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

Immune checkpoint inhibitors (ICI), including those that block PD-1/PD-L1, have revolutionized therapy for patients with non-small cell lung cancer. However, most patients demonstrate no clinical benefit or acquire resistance, even when tumors express PD-L1. This highlights the critical need to dissect tumor survival dependencies to overcome resistance. Using our Kras/p53-driven lung cancer models that demonstrate acquired or intrinsic resistance to ICI, we performed single-cell RNA sequencing and focused on predicted upstream regulators of differentially expressed genes 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, and that this was associated with significant upregulation of a multitude of miR-29 targets. Furthermore, we found expression of Enpp2/ATX, a gene encoding an immunosuppressive molecule, was modulated due to miR-29 loss. Re-expression of miR-29 in anti-PD-1 resistant models reduced ATX expression in tumor cells, diminished the fibrotic microenvironment, and increased CD8+ T-cell infiltration. These alterations promoted response to ICI in an anti-PD-1 resistant model by rewiring the tumor immune microenvironment, specifically through increased CD8+ T-cell infiltration, reduction of suppressive Ly6C+ monocytes, and a concomitant increase in pro-inflammatory macrophages. Additional analysis of publicly available RNA-sequencing data revealed tumors from lung adenocarcinoma patients with high miR-29 had increased CD8A and decreased CD14 expression, and broad enrichment in immunoregulatory pathways. Together, these data provide evidence that the miR-29 family regulates the tumor microenvironment, including antitumor immune-related pathways in lung cancer, through control of ATX among other target genes, with implications for ICI response.