Loss of miR-29a/b1 cluster reprograms the tumor microenvironment and contributes to immunosuppression in lung cancer

Immune checkpoint inhibitors (ICI), like those that block PD-1/PD-L1, have revolutionized oncological therapy for patients with non-small cell lung cancer (NSCLC). However, most patients demonstrate no clinical benefit or acquire resistance, even in PD-L1+ populations, highlighting the critical need to dissect tumor survival dependencies to overcome resistance. Using our unique Kras/p53-driven lung cancer models which demonstrate acquired or intrinsic resistance to ICI, we performed single cell RNA-sequencing and focused on predicted upstream regulators of differentially expressed genes (DEGs) in the malignant cell cluster of resistant tumors. We found that the micro-RNA, miR-29, was downregulated in tumors with anti-PD-1 resistance, leading to significant upregulation of a multitude of miR-29 targets. Interestingly, we found that the immunosuppressive molecule Enpp2/ATX was one of these genes modulated due to miR-29 loss, and re-expression of miR-29 in anti-PD-1 resistant models diminished ATX tumor expression, diminished the fibrotic microenvironment, and increased CD8 infiltration. Additionally, analysis of lung adenocarcinoma patients revealed miR-29-high patients had increased CD8A expression and enrichment in immunoregulatory pathways. Together, these data provide evidence that the miR-29 family broadly regulates the tumor microenvironment, including anti-tumor immune-related pathways in lung cancer, through control of ATX among many other target genes, with implications in ICI response.