ERK signaling promotes resistance to TRK kinase inhibition in NTRK fusion-driven glioma mouse models.

Pediatric-type high-grade gliomas frequently harbor gene fusions involving receptor tyrosine kinase genes, including neurotrophic tyrosine kinase receptor (NTRK) fusions. Clinically, these tumors show high initial response rates to tyrosine kinase inhibition but ultimately recur due to the accumulation of additional resistance-conferring mutations. Here, we develop a series of genetically engineered mouse models of treatment-naive and -experienced NTRK1/2/3 fusion-driven gliomas. All tested NTRK fusions are oncogenic in vivo. The NTRK variant, N-terminal fusion partners, and resistance-associated point mutations all influence tumor histology and aggressiveness. Additional tumor suppressor losses greatly enhance tumor aggressiveness. Treatment with TRK kinase inhibitors significantly extends the survival of NTRK fusion-driven glioma mice, but fails to fully eradicate tumors, leading to recurrence upon treatment discontinuation. Finally, we show that ERK activation promotes resistance to TRK kinase inhibition and identify MEK inhibition as a potential combination therapy. These models will be invaluable tools to study therapy resistance of NTRK fusion tumors.

ERK signaling promotes resistance to TRK kinase inhibition in NTRK fusion-driven glioma mouse models.

Pediatric-type high-grade gliomas frequently harbor gene fusions involving receptor tyrosine kinase genes, including neurotrophic tyrosine kinase receptor (NTRK) fusions. Clinically, these tumors show high initial response rates to tyrosine kinase inhibition but ultimately recur due to the accumulation of additional resistance-conferring mutations. Here, we developed a series of genetically engineered mouse models of treatment-naïve and -experienced NTRK1/2/3 fusion-driven gliomas. Both the TRK kinase domain and the N-terminal fusion partners influenced tumor histology and aggressiveness. Treatment with TRK kinase inhibitors significantly extended survival of NTRK fusion-driven glioma mice in a fusion- and inhibitor-dependent manner, but tumors ultimately recurred due to the presence of treatment-resistant persister cells. Finally, we show that ERK activation promotes resistance to TRK kinase inhibition and identify MEK inhibition as a potential combination therapy. These models will be invaluable tools for preclinical testing of novel inhibitors and to study the cellular responses of NTRK fusion-driven gliomas to therapy.

Kinetic evidence for multiple aggregation pathways in antibody light chain variable domains.

Aggregation of antibody light chain proteins is associated with the progressive disease light chain amyloidosis. Patient-derived amyloid fibrils are formed from light chain variable domain residues in non-native conformations, highlighting a requirement that light chains unfold from their native structures in order to aggregate. However, mechanistic studies of amyloid formation have primarily focused on the self-assembly of natively unstructured peptides, and the role of native state unfolding is less well understood. Using a well-studied light chain variable domain protein known as WIL, which readily aggregates in vitro under conditions where the native state predominates, we asked how the protein concentration and addition of pre-formed fibril "seeds" alter the kinetics of aggregation. Monitoring aggregation with thioflavin T fluorescence revealed a distinctly non-linear dependence on concentration, with a maximum aggregation rate observed at 8 µM protein. This behavior is consistent with formation of alternate aggregate structures in the early phases of amyloid formation. Addition of N- or C-terminal peptide tags, which did not greatly affect the folding or stability of the protein, altered the concentration dependence of aggregation. Aggregation rates increased in the presence of pre-formed seeds, but this effect did not eliminate the delay before aggregation and became saturated when the proportion of seeds added was greater than 1 in 1600. The complexity of aggregation observed in vitro highlights how multiple species may contribute to amyloid pathology in patients.

Kinetic evidence for multiple aggregation pathways in antibody light chain variable domains.

Aggregation of antibody light chain proteins is associated with the progressive disease light chain amyloidosis. Patient-derived amyloid fibrils are formed from light chain variable domain residues in non-native conformations, highlighting a requirement that light chains unfold from their native structures in order to aggregate. However, mechanistic studies of amyloid formation have primarily focused on the self-assembly of natively unstructured peptides, and the role of native state unfolding is less well understood. Using a well-studied light chain variable domain protein known as WIL, which readily aggregates in vitro under conditions where the native state predominates, we asked how the protein concentration and addition of pre-formed fibril "seeds" alter the kinetics of aggregation. Monitoring aggregation with thioflavin T fluorescence revealed a distinctly non-linear dependence on concentration, with a maximum aggregation rate observed at 8 µM protein. This behavior is consistent with formation of alternate aggregate structures in the early phases of amyloid formation. Addition of N- or C-terminal peptide tags, which did not greatly affect the folding or stability of the protein, altered the concentration dependence of aggregation. Aggregation rates increased in the presence of pre-formed seeds, but this effect did not eliminate the delay before aggregation and became saturated when the proportion of seeds added was greater than 1 in 1600. The complexity of aggregation observed in vitro highlights how multiple species may contribute to amyloid pathology in patients.