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 leveraged two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibited outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing. This resulted in the expression of ALK variants, i.e. short transcripts (ST), which we named ALK-ST1 (consisting of exons 1-2:18–29), ALK-ST2 (1:18–29), ALK-ST3 (18–29), and ALK-ST4 (1–5:12–17). To systematically investigate the oncogenic capacity of these ALK variants, we stably expressed them in p53-deficient MCF10A human mammary epithelial cells and performed different transformation assays. These experiments demonstrated that ALK-ST1, ALK-ST2, and ALK-ST3 are oncogenic variants, while ALK-ST4 could not transform MCF10A cells. We confirmed protein expression of ALK-ST1, ALK-ST2, and ALK-ST3 by western blotting, which was not possible for ALK-ST4 due to lack of a specific antibody that binds to the N-terminus of ALK that is lost in ALK-ST4. We therefore performed mass spectrometry-based label-free quantitative proteomics on lysates from MCF10A cells stably expressing empty vector (EV), wildtype ALK (ALK-WT), or ALK-ST4 to confirm its expression.