(P 465) JAK/STAT PATHWAY CO-OPERATES WITH CIC-FUSIONS TODRIVE CIC-REARRANGED SARCOMAS

Objective: CIC-rearranged sarcoma (CRS) is an rare disease driven by a specific fusion protein involving the CIC gene. Occurrence in the brain is 3% in all CRS patients and these tumors frequently metastasize to the brain. The most common rearrangement is with the double homeobox 4 (DUX4) transcription factor (CIC-DUX4), and others, such as CIC-NUTM1 fusions. However, the molecular mechanisms by which CIC-fusions drive CRS remain unknown. Preliminary data shows that CIC-DUX4/NUTM1 fusions activate JAK and its downstream effector STAT1/3.

Methods: CRS and fusion-negative sarcoma cell lines were treated with JAK1 inhibitors (Ruxolitinib, Solicitinib). STAT1/3activation and ETV1/4/5 expression were assessed by Western blot and qPCR. Luciferase assays measured ETV5 promoter activity. ChIP was used to examine STAT1/3 binding and histone acetylation. CRS xenografts in mice were treated with Ruxolitinib to evaluate in vivo effects on tumor growth and target gene expression

Results: Patient-derived CRS cell lines showed elevated levels of JAK1/STAT1/3 activation compared to fusion-negative sarcoma lines. Inhibition of JAK1 using Ruxolitinib and Solicitinib reduced STAT1/3 phosphorylation,downregulated ETV1/4/5 expression at both mRNA and protein levels, and diminished ETV5 promoter activity, cell proliferation, and tumorigenicity. Although the mechanism by which CIC-fusions activate oncogenic targets is still under investigation, histone acetylation appears to play a central role. STAT1/3 interacts with p300/CBP to enhance transcription, and STAT1 is necessary for p300 acetyltransferase activity.Ruxolitinib significantly reduced histone acetylation at ETV1/4/5 promoters in hMSC cells expressing CIC-DUX4/NUTM1, as well as in CRS cell lines. Unlike the p300 inhibitor C646, which causes global hypoacetylation, Ruxolitinib’s effects were promoter-specific, indicating its potential as a more targeted and less toxic therapeutic option. Importantly, we found that STAT1/3 binds to ETV1/4/5 promoters only in the presence of CIC-fusions. Luciferase assays confirmed that STAT1/3 alone cannot activate ETV5 transcription without CIC-fusions, revealing a novel cooperative mechanism. In vivo, Ruxolitinib treatment of CRS xenografts led to significant reductions in tumor volume, STAT1/3 activation, and ETV1/4/5 expression.

Conclusion: These findings support JAK1/STAT1/3 inhibition as a promising therapeutic strategy for CRS and warrant further preclinical investigation.