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. Overall design: Kras/p53 mutant murine lung cancer cells were implanted in vivo. The 344SQ are the parental line and the 140P (PD1R1) samples were derived from the 344SQ cells after in vivo treatment and resistance to anti-PD-1 therapy. The 344SQ and PD1R1 tumors were collected after 4 and 6 weeks of in vivo growth (T1 and T2) and analyzed by scRNA-seq for differentially expressed genes between the two models specifically in the malignant compartment.