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 ICIs, we performed single-cell RNA sequencing (scRNA-seq) 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 that expression of Enpp2/ATX, a gene encoding an immunosuppressive molecule, was modulated due to miR-29 loss. Reexpression 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 ICIs 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 proinflammatory macrophages. Additional analysis of publicly available RNA-seq data revealed that tumors from patients with lung adenocarcinoma 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.