The desmoplastic small round cell tumor (DSRCT) remains one of the most lethal adolescent/young adult (AYA) sarcomas. The EWSR1-WT1 gene fusion is the primary and defining genetic driver alteration in this sarcoma but a better understanding of EWSR1-WT1 oncogenic mechanisms is needed to identify novel, rational therapeutic strategies that will be more effective. We propose to address these knowledge gaps by establishing the first genetically engineered mouse model of DSRCT and to define its pathobiology more fully by harnessing two recent novel observations in DSRCT, namely induction of neotranscripts and recurrent ARID1A loss as a frequent additional genetic alteration in these cancers. Aim 1: Developing a mouse model of DSCRT by germline and somatic genome editing. We will use the in vivo somatic chromosomal engineering method pioneered by our laboratory (A.V.) to induce the EWSR1-WT1 translocation in mice, based on co-expression of Cas9 and two gRNAs targeting the desired translocation breakpoints. As p53 is mutated in approximately 10% of DSRCT cases, we will induce the translocation concomitantly with p53 deletion in a cohort of mice to enhance tumor development. Prompted by the recent finding that ARID1A mutations are commonly observed in DSRCT patients (see Aim 3), we will also test the consequences of Arid1a loss on DSRCT initiation and progression in vivo. Aim 2. Defining the landscape of EWSR1-WT1-associated neotranscripts in DSRCT. We (J.W., O.D.) have recently reported that, in addition to dysregulating the expression of many target genes, oncogenic fusions such as EWSR1-WT1 also drive the expression of completely novel sequences which we have termed neotranscripts (J.W., O.D.). These are unannotated multi-exonic, polyadenylated RNAs not expressed in any healthy tissue but directly induced by the altered localization patterns and co-factor associations of the oncogenic fusion protein. A subset of neotranscripts are actively translated into protein products, representing potential neoantigens. We will build upon our initial discovery of these sequences and use them as a model system for dissecting EWSR1-WT1 mediated gene regulation as well as determining what role these neotranscripts or their encoded proteins play in tumor biology. Aim 3. Determining the role of recurrent ARID1A loss in DSRCT. While DSRCT displays one of the lowest somatic mutation rates of any human solid cancer, intriguingly, inactivation of ARID1A, a subunit of the canonical BAF complex, is the most common secondary mutation in DSRCT (about 6-12% of cases), being more common than even TP53 alterations (M.L.). We propose to use isogenic models of ARID1A loss in DSRCT cell lines to define the phenotypic and epigenetic effects of this recurrent secondary alteration in DSCRT and use chemical and functional genomic screens to identify novel vulnerabilities associated with ARID1A loss which may also yield more general insights into DSRCT pathobiology.