Spontaneous expression of the CIC::DUX4 fusion oncoprotein from a conditional allele potently drives sarcoma formation in genetically engineered mice

Abstract

CIC::DUX4 sarcoma (CDS) is a rare but highly aggressive undifferentiated small round cell sarcoma driven by a fusion between the tumor suppressor Capicua (CIC) and DUX4. Currently, there are no effective treatments and efforts to identify and translate better therapies are limited by the scarcity of patient tumor samples and cell lines. To address this limitation, we generated three genetically engineered mouse models of CDS (Ch7CDS, Ai9CDS, and TOPCDS). Remarkably, chimeric mice from all three conditional models developed spontaneous soft tissue tumors and disseminated disease in the absence of Cre-recombinase. The penetrance of spontaneous (Cre-independent) tumor formation was complete irrespective of bi-allelic Cic function and the distance between adjacent loxP sites. Characterization of soft tissue and presumed metastatic tumors showed that they consistently expressed the CIC::DUX4 fusion protein and many downstream markers of the disease credentialing the models as CDS. In addition, tumor-derived cell lines were generated and ChIP-seq was preformed to map fusion-gene specific binding using an N-terminal HA epitope tag. These datasets, along with paired H3K27ac ChIP-sequencing maps, validate CIC::DUX4 as a neomorphic transcriptional activator. Moreover, they are consistent with a model where ETS family transcription factors are cooperative and redundant drivers of the core regulatory circuitry in CDS.

Figure 1.

Fusion of a human DUX4 C-terminal domain to endogenous Cic is sufficient to generate small round cell sarcomas. a) Schematic of the Ch7CDS allele; Cre-loxP recombination creates a CIC::DUX4 gene fusion at the endogenous Cic allele on chr 7. b) Gross images of soft tissue and visceral organ tumors from chimeric Ch7CDS mice. c) Kaplan-Meier survival curve of chimeric Ch7CDS animals. d) DNA gel of amplification products from PCR across the loxP sites in tails, tumors, and tumor-derived cell lines (n=2 each). In the tumor and tumor-derived cell lines, a ~800bp PCR product is amplified consistent with loxP recombination. e) Representative images from an H&E stained slide (scale bar 50μm) and immunohistochemistry (IHC) panel (scale bar 100μm) on spontaneous tumors from Ch7CDS mice.

Figure 2.

CIC haploinsufficiency is not required for CIC::DUX4 sarcomagenesis. a) Schematic of the Ai9CDS allele; Cre-loxP recombination removes a stop cassette and permits expression of an HA-CIC::DUX4 fusion gene at the Rosa26 locus. b) Kaplan-Meier survival curve of chimeric Ai9CDS animals. c) DNA gel of amplification products from PCR across the loxP sites in tails, tumors, and tumor-derived cell lines (n=2 each). d) Schematic of the TOPCDS allele; Cre-loxP recombination removes a stop cassette (containing multiple polyA sequences in tandem) and permits expression of an HA-CIC::DUX4 fusion gene at the Rosa26 locus. e) Kaplan-Meier survival curve of chimeric TOPCDS animals. f) DNA gel of amplification products from PCR across the loxP sites in tails, tumors, and tumor-derived cell lines (n=2 each). g) Sanger sequencing alignments of the PCR amplicons from Figure 2f revealing imprecise LSL deletions.

Figure 3.

Mouse tumors express CIC::DUX4 and a transcriptional signature consistent with CDS. a) Representative images from HA-tag (top) and DUX4 (middle) and ETV (bottom) IHC in primary and presumptive metastatic tumors from Ch7CDS, Ai9CDS, and TOPCDS mice (scale bar 100μm). b) Western blot on a Cre-expressing control cell line and tumor-derived cell lines probed for the HA-tag, DUX4 CTD, and Cre recombinase. DUX4 confirms the expression of a ~260kD protein in all three CDS cell lines corresponding to the predicted size of the CIC::DUX4 fusion. Anti-HA is specific to the two epitope-tagged cell lines, however, all three CDS cell lines are negative for Cre. c) Volcano plots showing differentially expressed genes in CDS tumors compared to KP (K-ras^G12D, p53^fl/fl) mouse tumors. Red dots correspond to 65 genes highly and specifically expressed CIC::DUX4 target genes selected from human datasets^,,. d) Fractional bar charts depicting the results of SCDC analysis on bulk RNA-sequencing from limb muscle (LM), KP tumor (KP) and CDS tumors revealing the relative composition of 12 cell types curated from the Tabula Muris atlas . e) Tumor classification results from OTTER, an ensemble convolutional neural network classifier for human tumors based on bulk transcriptomes, after adaptations for mouse.

Figure 4.

CIC::DUX4 behaves as a neomorphic transcriptional activator. a) Pie charts corresponding to the genomic location of all HA-CIC::DUX4 peaks in tumor cell lines from Ai9CDS and TOPCDS mice. b) Top enriched KEGG pathways based on all genes associated with a HA-CIC::DUX4 peak. c) Dot plot (top) showing the top 25 most enriched de novo motifs identified in 2,410 shared HA-CIC::DUX4 peaks. The top four de novo motifs match the predicted binding sites for CIC (AATG/CATT) and ETS-family transcription factors (TTCC/GGAA). d) Fractional bar chart showing the genomic location all 2,410 shared HA-CIC::DUX4 peaks broken down by motif type. e) Representative genes associated with promoter HA-CIC::DUX4 peaks containing one, both, or neither motif. f) Min/max box plots showing the log2 fold change of all genes associated with promoter HA-CIC::DUX4 peaks as a function of the motif they contain. Center line corresponds to group mean; p-values calculated using unpaired two-sided student t-test. g) Ranked heatmaps showing relative read coverage from HA-CIC::DUX4 and H3K27ac ChIP across all 5,057 HA-CIC::DUX4 peaks in TOPCDS cells. h) Heatmap displaying the log2 fold change (mouse CDS tumor/mouse KP sarcoma) for all 11 transcription factors predicted to be core regulators of the CDS transcriptional circuitry. On the bottom row, log2 fold change in human CDS relative to ES is included. i) Summary of the results from CRCmapper highlighting the interconnected auto-regulatory loop between CIC::DUX4 and ETS transcription factors.