(P 301) GENOME-WIDE CRISPR/CAS9 VIABILITY SCREENS UNCOVER NOVEL GENETIC DEPENDENCIES IN CIC::DUX4 SARCOMA

Objective: CIC::DUX4 sarcoma (CDS) is an intractable and lethal soft tissue sarcoma affecting adolescents and young adults. Driving the cancer is a lone recurrent gene fusion occurring between CIC and DUX4 giving rise to an exceptionally potent and transcriptionally active oncoprotein. Compared to other small round cell sarcomas like Ewing sarcoma (ES), CIC::DUX4 fusion-positive sarcomas have a poor prognosis characterized by a high rate of metastasis and resistance to conventional chemotherapies. Despite knowledge of chemo-resistance, most patients continue to receive ES-tailored regimens due to our limited knowledge of the mechanisms underlying tumor initiation and maintenance. To better define these mechanisms and to identify cell-type specific genetic dependencies for the development of targeted therapies, genome-wide CRISPR/Cas9 viability screens were performed.

Methods: All publicly available human CDS cell lines were collected and authenticated using RNA-sequencing, DNA-sequencing, ChIP-sequencing, and ATAC-sequencing. To identify CDS-specific genetic dependencies, genome-wide CRISPR/Cas9 viability screens were performed. First, stable Cas9-expressing lines were created by transducing them with a lentiviral Cas9 expressing construct. Drug-selected and validated Cas9-expressing cell lines were expanded and transduced with a pooled genome-wide lentiviral sgRNA library (MinLib) using a multiplicity of infection of < 0.3 to ensure each cell received only one sgRNA. Following puromycin selection, the cells were cultured for 14-18 population doublings and passaged every 2-3 days to maintain 1000X coverage (approximately 40 million cells/replicate for 20,000 genes and 2 sgRNAs/gene). The screens were conducted in technical triplicate to enable robust statistical identification of gene hits. At the completion of the screen, the cells were harvested, and an optimized PCR protocol was used to ligate sequencing adaptors and amplify the sgRNA barcodes for multiplex sequencing. Sequencing was performed by the Duke Sequencing and Genomics Core Facility. Raw sequencing reads were processed and converted to gene counts using MAGeCK. To quantify gene essentiality (i.e. Beta-score), the difference in relative sgRNA abundance between time zero (T0) and time final (TF) was calculated for each replicate and then statistically analyzed.

Results: To date, we have completed screens for two of the eight authenticated CDS cell lines including Kitra-SRS and NCC-CDS-X3C1. In both screens, the tumor suppressors MEN1 and TP53 emerged as the top ranking positively selected gene. Moreover, nearly all 1,671 common essential genes (CEGs) identified by DepMap were strongly and consistently selected against. In each cell line, the median Beta-score for all CEGs was used to set the essentiality threshold (Kitra: -0.44; X3C1:-0.70). In total, 356 and 319 uniquely essential genes were identified, respectively. Of those, 178 (~50%) were found to be essential in both CDS cell lines including NONO, TERF1, and other notable genes. Interestingly, while sgRNAs targeting the CIC::DUX4 oncogene were significantly negatively selected in the X3C1 cell line (beta score: -1.4), as expected, the loss of viability was much less significant in Kitra (beta score: -0.29). Conversely, sgRNAs targeting ETV4, a direct downstream target of CIC::DUX4 and effector of MAPK signaling were strongly negatively selected in Kitra (beta score: -0.63) but, instead, positively selected in X3C1 (beta score: 0.40).

Conclusion: Genome-wide Crispr/Cas9 viability screens are an unbiased way of identifying tumor-specific genetic vulnerabilities. As well as revealing important insights into the genes and mechanisms governing tumor maintenance, they can reveal potential targets for the development of precision cancer therapeutics. Here, to our knowledge, we present the first genome-wide CRISPR/Cas9 screens in CDS cell lines. As expected, loss of the tumor suppressors MEN1 and TP53 improved cell viability by promoting unchecked clonal expansion. On other hand, loss of ERCC3 and other components of the DNA repair and core transcriptional machinery resulted in cell death thus decreasing cell viability. Unexpectedly, we observed that deletion of the CIC::DUX4 fusion gene was reasonably well tolerated in the Kitra cell line which was also uniquely intolerant to ETV4 loss. Given the high redundancy of ETS binding sites at CIC::DUX4 bound regions described previously, we hypothesize that ETV4 can function as a cooperative or independent regulator of the oncogenic CDS transcriptional program. Future work will examine the mechanisms of CIC::DUX4 escape and focus on the identification/validation of the most clinically significant genetic dependencies unique to CDS.