ID #470 Histone crotonylation is a novel subgroup difference in atypical teratoid rhabdoid tumors

Background Atypical teratoid rhabdoid tumors (ATRTs) are aggressive tumors of the central nervous system with a marked predilection for young children. While all ATRTs are driven by aberrant chromatin remodeling resulting from SWI/SNF inactivation, the three molecular subgroups- ATRT-TYR, ATRT-SHH, and ATRT-MYC- are defined by transcriptomic and DNA methylation profiling, suggesting a unique oncogenic mechanism. Crotonylation is a novel post-translational modification first discovered on histones that has emerged as a major regulator of gene expression implicated in many cancer types. Methods We utilized in vitro patient-derived ATRT neurosphere models, crotonylation immunoblotting, functional genomics, and high-resolution mass spectrometry to determine whether crotonylation differs between ATRT subgroups. Results ATRT-SHH cells harbor increased crotonylation at lysine (K) residues K14, K18, K23, and K27 in histone H3 compared to ATRT-MYC cells. Analysis of RNA expression across pediatric brain tumors demonstrates that five genes associated with known histone crotonyltransferases are significantly upregulated ATRT-SHH compared to ATRT-MYC. Together, these findings suggest that crotonylation is enhanced in ATRT-SHH and regulates gene expression to drive ATRT-SHH signaling pathways. We then individually deleted the five crotonyltransferase genes in ATRT-SHH cells to determine if they are necessary for crotonylation in ATRT-SHH and if crotonylation is required for ATRT-SHH cell viability. To establish if sodium crotonate (NaCr), a crotonylation precursor, is sufficient to enhance histone crotonylation in ATRT-MYC, we cultured ATRT-MYC cells in medium containing NaCr and monitored for increased histone crotonylation. Transcriptomic profiling, currently underway, will determine if the increase in histone crotonylation is sufficient to induce the ATRT-SHH signaling program. Lastly, to determine if crotonylation is globally increased in ATRT-SHH cells, all crotonylated proteins will be identified via mass spectrometry with a pan-KCr antibody. Conclusion Histone crotonylation is a subtype difference in ATRT and represents a driver of ATRT-SHH pathogenesis which may be amenable to subtype specific therapeutic interventions.