Alterations in the PI3K/AKT pathway occur in up to 70% of melanomas and are associated with disease progression. The three AKT paralogs are highly conserved but data suggest they have distinct functions. Activating mutations of AKT1 and AKT3 occur in human melanoma but their role in melanoma formation and metastasis remains unclear. Using an established melanoma mouse model, we evaluated E17K, E40K, and Q79K mutations in AKT1, AKT2, and AKT3 and show that mice harboring tumors expressing AKT1 E17K had the highest incidence of brain metastasis and lowest mean survival. Tumors expressing AKT1 E17K displayed elevated levels of focal adhesion factors and enhanced phosphorylation of focal adhesion kinase (FAK). AKT1 E17K expression in melanoma cells increased invasion and this was reduced by pharmacologic inhibition of either AKT or FAK. These data suggest that the different AKT paralogs have distinct roles in melanoma brain metastasis and that AKT and FAK may be promising therapeutic targets. Implications: This study suggests that AKT1 E17K promotes melanoma brain metastasis through activation of FAK and provides a rationale for the therapeutic targeting of AKT and/or FAK to reduce melanoma metastasis.

In vitro polymerized type I collagen hydrogels have been used extensively as a model system for three-dimensional (3D) cell and tissue culture, studies of fibrillogenesis, and investigation of multiscale force transmission within connective tissues. The nanoscale organization of collagen fibrils plays an essential role in the mechanics of these gels and emergent cellular behavior in culture, yet quantifying 3D structure with nanoscale resolution to fully characterize fibril organization remains a significant technical challenge. In this study, we demonstrate that a new imaging modality, focused ion beam scanning electron microscopy (FIB-SEM), can be used to generate 3D image datasets for visualizing and quantifying complex nanoscale organization and morphometry in collagen gels. We polymerized gels at a number of concentrations and conditions commonly used for in vitro models, stained and embedded the samples, and performed FIB-SEM imaging. The resulting image data had a voxel size of 25 nm, which is the highest resolution 3D data of a collagen fibril network ever obtained for collagen gels. This resolution was essential for discerning individual fibrils, fibril paths, and their branching and grouping. The resulting volumetric images revealed that polymerization conditions have a significant impact on the complex fibril morphology of the gels. We segmented the fibril network and demonstrated that individual collagen fibrils can be tracked in 3D space, providing quantitative analysis of network descriptors such as fibril diameter distribution, length, branch points, and fibril aggregations. FIB-SEM 3D reconstructions showed considerably less lateral grouping and overlap of fibrils than standard 2D SEM images, likely due to artifacts in SEM introduced by dehydration. This study demonstrates the utility of FIB-SEM for 3D imaging of collagen gels and quantitative analysis of 3D fibril networks. We anticipate that the method will see application in future studies of structure-function relationships in collagen gels as well as native collagenous tissues.

NTRK1 gene fusions are actionable drivers of numerous human malignancies. Here, we show that expression of the TPR-NTRK1 fusion kinase in immortalized mouse pancreatic ductal epithelial (IMPE) (pancreas) or mouse lung epithelial (MLE-12) cells is sufficient to promote rapidly growing tumors in mice. Both tumor models are exquisitely sensitive to targeted inhibition with entrectinib, a tropomyosin-related kinase A (TRKA) inhibitor. Initial regression of NTRK1-driven tumors is driven by induced expression of BIM, such that BIM silencing leads to a diminished response to entrectinib in vivo. However, the emergence of drugresistant disease limits the long-term durability of responses. Based on the reactivation of RAF>MEK>ERK signaling observed in entrectinib-treated tumors, we show that the combination of entrectinib plus the MEK1/2 inhibitor cobimetinib dramatically forestalls the onset of drug resistance in vivo. Collectively, these data provide a mechanistic rationale for rapid clinical deployment of combined inhibition of TRKA plus MEK1/2 in NTRK1-driven cancers.