Myodifferentiation of Rhabdomyosarcoma

Rhabdomyosarcoma is an aggressive pediatric soft tissue malignancy thought to originate from muscle progenitor cells that failed to reach terminal differentiation. A common genetic alteration in rhabdomyosarcoma pathogenesis is a chromosomal translocation that fuses the DNA binding domain of PAX3 to the transcriptional activation domain of FOXO1, resulting in a chimeric transcription factor PAX3-FOXO1. This fusion oncoprotein leads to a myogenic differentiation block, promotes cellular proliferation, and oncogenic transformation. While rhabdomyosarcoma cells express several early myogenic markers such as MYOD1 and desmin, late markers of differentiation are absent and rhabdomyosarcoma cells do not fuse or form functional muscles. We set out to identify factors that cooperate with PAX3-FOXO1 to sustain the myodifferentiation block, the rationale being that perturbations of these gene products could lead to the identification of novel, mechanism-based targets for this disease. To this end, we developed a CRISPR/Cas9 phenotypic screen to identify genetic knockouts that phenocopy loss of PAX3-FOXO1 and lead to rhabdomyosarcoma myodifferentiation using intracellular staining for an endogenous reporter and fluorescence activated cell sorting. Using this approach, we identified NFYA, NFYB, and NFYC, all members of the heterotrimeric transcription factor complex NF-Y, at the leading edge of knockouts that re-engage forward myodifferentiation and restore lineage fates in this disease. We validated the results of the pooled screen with individual gene knockouts and demonstrated that cells depleted of NF-Y closely resemble cells depleted of PAX3-FOXO1 transcriptome-wide and at a single cell level. The myodifferentiation response following loss of NF-Y is conserved among fusion positive rhabdomyosarcoma models. We characterized genome-wide chromatin occupancy of NF-Y in rhabdomyosarcoma cells using CUT&RUN and showed that it binds a number of cancerpromoting and differentiation-inhibiting genes, including the PAX3 promoter that drives expression of PAX3-FOXO1. Mutagenesis of the NF-Y recognition motif or targeted displacement of NF-Y from the PAX3 promoter partially recapitulates the effects of NF-Y knockout. Since NF-Y binds primarily gene promoters and PAX3-FOXO1 binds enhancers, yet they control an overlapping set of genes, we examined genomic interactions in 3D space and uncovered a subset of genes where NF-Y and PAX3-FOXO1 participate in coordinated long-range interactions, including SIX1, a recently described master regulator of rhabdomyosarcoma differentiation state. Taken together, we conclude that NF-Y is a key transcription factor complex, whose function is coordinated with PAX3-FOXO1 to drive rhabdomyosarcoma pathogenesis