Expanding The Tlx1 Regulome In T-Cell Acute Lymphoblastic Leukemia Towards Long Noncoding Rnas

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that results from the malignant transformation of T-cell precursors and affects children, adolescents and adults. In T-ALL, genetic lesions in several possible oncogenes and tumor suppressors have been shown to cooperatively contribute to leukemogenesis. The TLX1 (T-cell leukemia homeobox protein-1, HOX11) oncoprotein is aberrantly expressed in in 5-10% of pediatric patients and 30% of adult T-ALL patients due to chromosomal translocations. Although many downstream protein coding targets genes of TLX1 have been identified, the non-coding network downstream of TLX1 remains elusive. In this study we expand the TLX1 regulome towards long non-coding RNAs (lncRNAs). Experimental procedures: We measured the transcriptional response of all protein coding genes and lncRNAs following TLX1 knock down in the ALL-SIL cell line by polyA and total RNA-sequencing. In addition, similar mRNA-lncRNA expression profiles of 64 primary T-ALL patient samples were generated which included five TLX1+ cases. To establish the direct transcriptional TLX1 targets, we generated TLX1 and H3K27ac ChIP-sequencing data from ALL-SIL leukemic cells. Results: We confirm direct regulation of previously established protein coding gene targets and de novo TLX1 motif discovery also identified RUNX1 as an important mediator of the global TLX1 transcriptional network (Della-Gatta et al., Nature Medicine, 2012). Complementary to these data, our analysis for the first time establishes the TLX1 driven lncRNAome in thymocyte derived leukemic cells. Remarkably, the majority of TLX1 controlled lncRNAs were upregulated suggesting that they may be implicated in the TLX1 driven repression of protein coding gene expression. Notably, an important subset of these candidates is clearly associated with H3K27ac marked super-enhancer regions. Finally, pairwise mRNA-lncRNA correlation analysis allowed functional annotation of TLX1 targeted lncRNAs. To functionally interrogate candidate TLX1 regulated lncRNAs, LNA-mediated lncRNA knockdown experiments are currently performed as well as 4C-seq to explore the regulatory interactions in which these lncRNAs are involved. Conclusion: We present the first landscaping of the genome-wide binding pattern of TLX1 and provide evidence for a previously unestablished role of lncRNAs in the TLX1 regulatory network. Citation Format: Kaat Durinck, Wouter Van Loocke, Inge Van de Walle, Joni Van der Meulen, Pieter-Jan Volders, Nadine Van Roy, Yves Benoit, Bruce Poppe, Pieter Mestdagh, Jo Vandesompele, Pieter Rondou, Tom Taghon, Jean Soulier, Pieter Van Vlierberghe, Frank Speleman. Expanding the TLX1 regulome in T-cell acute lymphoblastic leukemia towards long noncoding RNAs. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A28.