KEAP1-mutant lung cancers weaken anti-tumor immunity and promote an M2-like macrophage phenotype.
Considerable advances have been made in lung cancer therapies, but there is still an unmet clinical need to improve survival for lung cancer patients. Immunotherapies have improved survival, although only 20-30% of patients respond to these treatments. Interestingly, cancers with mutations in KEAP1, the negative regulator of the NRF2 cytoprotective pathway, are resistant to immune checkpoint inhibition and correlate with decreased immune cell infiltration. NRF2 is known for promoting an anti-inflammatory phenotype when activated in immune cells, but the study of NRF2 activation in cancer cells has not been adequately assessed. The objective of this study was to determine how lung cancer cells with constitutive NRF2 activity interact with the immune microenvironment to promote cancer progression. To assess this, we generated CRISPR-edited mouse lung cancer cell lines by knocking out KEAP1 or NFE2L2 genes. We also utilized publicly available single cell data through the Gene Expression Omnibus to investigate tumor/immune cell interactions. We show here that KEAP1-mutant cancers promote immunosuppression of the tumor microenvironment. Our data suggests KEAP1-deletion is sufficient to alter the secretion of cytokines, increase expression of immune checkpoint markers on cancer cells, and alter recruitment and differential polarization of immunosuppressive macrophages that ultimately lead to T cell suppression. Overall design: RNA sequencing was used to evaluate the transcriptomic changes related to NRF2 activation in LL2 lung cancer cells. Specifically, we used CRIPSR to functionally delete KEAP1 thereby inducing NRF2 transcriptional activity. We also created a second cell line by knocking out the NRF2 protein. We isolated 3 clonally derived KEAP1-mutant and 1 NRF2-mutant cell line. We then compared the new cell lines to the WT parental line. mRNA was isolated from the cell lines (3 biologic replicates per group) for comparative gene expression analysis.