Genetically Engineered Mouse Models of CIC-DUX4 Sarcoma Reveal an Unexpected Sensitivity to Histone Deacetylase (HDAC) Inhibition

Abstract

Undifferentiated small round cell sarcomas constitute a highly aggressive subtype of soft tissue sarcoma that affects adolescents and young adults. Frequently, these cancers are caused by a gene fusion between the DNA binding domain of capicua (CIC), a canonical tumor suppressor that normally acts as a transcriptional repressor, with the transcriptional activation domain of double homeobox 4 (DUX4). Given the rare nature of CIC-DUX4 sarcoma (CDS), obtaining patient tissues to study the mechanisms of disease is very challenging. Moreover, conducting clinical trials to identify radiosensitizers or other novel treatments is not possible underscoring the need for a primary animal model. Here, we report the development and characterization of the first genetically engineered mouse model of CDS and the use of this model to investigate novel therapies. Using Cre-loxP technology, three genetically engineered mouse models of CDS were developed. The resulting tumors were characterized by hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and RNA-sequencing. Cell lines from multiple primary and metastatic tumors were derived and used for Chromatin Immunoprecipitation (ChiP) sequencing, Rapid Immunoprecipitation mass spectrometry of Endogenous proteins (RIME), and targeted drug screening. In all three models, spontaneous (Cre-independent) recombination occurred in chimeric animals resulting in the formation of soft tissue tumors, widespread metastasis to the lungs, liver, and brain, and 100% lethality within 4-8 weeks of life. The tumors histologically resembled human CDS and stained positive for an HA (hemagglutinin) epitope tag confirming transgene expression. Of 108 chimeric pups (generation 0, G0), only one male bred before succumbing to metastatic disease. G2 progeny now exhibit resistance to spontaneous recombination and tumor development enabling reliable propagation of one of the models for experimentation. Using tumor derived cell lines for integrative analyses of multi-omics datasets, we find evidence for a model where CIC-DUX4 functions as a neomorphic and oncogenic transcriptional activator, which cooperates with ETS family transcription factors to drive the CDS transcriptional program. Lastly, small molecule epigenetic drug screens in mouse and human CDS cell lines point to an unexpected sensitivity to HDAC inhibitors, which we hypothesize may be attributable to ETS factor hyperactivity. Genetically engineered mouse models of CIC-DUX4 sarcoma form tumors that recapitulate the human disease. Using mouse tumors and tumor-derived cell lines, we identify a mechanistic role and potential therapeutic vulnerability that converges on the ETS family transcription factors.