Expression of the CIC-DUX4 fusion oncoprotein mimics human CIC-rearranged sarcoma in genetically engineered mouse models

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 tissues and patients. 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 tumors and widespread metastasis in the absence of Cre-recombinase. The penetrance of spontaneous (Cre-independent) tumor formation was complete irrespective of bi-allelic CIC function and loxP site proximity. Characterization of primary and metastatic mouse tumors showed that they consistently expressed the CIC-DUX4 fusion protein as well as other downstream markers of the disease credentialing these 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-seq 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.

Keywords: CIC-DUX4; autochthonous model; fusion oncoprotein; mouse model; rare disease; sarcoma; soft tissue sarcoma.

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 primary and metastatic 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 stain (scale bar 50um) and immunohistochemistry (IHC) panel (scale bar 100um) 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).

Figure 3.

Mouse tumors express CIC-DUX4 and a transcriptional signature consistent with CDS. a) Representative images from HA-tag (top) and DUX4 (bottom) IHC in primary and metastatic tumors from Ch7CDS, Ai9CDS, and TOPCDS mice (scale bar 100um). 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 (Kras^G12D, p53^fl/fl) tumors. Red dots correspond to 65 genes highly and specifically expressed CIC-DUX4 target genes selected from human datasets^,.

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) Ranked heatmaps showing relative read coverage from HA-CIC-DUX4 and H3K27ac ChIP across all 5,057 HA-CIC-DUX4 peaks in TOPCDS cells. e) Heatmap displaying the log2 fold change (tumor/KP) 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. f) Summary of the results from ‘CRCmapper’ highlighting the interconnected auto-regulatory loop between CIC-DUX4 and ETS transcription factors.