FANCM is Required for the PAX3::FOXO1-Driven Oncogenic Program in Rhabdomyosarcoma

Abstract

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric soft tissue sarcoma driven by the PAX3::FOXO1 fusion protein. Despite multipronged treatment approaches, survival rates for patients with fusion-positive (FP) ARMS remain poor, underscoring the need for novel therapeutic strategies. Here, we employ sequential multimodal CRISPR/Cas9 genetic screens to reveal FANCM, a key component of the Fanconi Anemia pathway, as a critical dependency. FANCM loss selectively impairs ARMS cell growth, reduces PAX3::FOXO1 levels, and induces myogenic differentiation. Mechanistically, FANCM depletion exacerbates replication stress, particularly at PAX3::FOXO1 target gene loci, which leads to DNA damage signaling and selective downregulation of the fusion-driven oncogenic program. CRISPR exon-tiling screens highlight FANCM's helicase and DNA-binding domains as essential for its FP-specific dependency, linking FANCM-mediated replication fork binding and downstream ATR signaling to FP ARMS survival. This study positions FANCM as a promising therapeutic target and introduces a novel paradigm for exploiting replication stress vulnerabilities in oncofusion-driven malignancies.