MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to repress target mRNAs. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter highly complementary targets, which can trigger a process termed target-directed microRNA degradation (TDMD). During TDMD, miRNAs undergo tailing and trimming, suggesting that this is an important step in the decay mechanism. Here, we identified a cullin-RING ubiquitin ligase (CRL), containing the substrate adapter ZSWIM8, that mediated TDMD. The ZSWIM8 CRL interacted with AGO proteins, promoted TDMD in a tailing and trimming-independent manner, and regulated miRNA expression in multiple cell types. These findings suggest a model in which the ZSWIM8 ubiquitin ligase mediates TDMD by directing proteasomal decay of miRNA-containing complexes engaged with highly complementary targets.

Alveolar rhabdomyosarcoma is a pediatric soft-tissue sarcoma caused by PAX3/7-FOXO1 fusion oncogenes and is characterized by impaired skeletal muscle development. We developed human PAX3-FOXO1 -driven zebrafish models of tumorigenesis and found that PAX3-FOXO1 exhibits discrete cell lineage susceptibility and transformation. Tumors developed by 1.6–19 months and were primitive neuroectodermal tumors or rhabdomyosarcoma. We applied this PAX3-FOXO1 transgenic zebrafish model to study how PAX3-FOXO1 leverages early developmental pathways for oncogenesis and found that her3 is a unique target. Ectopic expression of the her3 human ortholog, HES3, inhibits myogenesis in zebrafish and mammalian cells, recapitulating the arrested muscle development characteristic of rhabdomyosarcoma. In patients, HES3 is overexpressed in fusion-positive versus fusion-negative tumors. Finally, HES3 overexpression is associated with reduced survival in patients in the context of the fusion. Our novel zebrafish rhabdomyosarcoma model identifies a new PAX3-FOXO1 target, her3/HES3, that contributes to impaired myogenic differentiation and has prognostic significance in human disease.

In mice and humans with cancer, intravenous 13 C-glucose infusion results in 13 C labeling of tumor tricarboxylic acid (TCA) cycle intermediates, indicating that pyruvate oxidation in the TCA cycle occurs in tumors. The TCA cycle is usually coupled to the electron transport chain (ETC) because NADH generated by the cycle is reoxidized to NAD + by the ETC. However, 13 C labeling does not directly report ETC activity, and other pathways can oxidize NADH, so the ETC’s role in these labeling patterns is unverified. We examined the impact of the ETC complex I inhibitor IACS-010759 on tumor 13 C labeling. IACS-010759 suppresses TCA cycle labeling from glucose or lactate and increases labeling from glutamine. Cancer cells expressing yeast NADH dehydrogenase-1, which recycles NADH to NAD + independently of complex I, display normalized labeling when complex I is inhibited, indicating that cancer cell ETC activity regulates TCA cycle metabolism and 13 C labeling from multiple nutrients.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer characterized by a misregulation of skeletal muscle developmental pathways. To date, identified oncogenic drivers predominantly include RAS mutations or chromosomal translocations and gene fusions between PAX3 or PAX7 and FOXO1. RNAseq analysis of sarcomas with non-canonical gene fusions has identified new potential genetic drivers of tumorigenesis that have not been rigorously functionally validated for their transformation capacity and biological activity. One new fusion is a chromosomal translocation and inversion between chromosomes 6 and 8, which acts to juxtapose two transcriptional co-activators, VGLL2 and NCOA2. This VGLL2-NCOA2 fusion was identified in congenital rhabdomyosarcoma clinical cohorts by us and others, and characterizes aggressive RMS that express MYOD and MYOG histological markers. However, evidence of VGLL2-NCOA2 transformation capacity has not been verified, hindering insights into its functional contributions to tumorigenesis. Here, we interrogate the function ofVGLL2-NCOA2 using complementary genomic patient data and zebrafish model systems. We utilized the Tol2 transposon system to express mosaic human VGLL2-NCOA2 during early development, and found that VGLL2-NCOA2 is sufficient for tumorigenesis, and results in aggressive tumors with high penetrance by 75 days of age in zebrafish. Further, the histology of zebrafish tumors resembles the human disease, and tumors express markers indicative of RMS such as myog and desma. A cross-species RNAseq of patient and zebrafish VGLL2-NCOA2 RMS tumors highlights a significant enrichment and overlap between gene expression signatures. Finally, mapping the gene expression signatures of VGLL2-NCOA2 zebrafish RMS tumors along the spectrum of zebrafish embryogenesis indicates a clustering with developmental stages corresponding to early somitogenesis, highlighting their arrested developmental nature. Hence, we have generated the first animal model of human VGLL2-NCOA2 tumorigenesis, and have applied this model to understand the biology and identify potential therapeutic targets for this newly identified disease. Citation Format: Genevieve C. Kendall, Sarah Watson, Lin Xu, Collette LaVigne, Whitney Murchison, Dinesh Rakheja, Franck Tirode, Olivier Delattre, James Amatruda. Genetic models reveal that the novel VGLL2-NCOA2 fusion oncogene leverages embryonic programs for sarcomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 872.

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