Targeting NTRK1 Enhances Immune Checkpoint Inhibitor Efficacy in NTRK1 Wild-Type Non-Small Cell Lung Cancer

Abstract

Treatment of non-small cell lung cancer (NSCLC) has drastically changed in recent years owing to the robust anticancer effects of immune checkpoint inhibitors (ICI). However, only 20% of the patients with NSCLC benefit from ICIs, highlighting the need to uncover the mechanisms mediating resistance. By analyzing the overall survival (OS) and mutational profiles of 424 patients with NSCLC who received ICI treatments between 2015 and 2021, we determined that patients carrying a loss-of-function mutation in neurotrophic tyrosine kinase receptor 1 (NTRK1) had a prolonged OS when compared with patients with wild-type NTRK1. Notably, suppression of the NTRK1 pathway by knockdown or entrectinib treatment significantly enhanced ICI efficacy in mouse NSCLC models. Comprehensive T-cell population analyses demonstrated that stem-like CD4+ T cells and effector CD4+ and CD8+ T cells were highly enriched in anti-PD-1-treated mice bearing tumors with decreased NTRK1 signaling. RNA sequencing revealed that suppression of NTRK1 signaling in tumor cells increased complement C3 expression, which enhanced the recruitment of T cells and myeloid cells and stimulated M1-like macrophage polarization in the tumor. Together, this study demonstrates a role for NTRK1 signaling in regulating cross-talk between tumor cells and immune cells in the tumor microenvironment and provides a potential therapeutic approach to overcome immunotherapy resistance in patients with NSCLC with NTRK1 wild-type. Significance: Inhibition of NTRK1 signaling confers sensitivity to immunotherapy by enhancing complement C3-mediated T-cell and macrophage functions, leading to improved responses to immune checkpoint inhibitors in patients with lung cancer with NTRK1 mutations.

Figure 1.. Mutational and survival analyses of ICI treated NSCLC patients.

(A) Diagram of how patient population was selected. (B) Association between clinical factors and patients’ overall survival (OS) examined by multivariate analyses by logistic regression. (C) OS of patients with or without NTRK1 or MAP2K1 mutations by Mantel-Cox test. (D) Lollipop analysis showing the position and types of NTRK1 mutations. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 2.. Knockdown of NTRK1 enhances anti-PD-1 efficacy in vivo.

(A) Tumor volume of mice inoculated with LL/2 (left, n=10) and CMT167 (right, n=5) cells with or without KD of NTRK1 and anti-PD-1 treatment. The KD efficiency of NTRK1 by shRNA were confirmed by Western Blot. (B) Immunocytochemistry analyses of CD4⁺ (red) CD8⁺ (green) T cells in the tumor tissues from mice inoculated with LL/2 (left) or CMT167 (right) with or without KD of NTRK1 treated with indicated treatments. Scale bar: 20μm. Quantification of (C) effector CD4⁺ T cells, (D) stem-like CD4⁺ T cells, and (E) effector CD8⁺ T cells in the spleen and TDLNs of tumor bearing mice inoculated with LL/2 (left) and CMT167 (right) with or without KD of NTRK1 and anti-PD-1 treatment. Scale bar: 20μm. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 3.. Entrectinib promotes anti-PD-1 response in NTRK1 wild-type tumors.

(A) Tumor volume of mice inoculated with LL/2 (left) and CMT167 (right) cells with or without receiving Entrectinib or anti-PD-1 treatment. (n=5) (B) Quantification of CD4⁺ (red) CD8⁺ (green) T cells in the tumor tissues from LL/2 (left) and CMT167 (right) inoculated mice treated with indicated treatments. Scale bar: 20μm. Quantification of (C) effector CD4⁺ T (D) stem-like CD4⁺ T cells and (e) effector CD8⁺ T cells in the spleen and TDLNs of tumor bearing mice inoculated with LL/2 (left) and CMT167 (right) with or without Entrectinib and anti-PD-1 treatment. Scale bar: 20μm *p < 0.05, **p < 0.01, ***p < 0.001

Figure 4.. Entrectinib treatment modulates immune microenvironment.

(A) PCA results of LL/2 tumor samples (n=3–4) from mice received indicated treatments. (B) IPA results of immune related functions that were enriched in Entrectinib treated LL/2 tumor compare to IgG treated tumor. (C) Immunocytochemical analyses of M1 macrophages in tumors with or without KD of NTRK1(left) and tumors with or without treatment of Entrectinib. (D) IPA results of enriched T cell related functions in combination treated tumors compared to Anti-PD1 monotherapy treated groups. (E) mMCP-counter and QuanTiseq analyses of T cell populations in tumors from LL/2 tumor bearing mice treated with indicated treatments. (F) DCQ analyses of different T cell populations within the tumors derived from indicated groups. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 5.. Suppression of NTRK1 promotes Complement C3 expression in tumor cells.

(A) IPA results of upregulated immune related activities in LL/2 cells with KD of NTRK1. (B) Volcano Plot of upregulated immune related secretory or extracellular genes in LL/2-shNTRK1 cells. Fold changes (log) of genes between the shscramble and shNTRK1 were plotted on the xaxis, and the adjusted p-value (−1*log10 scale) was plotted on the y-axis. (C) mRNA expression of NTRK1 target genes identified from figure 5B were examined by qRT-PCR in LL/2 and CMT167 cells. (D) mRNA expressions of CCN1, C3, CXCL1 and CCL2 in responders (n=458) and non-responders (n=809) of patients who received anti-PD-1 treatments using ROC plotter. (E) C3 protein was examined by WesternBlot in LL/2 and CMT167 cells with or without KD of NTRK1. (F) WesternBlot analyses of C3 expression in LL/2 and CMT167 cells treated with different doses of Entrectinib. C3 levels in LL/2 and CMT167 cells treated with various doses of (G) LY294002 and (H) PD98059 were measured by WesternBlot. (I) Representative images of C3 immunohistochemical staining in NSCLC samples from patients with or without NTRK1 mutations. Scale bar: 20μm (J) Quantification of C3 staining intensity of patients’ samples. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 6.. Knockout of C3 mitigates the ICI response in NTRK1 KD cells.

(A) Tumor volume of mice inoculated with LL/2-shNTRK1 (left) and CMT167-shNTRK1 (right) cells with or without KO of C3 (n=5). KO of C3 in both cell lines were confirmed by Western Blot. (B) Quantification of CD4⁺ (red) CD8⁺ (green) T cells in the tumor tissues from IgG or anti-PD-1 treated mice inoculated with LL/2-shNTRK1 (left) or CMT167-shNTRK1 (right) with or without KO of C3. Scale bar: 20μm Quantification of (C) effector CD4⁺ T cells, (D) effector stem-like CD4⁺ T cells, and (E) effector CD8⁺ T cells in the spleen and TDLNs of tumor bearing mice inoculated with LL/2-shNTRK1 (left) and CMT167-shNTRK1 (right) with or without KO of C3 and anti-PD-1 treatment. Scale bar: 20μm *p < 0.05, **p < 0.01, ***p < 0.001