Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Here, we show that the zinc finger transcription factor CASZ1 up-regulates MYOD signature genes and induces skeletal muscle differentiation in normal myoblasts and ERMS. The oncogenic activation of the RAS-MEK pathway suppresses CASZ1 expression in ERMS. ChIP-seq, ATAC-seq and RNA-seq experiments reveal that CASZ1 directly up-regulates skeletal muscle genes and represses non-muscle genes through affecting regional epigenetic modifications, chromatin accessibility and super-enhancer establishment. Next generation sequencing of primary RMS tumors identified a single nucleotide variant in the CASZ1 coding region that potentially contributes to ERMS tumorigenesis. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis.

DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N2-2′-deoxyguanosine (N2-dG) and N6-2′-deoxyadenosine (N6-dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N2-dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.

Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Zinc finger transcription factor CASZ1 is a tumor suppressor and orchestrates cell-fate specification, commitment, and differentiation in many cell types during development. CASZ1 is expressed during skeletal myogenesis; however, whether CASZ1 plays a role in skeletal muscle cell differentiation and RMS pathogenesis is unknown. Here we uncover that CASZ1 crosstalks with myogenic regulatory factors Myf5, MyoD, and Myog to regulate myogenesis in C2C12 cells. Public transcriptome data show that CASZ1 mRNA levels are lower in ERMS compared to normal muscle (p<0.001). CASZ1 levels decrease upon oncogenic activation of RAS-MEK pathway and increase when ERMS undergoes terminal differentiation after MEK inhibitor trametinib (MEKi) treatment. Analysis of super-enhancer landscape in MEKi-induced differentiated ERMS cells reveals a core transcriptional regulatory circuitry (CRC) including CASZ1, MYOD, and MYOG. Loss of CASZ1 attenuates MEKi-induced myogenic programming in ERMS. Restoration of CASZ1 in ERMS cells induces muscle differentiation. ChIP-seq, ATAC-seq, and RNA-seq results show that CASZ1 directly upregulates expression of skeletal muscle differentiation genes and represses non-muscle genes through affecting chromatin accessibility, enhancer maintenance, and super-enhancer activity. Next-generation sequencing of primary RMS tumors identified a nonsynonymous single-nucleotide variant causing an arginine-to-cysteine switch (R25C). When evaluated in 293T and ERMS cells, R25C impairs CASZ1 nuclear localization and fails to activate target genes’ transcription. Tumor xenograft studies show that CASZ1 suppresses RMS tumor growth (p<0.005), while the CASZ1-R25C mutant has no effect. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis. Citation Format: Zhihui Liu, Xiyuan Zhang, Haiyan Lei, Norris Lam, Oliver Yockey, Max Xu, Arnulfo Mendoza, Jun S Wei, Javed Khan, Marielle E. Yohe, Jack F. Shern, Carol J. Thiele. Mutant RAS represses CASZ1, a novel regulator of MYOD and MYOG, to inhibit embryonal rhabdomyosarcoma differentiation [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B42.