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. 2021 Apr 6;10:e66788. doi: 10.7554/eLife.66788

Figure 4. NKX2-1 status modulates response to MAPK pathway inhibitors.

(A) Representative H and E and phospho-ERK1/2 immunostaining photomicrographs of paraffin-embedded lung sections from BrafLSL-V600E/+;Trp53f/f;Nkx2-1f/+;Rosa26LSL-tdTomato/LSL-tdTomato and BrafLSL-V600E/+;Trp53f/f;Nkx2-1f/f;Rosa26LSL-tdTomato/LSL-tdTomato mice that were treated with control chow (BP C/BPN C) or chow containing PLX4720 (200 mg/kg) and PD0325901 (7 mg/kg) inhibitors (BP Tx/BPN Tx) for 2 weeks starting at 6 weeks post-tumor initiation with PGK-Cre lentivirus (5 × 103 pfu/mouse). Scale bar: 100 µm. (B, C) Quantitation of tumor burden in BP and BPN mice that were fed control chow or chow containing MAPK inhibitors starting at 6 weeks post-tumor initiation with PGK-Cre lentivirus (5 × 103 pfu/mouse). Graphs represent mean ± S.D. (B) Chow treatment lasted 2 weeks and lungs were harvested at the 8-week timepoint. BP C (n = 8), BP Tx (n = 8), BPN C (n = 9), BPN Tx (n = 9). *p<0.05 by Student’s t-test. Numbers indicated above graphs represent the fold reduction in tumor burden upon inhibitor-chow administration. (C) Chow treatment lasted 4 weeks and lungs were harvested at the 10-week timepoint. BP C (n = 6), BP Tx (n = 9), BPN C (n = 8), BPN Tx (n = 8). **p<0.01, ***p<0.001 by Student’s t-test. Numbers indicated above graphs represent the fold reduction in tumor burden with inhibitor-chow administration. (D) Global gene expression analyses were performed on RNAs from FACS-sorted tdTomato+ BP C (n = 5), BP Tx (n = 3), BPN C (n = 4), and BPN Tx (n = 5) murine lung tumor cells isolated at 7 weeks following initiation with Ad5-Spc-Cre adenovirus (5 × 108 pfu/mouse for BP and 8 × 108 pfu/mouse for BPN mice). Control and MAPK-inhibitor chow treatments were given for 1 week at 6 weeks post-adenoviral instillation. Shown is the principal-component analysis (PCA) plot of the top 500 most variable genes showing that the four experimental groups of lung tumors, BP C, BP Tx, BPN C, and BPN Tx, had distinct global patterns of gene expression. (E) UMAP plots showing relatedness of high-quality, tumor cell scRNA-seq profiles from BPN control (n = 2) and BPN MAPKi-treated (n = 2) mice. Tumor cluster designations are indicated (left). Control and treated cells indicated (middle). Nkx2-1 expression in scRNA-seq data indicating that clusters 4–8 represent incomplete recombinants (right). Single tumor cells were obtained by FACS-sorting tdTomato+ cells isolated at 7 weeks following initiation with Ad5-Spc-Cre adenovirus (8 × 108 pfu/mouse). Control and MAPK-inhibitor chow treatments were given for 1 week at 6 weeks post- adenoviral instillation. (F) Beeswarm plots of single cell sequencing data showing expression levels of Nkx2-1, Sftpc and Aurkb transcripts in tumor clusters 1–8.

Figure 4.

Figure 4—figure supplement 1. NKX2-1 status modulates response to MAPK pathway inhibitors.

Figure 4—figure supplement 1.

(A) Representative histology of BP and BPN tumors after 4 weeks of combined BRAFV600E and MEK inhibitor chow treatment, as well as at different timepoints following drug withdrawal demonstrating rapid tumor relapse. (B) Graphs comparing absolute expression levels of direct NKX2-1 targets (Sftpc and Sftpb) as well as the gastrointestinal marker Pdx1 in BP (n = 8) and BPN (n = 9) tumors, data obtained from RNA sequencing from whole tumors. (C) Whole-tumor RNA-seq analyses of BP and BPN tumors via Illumina Correlation Engine indicating that HNF4A-binding site geneset three becomes significantly enriched upon loss of Nkx2-1. (D) Volcano plots of whole-tumor RNA sequencing data from control and MAPK inhibitor-treated BP tumors (left) and BPN tumors (right) indicating differentially expressed genes (EdgeR) (see also Supplementary file 2, 3). Select highly differentially expressed genes are highlighted. (E) Left- UMAP visualization of single cell transcriptome relatedness indicating 14 distinct clusters. Right- Relative expression of stromal marker genes and feature complexity score used in selecting high complexity tumor clusters that lack stromal marker gene expression for further analysis (red box). * Lower than average complexity. Note that the cluster ID labels for tumor cell clusters used in Figure 4E (post-stromal cell exclusion) and E are different. (F) Sashimi plots visualizing raw RNA-seq densities along Nkx2-1 exons and splice junction for Nkx2-1-positive and Nkx2-1-negative single tumor cells from their alignment data. Exon 1a-2 junctions and exon two reads are only detected in cells expressing Nkx2-1 to variable degree indicating that these are incomplete recombinants (BP cells). (G) UMAP visualization of Hmga2 expression in tumor clusters 1–8. (H) Quantitation of MAPK activation levels in single cell RNA sequencing data using Mek18 transcriptional signature (Dry et al., 2010). BP indicates cells from clusters 4–8; BPN indicates cells from clusters 1–3 (see Figure 4E, F). (C) Control, Tx = BRAFi/MEKi treatment.