Cell cycle regulation by PAX3-FOXO1 via WEE1 kinase in fusion-positive rhabdomyosarcoma

Introduction Fusion-positive rhabdomyosarcoma (FPRMS) is a highly aggressive form of the most common childhood muscle cancer, rhabdomyosarcoma. A distinct genetic signature, the PAX3-FOXO1 (P3F) fusion gene, characterizes a subtype of the most common pediatric soft tissue sarcoma. We aim to understand the collective cell invasion by determining how leader-follower cellular cooperation, P3F signaling, and the cell-cycle checkpoint kinase WEE1 drive tumor growth and invasion, and whether inhibiting WEE1 can blunt these behaviors. Brief Experimental Procedures To understand the collective cell invasion of FPRMS, we utilize a 3D "mini-tumor" (spheroid) model that recapitulates leader cells at the invasive front and rapidly proliferating follower cells behind them. We paired this with live-cell reporters of cell-cycle checkpoint activity and proliferation. We analyzed the transcriptome of FPRMS cells with P3F knockdown (PFKD) to confirm whether P3F regulates cell cycle checkpoints and DNA replication. Additionally, we employ ChIP-seq to identify the P3F-regulated genes and pharmacological WEE1 inhibition. Results Our preliminary data show that follower cells exhibit higher P3F expression, increased proliferation, and elevated WEE1 activity relative to other regions, consistent with a fast-cycling compartment that mechanically supports leader-directed invasion. These findings suggest that P3F upregulates WEE1-dependent programs, which fuel follower growth and cooperative invasion. Additionally, we use P3F knockdown to demonstrate, via RNA-seq, the Negative enrichment of gene sets associated with the G1/S transition and DNA replication in PFKD FPRMS cells. PAX3-FOXO1 regulates cell cycle progression by S-phase arrest. Furthermore, P3F regulates cell cycle progression by reducing the expression of pCDK2 and the double-strand break regulatory gene gammaH2A.X. Conclusions Together, the data support a model in which P3F-driven WEE1 signaling sustains the follower population that powers collective invasion in FPRMS. Targeting WEE1 slows growth and invasion in our 3D model, suggesting that WEE1 inhibition is a therapeutic strategy worthy of further evaluation in patient-derived specimens and preclinical studies.