David Ryan Ormond

ENT reduces NSCLC cell proliferation in coculture with astrocytes. Proliferation was quantified as green area (μm2) using live-cell imaging. A, H358; (B) H2030Br3; (C) Lu65; and (D) PC9Br3 cells were cultured either in starvation media (2% cs-FBS) alone or over a 100% confluent monolayer of primary mouse astrocytes, in combination with vehicle (Veh; DMSO) or ENT (1 μmol/L). Data were analyzed by repeated-measures two-way ANOVA followed by Fisher LSD test (n = 5 wells per treatment). P values shown are the adjusted P values for Veh vs. Veh + Ent or Veh-mAst vs. ENT-mAst at the last time point.

ENT prevents brain metastatic colonization of H2030Br3 cells. A, Experimental design: NSG female mice were randomized to receive vehicle (Veh) or ENT (60 mg/kg, twice daily (BID), orally (PO)]. Treatment began 3 days prior to the intracardiac injection of 200,000 H2030Br3 GFPLuc cells. Metastatic progression was monitored weekly by IVIS imaging, and mice were euthanized 4 weeks after inoculation. B, (Left) Mean ± SEM of total head flux by treatment; (middle and right) individual head flux values for ENT- and Veh-treated mice, respectively. C, Total ex vivo flux in the brain with representative IVIS images. D, Each dot represents the median number of micrometastases (300 μm) per mouse, Mann–Whitney test; representative hematoxylin and eosin images of macro and micro metastases forming a cluster are shown. E, (Left) Mean ± SEM of total extracranial flux by treatment; (middle and right) individual extracranial flux values for ENT-treated and Veh-treated mice. Group sizes are as follows: Veh = 18 and ENT = 9. F, Total ex vivo flux in lungs with representative IVIS images. G, Total ex vivo flux in livers with representative IVIS images. Statistical analysis: In vivo IVIS data were analyzed using two-way repeated-measures ANOVA; P values show adjusted P values for the last time point. Ex vivo data were analyzed using the Kruskal–Wallis test followed by Fisher LSD post hoc test.

List of antibodies, sources, and working dilutions used in this study

The central nervous system (CNS) is a common site of metastasis for both non–small cell and small cell lung cancer, yet the therapeutic strategies to prevent and decrease lung cancer brain metastases (BM) remain limited. Tyrosine kinase inhibitors have shown promising results in increasing overall response in BM, owing to their brain penetrance and increased effectiveness; however, their use is limited to a small group of tumors carrying specific oncogenic drivers. Among these, inhibitors targeting neurotrophic tyrosine receptor kinases (NTRK) are showing promising effects in reducing CNS metastases in cancers driven by gene rearrangements of these drugs’ targets. However, wild-type NTRKs are susceptible to activation by their canonical ligands, which are expressed throughout the BM niche and can, in a paracrine manner, activate NTRK function in cancer cells. In this study, we show that NTRKs are expressed in primary tumors, BM, and lung cancer cells with various driver mutations expressing wild-type NTRK2 (WT-TrkB). We demonstrate that WT-TrkB activates downstream signaling and proliferation in response to exogenous brain-derived neurotrophic factor (BDNF) and conditioned media from reactive astrocytes known to secrete BDNF in the brain niche. Importantly, the FDA-approved NTRK inhibitor entrectinib blocked BDNF and astrocyte-induced survival pathways in multiple lung cancer cell lines, decreased their proliferation in vitro, and effectively prevented BM colonization and progression in vivo without significant effects on extracranial disease. Thus, these studies suggest that brain-dependent activation of NTRK is critical for BM of WT-NTRK+ lung cancers; therefore, NTRK inhibitors can be used to target nonfusion NTRK function to prevent or decrease BM. Significance:These studies demonstrate that NTRK wild-type receptors are important drivers of brain metastatic colonization and progression in different subtypes of lung cancer, independent of their driver alterations. Thus, they provide a rationale to expand the use of FDA-approved NTRK inhibitors with brain penetrance for the prevention of CNS metastases.

ENT blocks TrkB activation and proliferation of NSCLC TrkB+ cells lacking NTRK driving mutations. A, Western blot of NSCLC cells plated in phenol red–free media with 2.5% cs-FBS and pretreated for 2 hours with DMSO [vehicle (Veh)], ENT, LOXO101 (LOXO), cyclotraxin (Cyclo), or ANA12 (all drugs at 1 μmol/L), followed by stimulation with 50 ng/mL BDNF for 10 minutes. Tubulin served as a loading control. Densitometric values were calculated as phospho-TrkB/tubulin, total TrkB/tubulin, phospho-AKT/total AKT, and phospho-ERK/total ERK ratios and expressed relative to the Veh control group. B, Proliferation measured as percent confluency (mean ± SEM) using Incucyte live-cell imaging (n = 5 per treatment). Cells were plated in phenol red–free media with 2.5% cs-FBS and treated with Veh, 50 ng/mL BDNF alone, or BDNF combined with 1 μmol/L tyrosine kinase inhibitors. Data were analyzed by repeated-measures two-way ANOVA with post hoc multiple-comparison corrections.

Effects of genetic or pharmacological TrkB inhibition on astrocyte-conditioned media-induced proliferation and signaling in NSCLC cells

ENT decreases AKT and ERK activation and proliferation induced by astrocyte-conditioned media (Ast-CM) in NSCLC cells. A, Western blot of NSCLC cells serum-starved overnight, pretreated for 2 hours with 1 μmol/L ENT or LOXO101 (LOXO), then stimulated with Ast-CM (10×) for 30 minutes. Tubulin served as a loading control. Densitometric values were calculated as phospho-TrkB/tubulin, total TrkB/tubulin, phospho-AKT/total AKT, and phospho-ERK/total ERK ratios and were expressed relative to the vehicle (Veh) control group. B, Proliferation expressed as percent confluence (mean ± SEM) in NSCLC cells cultured in serum-starved media and treated with Ast-CM (10×) plus Veh (DMSO) or ENT (1 μmol/L). Data were analyzed by repeated-measures two-way ANOVA followed by Fisher Least Significant Difference (LSD) test (n = 5 wells per treatment).

ENT reduces brain metastatic burden in a late-stage model of BMs. A, Experimental design: NSG female and male mice were injected intracardially with 200,000 H2030BrM3 GFPLuc cells. Head IVIS signal was monitored every 10 days. Mice showing a head signal at least twice the initial level were randomized to receive vehicle (Veh) or ENT (60 mg/kg, twice daily, orally). Group sizes are as follows: female (ENT) = 6, male (ENT) = 6, female (Veh) = 5, and male (Veh) = 7. Treatment lasted 14 days, and metastatic burden was monitored weekly. B, Total head flux for both sexes combined. C, Total extracranial flux for both sexes combined. In vivo IVIS data were analyzed using two-way repeated-measures ANOVA followed by Fisher LSD test for multiple comparisons. P values shown are adjusted P values for the last time point. D, Representative images of full brain sagittal sections from Veh and ENT-treated mice. Dotted lines mark the pan-cytokeratin positive (panCK+, gray tumor areas. Boxes show 5× magnification of p-TrkB (red), panCK (green), DAPI (nuclei), and merged images. Scale bars, 1,000 μm. E, Quantification of p-TrkB in 10 images/mice from 2 individual mice per group, at 40×. PanCK+ mask was generated to define tumor areas and converted into a region of interest; pTrkB mean fluorescence intensity was measured within panCK+ areas only. Representative images of fields were used for quantification. Scale bars, 50 μm.

TrkB is expressed in lung cancer cells and activated by exogenous BDNF. A, Gene expression levels of NTRK1, NTRK2, and NTRK3 in lung adenocarcinoma (LUAD) tumor samples from TCGA. Normalized gene counts [fragments per kilobase of transcript per million mapped reads (FPKM)] were obtained using the TCGA biolinks R package and analyzed in GraphPad Prism 10.6.1. One-way ANOVA with Tukey multiple comparisons. B,NTRK1, NTRK2, and NTRK3 expression between PTs and matched BM. Q3-normalized values were paired by patient ID (n = 23). Paired comparisons were performed using the Wilcoxon signed-rank test. C, IHC showing TrkB expression in a small cohort (n = 9) of NSCLC-BM; scale bars, 100 μm. D, Western blot showing expression of TrkB, TrkA, TrkC, and BDNF in NSCLC cell lines. Mouse astrocytes were used as a positive control for TrkA and TrkC. E, Relative mRNA expression of mouse and human BDNF in astrocytes (Ast), neurons (Neu), oligodendrocyte precursor cells (OPC), endothelial cells (Endo), microglia (Mg), macrophages (MΦ), oligodendrocytes (OL), mature oligodendrocytes (MOL), and newly formed oligodendrocytes (NFOL), data from Brain RNAseq.org. F, Immunofluorescence expression of BDNF in GFAP+ astrocytes surrounding lung cancer BM in a xenograft model. Scale bars, 50 μm. G, Western blots show the time course of BDNF-induced TrkB signaling in NSCLC cells serum-starved overnight and stimulated with 50 ng/mL BDNF. Tubulin served as a loading control in all blots. Molecular weight markers of 150 and 100 kD show full-length TrkB expression (110–140 kD depending on glycosylation levels).

Downregulation of TrkB impairs BDNF-TrkB signaling and decreases proliferation induced by astrocytic conditioned media