M mutations, respectively. In conclusion, we have devised a multi-assay approach to evaluate TRKB inhibition and guide discovery of selective ROS1 and ALK inhibitors, with the goal of minimizing adverse events and driving durable responses for patients.

Citation Format: Anupong Tangpeerachaikul, Joshua C. Horan, Henry E. Pelish. Evaluating TRKB activity of novel preclinical brain-penetrant ROS1 and ALK inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P247.