Abstract 5463: Germline DICER1 loss promotes rhabdomyosarcoma via innate immune system

Abstract DICER1 syndrome is a cancer predisposition syndrome where affected patients have increased risk of neoplasms including pleuropulmonary blastoma, Sertoli-Leydig cell tumor, and fusion-negative rhabdomyosarcoma (FN-RMS). This syndrome is defined by germline heterozygous loss of function mutations in the gene DICER1, but it remains unclear exactly how these mutations predispose children to develop cancer. One would expect the germline loss of one allele to provide a sensitized background in the tumor cell to acquire a second hit mutation. To gain insight into the role of Dicer1 loss in FN-RMS, we deleted the Dicer1 gene both globally in the germline and conditionally in the tumor cell in our previously established mouse model of FN-RMS. Surprisingly, we observed faster tumor onset and increased penetrance in mice with germline heterozygous Dicer1 loss (Dicer1+/-) but not in mice where Dicer1 loss was restricted to the tumor cells, demonstrating that Dicer1 functions as a non-cell autonomous haploinsufficient tumor suppressor. Single cell RNA sequencing (scRNA-Seq) revealed massive expansion of immature neutrophils in Dicer1+/- tumors, which were enriched for proteases associated with neutrophil extracellular traps (NETs). NETs are webs of chromatin and proteases released when neutrophils undergo NETosis in response to inflammatory stimuli. These NETs are known to cause significant damage to the local extracellular matrix (ECM) and have been implicated in tumor promotion. In addition to the neutrophil enriched immunophenotype, we found that Dicer1+/- tumors show significant ECM remodeling and are enriched for C5a, the potent neutrophil chemoattractant and NET-priming factor. Taken together, these results suggest NETosis is upregulated in Dicer1+/- FN-RMS tumors. Performing iTALK ligand-receptor pair analysis on the scRNA-Seq data revealed a putative interaction between a NET-derived ligand and EGFR and IGF1R on tumor cells, suggesting direct tumor promoting signaling from NET-to-tumor. We validated this novel FN-RMS promotion mechanism in vitro using human FN-RMS cell lines and observed that this NET-derived ligand promoted FN-RMS growth in a dose-dependent manner and could function through both EGFR and IGF1R signaling. Finally, we incorporated a Padi4-/- allele into our tumor model to genetically block NETosis and observed complete rescue of the accelerated tumor onset and increased penetrance observed in Dicer1+/- mice, demonstrating that NETosis promotes the growth of Dicer1+/- FN-RMS tumors. Importantly, these findings may be applicable to many DICER1 syndrome-associated cancers, as neutrophils which get converted to tumor promoters by heterozygous DICER1 loss are present in DICER1 syndrome patients regardless of tumor type. Citation Format: Randolph Larsen, Jason Hanna, Kristin Reed, Hongjian Jin, Wood Kimbrough, Kyna Vuong, Myron Evans, Casey Langdon, Catherine Drummond, Matthew Garcia, David Finkelstein, Patrick Schreiner, Jerold Rehg, Mark Hatley. Germline DICER1 loss promotes rhabdomyosarcoma via innate immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5463.