Epigenetic inactivation of oncogenic brachyury (TBXT) by H3K27 histone demethylase controls chordoma cell survival

Abstract

Equal contribution Corresponding authors Adrienne M Flanagan, a.flanagan@ucl.ac.uk, Telephone +44 (0) 20 7679 6304 and Udo Oppermann, udo.oppermann@sgc.ox.ac.uk, Telephone +44 (0) 1865 227308 2 2 AbstractThe expression of the transcription factor brachyury (TBXT) is normally restricted to embryonic development and its silencing after mesoderm development is epigenetically regulated. In chordoma, a rare tumour of notochordal differentiation, TBXT acts as a putative oncogene, and we hypothesised that its expression could be controlled through epigenetic inhibition. Screening of five chordoma cell lines revealed that only inhibitors of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (Jmjd3) reduce TBXT expression and lead to cell death, findings validated in primary patient-derived culture systems. Pharmacological inhibition of KDM6 demethylases leads to genome-wide increases in repressive H3K27me3 marks, accompanied by significantly reduced TBXT expression, an effect that is phenocopied by the dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Transcriptional profiles in response to a novel KDM6A/B inhibitor, KDOBA67, revealed downregulation of critical genes and transcription factor networks for chordoma survival pathways, whereas upregulated pathways were dominated by stress, cell cycle and pro-apoptotic response pathways.This study supports previous data showing that the function of TBXT is essential for maintaining notochord cell fate and function and provides further evidence that TBXT is an oncogenic driver in chordoma. Moreover, the data suggest that TBXT can potentially be targeted therapeutically by modulating epigenetic control mechanisms such as H3K27 demethylases.3 3

Frequent alterations in p16/CDKN2A identified by immunohistochemistry and FISH in chordoma

“…DNA methylation array data (450K or EPIC array, Illumina, CA, USA) of 35 chordoma samples have been published previously and EPIC array was performed on chordoma (UCH1, UM‐Chor, UCH7, MUG‐Chor) and U2OS cell lines. Nucleic acid was prepared as previously reported .…”

An update of molecular pathology of bone tumors. Lessons learned from investigating samples by next generation sequencing

“…This research has resulted in the opening of a recent phase II clinical trial using Afatinib, a third generation EGFR inhibitor. Finally, cancer driver events have also been identified in chromatin remodeling genes including SETD2 , ARID1A , and PBRM1 raising the possibility that chordoma may be susceptible to epigenetic inhibitors …”

“…Finally, cancer driver events have also been identified in chromatin remodeling genes including SETD2, ARID1A, and PBRM1 raising the possibility that chordoma may be susceptible to epigenetic inhibitors. 49,59,60 Poorly differentiated chordoma was first described by Mobley et al as tumors with cohesive epithelioid morphology, marked pleomorphism, and mitotic activity. They express TBXT and cytokeratins but unlike conventional chordoma additionally reveal loss of SMARCB1 expression on immunohistochemistry.…”

Orthogonal targeting of KDM6A/B and HDACs mediates potent therapeutic effects in IDH1-mutant glioma

“…KDM5-specific inhibitors, such as KDM5-C70 and KDOAM25a 26,27 , or pan 2-OG inhibitor IOX-1, were also largely ineffective on IDH1 -mutant cells. On the other hand, KDOBA67, a similar chemical to GSK-J4 that inhibits KDM6 enzymes 28 , displayed similar efficacy with GSK-J4 either individually or in combination with Belinostat ( Figure 6C and 6D ).…”

“…KDM5-specific inhibitors 24 , KDM5-C70 and KDOAM25a, or pan-2-OG inhibitor IOX-1, were also ineffective on IDH1 -mutant cells ( Figure S5C-D ). In contrast, KDOBA67, another compound targeting KDM6 enzymes 25 , displayed similar efficacy compared to GSK-J4 ( Figure S5C-D ). Targeting KDM6A and/or KDM6B genes via CRISPR/Cas9 demonstrated that ablation of both KDM6A and KDM6B phenocopied the effects observed with GSK-J4 in IDH1 -mutant cells ( Figure S5E-G ).…”