An NKX2-1/ERK/WNT feedback loop modulates gastric identity and response to targeted therapy in lung adenocarcinoma.

Cancer cells often undergo lineage switching during their natural progression and in response to therapy. Lung adenocarcinomas (LUADs) exhibit a variety of differentiation states accompanied by dysregulation of lineage-specific transcription factors such as NKX2-1. Loss of NKX2-1 in human and murine LUAD leads to invasive mucinous adenocarcinoma (IMA), a subtype of lung cancer that exhibits pulmonary to gastric transdifferentiation. Human IMAs harbor a distinct spectrum of mutationally activated driver oncogenes compare to LUAD overall, suggesting that the pulmonary to gastric transdifferentiation induced by NKX2-1 loss plays a context-dependent role in LUAD progression. Using genetically engineered mouse models, we find that NKX2-1 is required for optimal BRAFV600E driven lung tumor initiation but is dispensable for growth of established lung tumors. NKX2-1-deficient, BRAFV600E driven tumors morphologically resemble human IMA, have high levels of ERK phosphorylation and exhibit a distinct response to treatment with combined BRAF/MEK inhibitors. Whereas NKX2-1-positive tumor cells enter quiescence when treated with BRAF/MEK inhibitors, residual NKX2-1-negative cells fail to exit the cell cycle in response to the same therapy. Additionally, BRAF/MEK inhibitors induce canonical WNT signaling in NKX2-1-negative lung tumor cells, which is accompanied by cell identity switching within the gastric lineage. Co-inhibition of MAPK and WNT pathways blocked elements of this lineage switch in vitro and interfered with cell cycle changes imposed by MAPK inhibition in vivo. Our data show that there is a complex and reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of LUAD identity and suggest that lineage switching induced by targeted therapies may confer new therapeutic vulnerabilities. Overall design: For bulk RNAseq, lung tumors were isolated from BRafCA/+;Trp53f/f;Nkx2-1f/+;Rosa26tdTomato/tdTomato mice (5 control and 3 MAPK inhibitor treated BP mice- a 4th sample was an outlier/of poor quaility and was excluded from analysis) and from BRafCA/+;Trp53f/f;Nkx2-1f/f;Rosa26tdTomato/tdTomato (4 control and 5 MAPK inhibitor treated BPN mice). Transcriptome profiles were generated by Illumina HiSeq 2500 sequencing. For single cell RNAseq, lung tumors from BRafCA/+;Trp53f/f;Nkx2-1f/f;Rosa26tdTomato/tdTomato (2 control BPN mice pooled into 1 sample and 2 MAPK inhibitor treated BPN mice pooled into 1 sample) were isolated. Transcriptome profiles were generated by NovaSeq 6000 with 2x150 PE mode sequencing.