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Key oncogenes can be misregulated in tumor cells by the acquisition of tumor-specific enhancers that direct gene expression through physical contacts (1,2). Identifying the full complement of enhancers regulating each oncogene could identify additional vulnerabilities in tumor expression programs, but remains challenging, because a given enhancer can regulate multiple genes and each of its targets may be genomically distant. Here we use DNA interaction data to construct gene regulatory landscapes for all expressed genes in a tumor cell and demonstrate that sets of enhancers can be assigned to these oncogenes using direct looping and insulated neighborhood data. A subset of these landscapes contains an exceptional amount of transcriptional apparatus, reminiscent of SEs (3), so we term this subset 3D-SEs. Well-characterized oncogenes, including c-Myc, acquire 3D-SEs in tumor cells where a c-Myc¬-associated linear SE was not identified, suggesting SE acquisition by c-Myc and other oncogenes has been underestimated. Each gene's regulatory network can also surprisingly extend beyond the confines of insulated neighborhoods and incorporate additional distal enhancers. Visualizing instances of 3D-SEs with microscopy demonstrates that they are components of liquid-liquid phase-separated bodies in cells, suggesting gene regulatory landscapes underpin these transcriptional condensates. The interactions comprising gene regulatory networks and their target genes thus extend previous interpretations of the targets of enhancers, signaling pathways, and disease-associated enhancer variants. References: 1. Hnisz, Abraham, Lee, et al. and Young, Cell 2013. 2. Mansour, Abraham, et al. and Young and Look, Science 2014. 3. Whyte, Orlando, Hnisz, Abraham, et al. and Young, Cell 2013. Citation Format: Brian J. Abraham, Yang Eric Guo, Denes Hnisz, Charles H. Li, Abraham S. Weintraub, Daniel S. Day, Jurian Schuijers, Tong Ihn Lee, Richard A. Young. Three-dimensional gene regulatory landscapes in normal and cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 971.

Oncogenic driver mutations are those that provide a proliferative or survival advantage to neoplastic cells resulting in clonal selection. Although most cancer causing mutations have been detected in the protein-coding regions of the cancer genome, driver mutations have recently also been discovered within noncoding genomic sequences. Thus, a current challenge is to gain precise understanding of how these unique genomic elements function in cancer pathogenesis, while clarifying mechanisms of gene regulation and identifying new targets for therapeutic intervention. Here we report a C-to-T single nucleotide transition that occurs as a somatic mutation in noncoding sequences 4 kb upstream of the transcriptional start site of the LMO1 oncogene in primary samples from patients with T-cell acute lymphoblastic leukaemia. This single nucleotide alteration conforms to an APOBEC-like cytidine deaminase mutational signature, and generates a new binding site for the MYB transcription factor, leading to the formation of an aberrant transcriptional enhancer complex that drives high levels of expression of the LMO1 oncogene. Since APOBEC-signature mutations are common in a broad spectrum of human cancers, we suggest that noncoding nucleotide transitions such as the one described here may activate potent oncogenic enhancers not only in T-lymphoid cells but in other cell lineages as well. Citation Format: Zhaodong Li, Brian Abraham, Alla Berezovskaya, Nadine Farah, Yu Liu, Theresa Leon, Adele Fielding, Shi Hao Tan, Takaomi Sanda, Abraham Weintraub, Benshang Li, Shuhong Shen, Jinghui Zhang, Marc Mansour, Richard Young, Thomas Look. APOBEC signature mutation generates an oncogenic enhancer that drives LMO1 expression in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5488. doi:10.1158/1538-7445.AM2017-5488

Transcriptional super-enhancers drive expression of oncogenes in many cancers and are being targeted with novel transcriptional and epigenetic therapeutics[1,2,3,4]. Super-enhancers are acquired by cancers through multiple mechanisms, including DNA translocation of an extant super-enhancer and focal amplification. We recently discovered a novel mechanism by which super-enhancers are nucleated in T cell acute lymphoblastic leukemias (T-ALLs)[5]. In this case, a small, monoallelic insertion creates a DNA binding site for a master transcription factor protein, which binds and recruits additional factors to nucleate the super-enhancer, which in turn drives high levels of the TAL1 oncogene. We describe here a method for unbiased identification of similar genomic insertions that nucleate potentially oncogenic regulatory elements in cancers. This approach uses data from genome-wide ChIP-Seq studies that map locations of enhancer-binding proteins to identify short DNA sequences missing from reference genomes. We have found and catalogued many additional genomic insertions in 80 additional cancers. An additional insertion has been functionally characterized and determined to be a bona fide enhancer-nucleating insertion that exists in patient genomes. I will describe new insights into the regulation of cancer that occur due to nucleation of novel regulatory elements. [1] Hnisz, Abraham, Lee, et al., Cell 2013 [2] Loven, Hoke, Lin, et al., Cell 2013 [3] Kwiatkowski, et al., Nature, 2014 [4] Wang, et al., Cell, 2015 [5] Mansour et al., Science 2014 Citation Format: Brian J. Abraham, Denes Hnisz, Abraham S. Weintraub, Nicholas Kwiatkowski, Charles H. Li, Sunniyat Rahman, Zhaodong Li, Tong Ihn Lee, A Thomas Look, Marc Mansour, Richard A. Young. Nucleation of transcriptional super-enhancers at tumor oncogenes by small insertions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2004.

Loss of function mutations in the plant homeodomain factor 6 (PHF6) are responsible for the Börjeson–Forssman–Lehmann syndrome, a familial X-linked intellectual disability disorder, and are observed in approximately 20% of adult T-cell acute lymphoblastic leukemias (T-ALLs) and 3% of adult acute myeloid leukemias (AMLs). However, mutations in B-cell lineage malignancies are notably absent. Interestingly, our recent work has uncovered PHF6 as a positive growth regulator in B-cell acute lymphoblastic leukemia (B-ALL) through a genome-scale in vivo loss-of-function screen. To identify the molecular mechanism by which PHF6 acts to promote B-ALL growth in vivo, we utilized CRISPR-Cas9 to delete Phf6 in murine B-ALL cells. Transplantation of Phf6 knockout cells (Phf6KO) into immunocompetent syngeneic recipients significantly extends disease latency and survival, therefore validating PHF6 as a bona fide positive growth regulator of B-ALL in vivo. Strikingly, these mice develop lymphoma-like disease with complete penetrance, characterized by significantly enlarged lymph nodes, decreased disease burden in the spleen and increased expression of the canonical T-cell marker CD4, suggesting that Phf6KO B-ALL cells transdifferentiated to cells resembling those of the T-cell lineage. To dissect the mechanism by which PHF6 regulates this lineage decision, we carried out a combination of RNA sequencing and chromatin immunoprecipitation (ChIP) analyses in Phf6WT and Phf6KO cells. RNA sequencing analysis revealed many differentially expressed genes in Phf6KO B-ALL cells , including gene sets involved in pathways important for B-cell development. ChIP-sequencing analysis of PHF6 and several histone marks (H3K27Ac, H3K27me3, H3K4me3) in Phf6WT B-ALL cells revealed that PHF6 and H3K27Ac signals co-localize close to the transcription start site of a significant proportion of differentially expressed genes. Transcription factor binding motif analysis revealed significant enrichment for several well-described master regulators of B-cell development, including PU.1, EGR-1, EBF-1, NF-κB and TCF3/TCF12. Notably, a number of these predicted transcription factors co-immunoprecipitated with PHF6 in Phf6WT B-ALL cells. These findings reveal an essential role for PHF6 in the maintenance of B-cell identity in B-ALL by activating, directly or indirectly, genes that are crucial for B-cell lineage commitment. Collectively, these results indicate that loss-of-function of PHF6 in B-ALL leads to transdifferentiation to the T-cell lineage, potentially explaining the apparent absence of PHF6 mutations in human B cell-lineage malignancies. Citation Format: Yadira M. Soto-Feliciano, Jordan ME Bartlebaugh, Yunpeng Liu, Francisco J. Sánchez-Rivera, Abraham S. Weintraub, Arjun Bhutkar, Tyler E. Jacks, Richard A. Young, Michael T. Hemann. The role of PHF6 in maintaining pre-B cell commitment in B-cell acute lymphoblastic leukemia. [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 A44.

Transcriptional super-enhancers drive expression of oncogenes in many cancers and are being targeted with novel transcriptional and epigenetic therapeutics (1,2,3,4). Super-enhancers are acquired in cancers through multiple mechanisms, including DNA translocation of an extant super-enhancer and focal amplification. We recently discovered a novel mechanism by which super-enhancers are nucleated in T cell acute lymphoblastic leukemias (T-ALLs) (5). In this case, a small, monoallelic insertion creates a DNA binding site for a master transcription factor protein, which binds and recruits additional factors to nucleate the super-enhancer, which in turn drives high levels of the TAL1 transcription factor. We describe here a method for unbiased identification of similar genomic insertions that nucleate potentially oncogenic regulatory elements in cancers. This approach uses data from genome-wide ChIP-Seq studies that map locations of enhancer-binding proteins to identify sequences missing from reference genomes. We have found many additional genomic insertions in many additional cancers. I will describe new insights into the regulation of cancer that occur due to nucleation of novel regulatory elements. 1 Hnisz, Abraham, Lee, et al., Cell 2013 2 Loven, Hoke, Lin, et al., Cell 2013 3 Kwiatkowski, et al., Nature, 2014 4 Chipumoro, et al., Cell, 2014 5 Mansour et al., Science 2014 Citation Format: Brian J. Abraham, Nicholas Kwiatkowski, Abraham S. Weintraub, Denes Hnisz, Nancy Hannett, Richard A. Young. Nucleation of transcriptional super-enhancers at tumor oncogenes. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR06.