Therapeutic targeting of YOD1 disrupts the PAX-FOXO1/N-Myc feedback loop in rhabdomyosarcoma
Wenwen Ying, Jiayi Yu, Xiaomin Wang, Jiayi Liu, Boyu Deng, Xuejing Shao, Jinhu Wang, Ting Tao, Ji Cao, Qiaojun He, Bo Yang, Yifan Chen, Meidan Ying
Wenwen Ying, Jiayi Yu, Xiaomin Wang, Jiayi Liu, Boyu Deng, Xuejing Shao, Jinhu Wang, Ting Tao, Ji Cao, Qiaojun He, Bo Yang, Yifan Chen, Meidan Ying
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Research Article Cell biology Oncology

Therapeutic targeting of YOD1 disrupts the PAX-FOXO1/N-Myc feedback loop in rhabdomyosarcoma

Abstract

Fusion-positive rhabdomyosarcoma (FP-RMS), driven by PAX-FOXO1 fusion oncoproteins, represents the subtype of RMS with the poorest prognosis. However, the oncogenic mechanisms and therapeutic strategies of PAX-FOXO1 remain incompletely understood. Here, we discovered that N-Myc, in addition to being a classic downstream target of PAX-FOXO1, can also activate its expression and form a transcriptional complex with PAX-FOXO1, thereby markedly amplifying oncogenic signaling. The reciprocal transcriptional activation of PAX3-FOXO1 and N-Myc is critical for FP-RMS malignancy. We further identified YOD1 as a deubiquitinating enzyme that stabilizes both PAX-FOXO1 and N-Myc. Knocking down YOD1 or inhibiting it with G5 could suppress FP-RMS growth both in vitro and in vivo, through promoting the degradation of both PAX-FOXO1 and N-Myc. Collectively, our results identify that YOD1 promotes RMS progression by regulating the PAX3-FOXO1/N-Myc positive feedback loop, and highlight YOD1 inhibition as a promising therapeutic strategy that concurrently reduces the levels of both oncogenic proteins.

Authors

Wenwen Ying, Jiayi Yu, Xiaomin Wang, Jiayi Liu, Boyu Deng, Xuejing Shao, Jinhu Wang, Ting Tao, Ji Cao, Qiaojun He, Bo Yang, Yifan Chen, Meidan Ying

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Figure 2

PAX-FOXO1/N-Myc positive feedback loop drives global transcriptional reprogramming in RMS.

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PAX-FOXO1/N-Myc positive feedback loop drives global transcriptional rep...
(A) Identification of a Hallmark gene signature coregulated by PAX3-FOXO1+N-Myc. GSEA was performed on transcriptomic data from RD cells comparing the PAX3-FOXO1+N-Myc group versus N-Myc– or P3F1-alone groups. Significantly enriched Hallmark pathways (FDR < 0.05, |NES| > 1) from both comparisons are displayed in the scatter plot. NES, normalized enrichment score. The coregulated signature was defined as the intersection of significant pathways from the 2 comparisons. n = 3. (B) Combined bar and line graph representing the 11 overlapping Hallmark pathways. Bars show the NES, and the line shows the –log10(FDR) for each pathway. (C) Heatmap displaying gene expression clusters. Cluster A denotes genes specifically activated in the PAX3-FOXO1+N-Myc group. n = 3. (D) Immunoprecipitations (IP) by anti-FLAG beads from HEK-293T cells transfected with vector or N-Myc-HA along with vector or P3F1-FLAG for 48 hours were analyzed by immunoblot (IB). (E) IPs by anti-FLAG beads from HEK-293T cells transfected with vector or P3F1-HA along with vector or N-Myc-Flag for 48 hours were analyzed by IB. (F) IPs by anti-FLAG beads from RD cells transfected with vector or N-Myc-HA along with vector or P3F1-FLAG for 48 hours were analyzed by IB. Data are representative of 3 independent experiments. (G) Immunofluorescence analysis showing subcellular colocalization of P3F1 (green) and N-Myc (red) in HeLa and RD cells. HeLa was used as a tool cell. Cells were infected with GFP-P3F1 and N-Myc-HA for 48 hours. Nuclei were stained with DAPI (blue). Colocalization was analyzed using ImageJ. Scale bar: 10 μm.