Multi-Omic and Functional Analysis for Classification and Treatment of Sarcomas with FUS-TFCP2 or EWSR1-TFCP2 Fusions

Linking clinical multi-omics analyses with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in truncated ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for CDK4/6- and PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling indicate a close relationship with undifferentiated sarcomas. Finally, in two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, providing insight into stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers. The RNA-seq and ACT-seq data from cell lines generated in this study have been deposited under this accession code. RNA-sequencing: MCF10A (1x10*6) or SCP-1 (5x10*6) cells stably transduced in triplicate (MCF10A) or three to nine replicates (SCP-1) with EV, FUS-TFCP2, EWSR1-TFCP2, FUS, EWSR1, or TFCP2 were seeded in 10- or 15-cm dishes, and RNA was isolated the next day using the RNeasy Mini Plus Kit (Qiagen). Library preparation and 125-nt paired-end read sequencing on an Illumina HiSeq 4000 system were performed at the DKFZ Genomics and Proteomics Core Facility. ACT-seq: MCF10A cells were transduced with two different lentiviral vectors to obtain four biological replicates expressing HA-FUS-TFCP2 or EV (R1–R4) and three biological replicates expressing HA-TFCP2 (R2–R4). For each replicate, ACT-seq was performed with HA-Tag (C29F4) rabbit mAb (Cell Signaling, #3724), HA-Tag (F-7) mouse mAb (Santa Cruz, sc-7392), anti-histone H3 (acetyl K27) antibody – ChIP grade (Abcam, ab4729), and rabbit IgG polyclonal antibody (Merck, #PP64) as negative control. Further details can be found in the Supplementary Information of the related manuscript.