MINI REVIEW published: 07 October 2020 doi: 10.3389/fimmu.2020.592087 The Genome in a Three-Dimensional Context: Deciphering the Contribution of Noncoding Mutations at Enhancers to Blood Cancer Llorenç Rovirosa 1, Alberto Ramos-Morales 1 and Biola M. Javierre 1,2* 13D Chromatin Organization Group, Josep Carreras Leukaemia Research Institute (IJC), Germans Trias i Pujol, Badalona, Spain, 2 Institute for Health Science Research Germans Trias i Pujol (IGTP), Badalona, Spain Associations between blood cancer and genetic predisposition, including both inherited variants and acquired mutations and epimutations, have been well characterized. However, the majority of these variants affect noncoding regions, making their mechanisms difficult to hypothesize and hindering the translation of these insights into patient benefits. Fueled by unprecedented progress in next-generation sequencing and computational integrative analysis, studies have started applying combinations of epigenetic, genome architecture, and functional assays to bridge the gap between Edited by: Ageliki Tsagaratou, noncoding variants and blood cancer. These complementary tools have not only The University of North Carolina at allowed us to understand the potential malignant role of these variants but also to Chapel Hill, United States differentiate key variants, cell-types, and conditions from misleading ones. Here, we Reviewed by: Panagiotis Ntziachristos, briefly review recent studies that have provided fundamental insights into our Northwestern Medicine, United States understanding of how noncoding mutations at enhancers predispose and promote Juan Ignacio Fuxman Bass, blood malignancies in the context of spatial genome architecture. Boston University, United States *Correspondence: Keywords: spatial genome architecture, 3D chromatin organization, DNA loops, noncoding mutations and Biola M. Javierre epimutations, enhancers, blood cancer, hematopoietic malignancies bmjavierre@carrerasresearch.org Specialty section: This article was submitted to INTRODUCTION: WHY WE NEED TO STUDY ENHANCERS B Cell Biology, AND SPATIAL GENOME ORGANIZATION TO UNDERSTAND a section of the journal BLOOD CANCER Frontiers in Immunology Received: 06 August 2020 Noncoding regions of the genome, which comprise more than 98% of the genome, have historically Accepted: 21 September 2020 been overlooked. However, more than 95% of the risk variants associated with genetically complex Published: 07 October 2020 diseases, such as blood cancer, map at noncoding regions and remain unexplored due to the lack of Citation: an obvious disease mechanism (1). In addition, the oncogenic potential of somatically acquired Rovirosa L, Ramos-Morales A and noncoding mutations has become increasingly evident (2). The mutational rate of the noncoding Javierre BM (2020) The Genome in a genome is significantly higher than its coding counterpart, although the selective pressure is weaker Three-Dimensional Context: unless the mutations have a functional effect that confers an advantage to cell survival (3). For this Deciphering the Contribution of Noncoding Mutations at reason, in the search for noncoding mutations it is important to find and focus on functional regions Enhancers to Blood Cancer. that impact gene regulation such as enhancers. Front. Immunol. 11:592087. Enhancers are frequent targets of genetic and epigenetic alterations in many diseases including doi: 10.3389/fimmu.2020.592087 blood cancer (1, 4–6). The human genome contains around one million enhancers, many of which Frontiers in Immunology | www.frontiersin.org 1 October 2020 | Volume 11 | Article 592087 Rovirosa et al. Noncoding Mutations in Blood Cancer are cell-type or stimulus-specific (7). They modulate the scientists identify target genes, thus helping us determine the disease activation of promoters and fine-tune transcription over large mechanism of enhancer mutations. genomic distances, independent of sequence orientation and In the following sections, we will review the current position (8). It is now generally accepted that long-range knowledge on the effect of noncoding mutations on enhancers enhancers are brought into close spatial proximity with the and genomic 3D structure in blood malignancies. We will promoters they regulate. This proximity is determined by the highlight the cognate clinical implications, not only in better DNA folding into loops, which are cell-type and stimulus- understanding oncogenesis, but also in the identification of specific (6, 9–11). potential new biomarkers and therapeutic strategies to improve Though enhancers have been relatively easy to identify—since disease diagnosis, monitoring and treatment. they harbor a high density of DNA motifs recognized by transcription factors (12–14), have specific histone modification and co-factor binding profiles (13, 15–17), and produce enhancer RNAs (18, 19) —their target genes are harder to pinpoint (7, 20). THE THREE-DIMENSIONAL CONTEXT Enhancers can be up to a few megabases away from their targets, OF THE GENOME PLAYS A MAJOR often jumping over several intervening genes or even being located ROLE IN THE DEVELOPMENT AND in the intron of a non-target gene (21). In addition, a given gene is PROGRESSION OF BLOOD CANCER frequently controlled by more than one enhancer, and an enhancer can control more than one gene (22, 23) (Figure 1). Enhancers Chromatin interactions are crucial for cellular health, and errors in cannot simply be assigned to the nearest gene, and most of their these interactions give rise to a broad range of diseases, including target genes in each cell-type and stimulus condition remain blood cancers (25–27). Cancer-associated alterations in chromatin unknown. For this reason, during the last years chromatin architecture increase proliferation and decrease differentiation conformation capture (3C) techniques, such as Hi-C (24), a capacity by blocking cell differentiation, altering the expression technique designed to identify the entire ensemble of of oncogenic or tumor suppressor genes, and/or creating unnatural chromosomal interactions within a cell population, have emerged cell states with deregulated expression of key developmental genes. to support that spatio-temporal chromatin organization not only Genomic regions brought into close proximity by the chromatin plays a key role in the function of enhancers, but will also help architecture have a higher frequency of producing translocations, FIGURE 1 | Enhancer–promoter interactions are complex and cell- and stimulus-specific. Frontiers in Immunology | www.frontiersin.org 2 October 2020 | Volume 11 | Article 592087 Rovirosa et al. Noncoding Mutations in Blood Cancer thus explaining the frequency of recurrent pathological conformational landscapes. Interestingly, some anti-leukemic translocations such as BCR-ABL and MYC-IGH (26, 27). In drugs partially reverse the altered topology of some genomic addition to chromosome-scale alterations, three-dimensional regions. For example THZ1, a covalent CDK7 inhibitor, has been chromatin organization works on a smaller scale to impact shown to abrogate MYC-enhancer aberrant contacts (44) and gene regulation by rewiring physical interactions between gene deter growth in T-ALL cell lines (60), potentially accounting for promoters and regulatory elements. This important spatial the anti-tumoral effect of these small-molecule inhibitors. organization is often altered in blood cancer through the Although we have just started understanding the role of genetic alteration or altered binding of the proteins involved in spatial-temporal genome architecture in blood cancer, there is establishing and maintaining chromatin loops. no doubt about the potential therapeutic opportunities it Architectural proteins, including CTCF and cohesins, play an encompasses. In order to understand changes in genomic important role in establishing and maintaining chromatin loops spatial architecture, we must first understand the causal (28, 29), facilitating communication of gene promoters with changes in the underlying DNA, resulting from both inherited some regulatory elements while reducing contacts with others variants and somatic mutations. (30–32). Cohesin complex mutations, which impair hematopoietic progenitor differentiation (33–35), occur in ~13% of acute myeloid leukemia (AML) patients (36–38). CTCF generally only binds non-methylated DNA sequences INHERITED GENETIC SUSCEPTIBILITY TO (39–42), so cancer-related methylation changes can impact BLOOD MALIGNANCY FROM THE THREE- genomic architecture broadly or at specific loci, although in DIMENSIONAL PERSPECTIVE some sites CTCF binding remains independent of DNA methylation suggesting that methylation may be one of many Blood cancer is not entirely explained by acquired genomic synergistic factors impacting transcription factor binding (43). A rearrangements, amplifications, deletions or mutations. In fact, recent study by Kloetgen and colleagues characterized global inherited genetic predisposition plays a key role (61–63). In fact, alterations in genome architecture in T-cell precursor acute survival rates and phenotype differ between racial and ethnic lymphoblastic leukemia (T-ALL) (44). The authors found that groups (64–66). The effect of genetic background in blood the lack of CTCF-mediated insulation at a specific locus allowed malignancies has been elegantly demonstrated by a recent the cancer-driver gene MYC to come into direct contact with a work from Young and colleagues in which they cross the Mll- distal super-enhancer, thus increasing this transcription factor’s AF9 knockin mouse with differing inbred strains (67). The expression, allowing MYC to turn on signal transduction authors show that genetic background not only impacts blood pathways leading to cell growth and proliferation. composition and survival rates, but also the type of malignancy, Interestingly, altered DNA methylation at enhancers in from ALL to AML or mixed phenotype acute leukemia. cancer is more closely related to changes in gene expression Risk loci frequently span noncoding regions (6, 68) and, than at promoters (45). Hypomethylated enhancers bind in fact, cluster at enhancers (1). The single-nucleotide transcription factors better than methylated ones (46–49), thus polymorphisms (SNPs) or structural variants (SVs) can alter promoting gene expression and potentially affecting genome the binding of transcription factors or structural proteins to architecture. Transcription factors can also influence spatial regulatory elements by modifying recognition motifs or chromatin organization, from ubiquitously expressed factors modulating accessibility, thus altering the expression of their such as YY1 (50) to cell-type specific factors including target genes (Figure 2). These inherited variants may either GATA1, LBD1, HSPs, KLF4, LacI, MYOD, and OCT4 (51–56). disrupt enhancer function, leading to reduced or lost target gene In addition to mutating these proteins, genetic or epigenetic expression, or increase enhancer function, leading to increased alterations can have profound effects on chromatin architecture target gene expression or even ectopic expression (5). In both by affecting the binding of these structural proteins (48, 57, 58). cases, if the binding of structural proteins is affected, the Acquired mutations, regardless of whether they directly affect enhancer–promoter interaction frequency shifts. However, if transcription, can alter spatial chromatin organization and transcription factor binding is altered, chromatin looping can chromatin states to ultimately promote cancer-specific either be compromised or not. transcriptional programs. This complex interplay is nicely Demonstrating this, Speedy and colleagues found that noncoding deciphered by Yun and colleagues who used an allelic series of genetic variants associated with chronic lymphocytic leukemia mutant mice to model normal, pre-malignant and AML states (CLL) predisposition map to CLL active chromatin, with evidence using the commonly co-occurring FLT3 and NPM1 mutations. of that chromatin state being CLL-specific or differentially Each mutation in isolation altered chromatin state, but together, regulated in normal B-cell differentiation (69). The risk alleles they synergized to cause global alterations in spatial chromatin disrupt SPI1, NFKB, and PAX5 binding motifs, or affect organization that produced a leukemia-associated transcriptional accessibility, suggesting a potential impact on enhancer activity. program (59), despite these mutations lacking direct influence on Using chromatin conformation data in parallel with expression transcription or epigenetics. For this reason, the development of data, they connected variants at enhancers with target gene therapeutic approaches against mutations also needs to take into promoters and observed that candidate genes are involved in account cancer-associated genome-wide epigenetic and B-cell biology including immune response, Wnt signaling and Frontiers in Immunology | www.frontiersin.org 3 October 2020 | Volume 11 | Article 592087 Rovirosa et al. Noncoding Mutations in Blood Cancer FIGURE 2 | Noncoding mutations and epimutations lead to enhancer loss- or gain-of-function. apoptosis. Although experimental validation is needed to oncogene CRLF2 in proximity to a distal enhancer, promoting completely understand the molecular mechanism by which CLL CRLF2-mediated constitutive activation of the JAK-STAT germline variants contribute to malignant transformation, this pathway, which has been implicated in leukemogenesis (73). study underlines the value of epigenetics and genome architecture This variant is not enough to cause cancer on its own, but it sets in interpreting genetic susceptibility. the stage for a driver mutation to tip the scales. Not only does The mechanistic insights acquired from inherited variants can GATA3 overexpression facilitate enhancer hijacking by oncogenes, be extrapolated to acquired mutations and epimutations. Indeed, but the authors propose that it also causes chromosomal instability germline risk variants co-segregate with acquired genomic and translocations, due to GATA3 binding sites clustering near abnormalities, although the interplay between these remains Philadelphia-like ALL translocation breakpoints. poorly understood (70, 71). A possible mechanism is that inherited variants alter the expression of pioneer transcription factors, which could synergize with the action of driver mutations ACQUIRED MUTATIONS DURING BLOOD to promote genome-wide gene deregulation and oncogenesis. Supporting this hypothesis, Yang and colleagues identified a MALIGNANCY TRANSFORMATION germline variant at the GATA3 intron that is strongly associated FREQUENTLY TARGET NONCODING with Philadelphia-like ALL (72). This enhancer gain-of-function REGULATORY ELEMENTS mutation upregulates GATA3 expression, which in turn, reprograms spatial genome architecture and chromatin Similar to inherited genetic variants, acquired mutations commonly accessibility genome-wide. This reprogramming puts the target enhancers, altering gene expression, in blood cancer (74). Frontiers in Immunology | www.frontiersin.org 4 October 2020 | Volume 11 | Article 592087 Rovirosa et al. Noncoding Mutations in Blood Cancer Mutations preferentially cluster at regulatory elements characteristic regulatory elements of naive B lymphocytes (85). Using to the cell-type from which the tumor originates (75). These structural data to connect cis-regulatory elements harboring mutations may be enhancer gain-of-function, enhancer loss-of- structural and point mutations to promoters, they identified function or enhancer hijacking. Of note, the last category can also putative noncoding driver mutations. These mutations alter be considered a gain-of-function and is frequently linked with major distal regulatory elements, leading to deregulation of target chromosomal rearrangements or insulator alteration (76, 77). gene transcription. Intriguingly, they also observed that coding Similar to mutations, epimutations such as DNA methylation and noncoding mutations often converged on the same genes. alteration, also target enhancers (45), and in principle, can affect For instance, MMP14 is a negative regulator of Notch signaling. It enhancers in similar ways (Figure 2). Silencers and other distal cis- plays a key role in normal B cell differentiation, the development regulatory elements can also be targeted by mutations. However, the of diffuse large B-cell lymphoma and patient survival (86, 87). narrow characterization of these elements and the incomplete Distal cis-regulatory elements of MMP14 are frequently deleted knowledge of their mechanism of action limit their study. in B-cell lymphoma, leading to MMP14 downregulation. On top Nevertheless, breakthroughs in genomics and molecular biology of that, coding mutations frequently affect the MMP14 gene in will put into play these elements in cancer genomics very soon. several malignancies, often resulting in loss-of-function. Many noncoding gain-of-function mutations, including This convergence needs to be investigated in other contexts, for copy number variation (duplications and deletions) and point example, whether recurrent coding mutations in blood mutations, have been associated with activating proto-oncogene malignancies, such as in histone modifiers and chromatin transcription in blood cancer. For instance, Herranz and remodeling factors, operate in parallel with noncoding mutations. colleagues identified a long-range enhancer controlled by Is there co-occurrence between the coding mutations of oncogenes NOTCH1 that has recurrent chromosomal duplications in and noncoding mutations at their regulatory elements in the same human T-ALL (78). This region is activated through a malignant cell or tumor type? If so, is this a synergy, an exclusion, mechanism of nucleosome eviction mediated by GATA3 (79), or a redundant event? Could a cancer-related reduction of a given it physically interacts with the MYC promoter 1.4 megabases transcription factor level be accompanied by a reduction of its away to induce MYC transcription, and it has a fundamental role binding at regulatory elements due to mutations or epimutations in the homeostasis of immature T cells. This gain-of-function at its recognition motifs? Or do tumoral cells use one or the amplification drives MYC expression downstream of NOTCH1 other mechanism exclusively to silence a given transcription factor? in T-ALL and helps to connect MYC and Notch signaling in Many questions remain open that the scientific community driving oncogenesis; a different mechanism but with the same will have to address using the fast-growing number of effect as the previous mentioned study in which an impaired methodological breakthroughs. CTCF binding (due to reduced chromatin accessibility) leads to Noncoding loss-of-function mutations at enhancers also MYC enhancer hijacking (44). Regarding deletions, Liu and contribute to cancer pathology by silencing tumor-suppressor colleagues identified a recurrent deletion of a noncoding region genes, although these are less well characterized. These in T-ALL using cis-X, a computational method for identifying noncoding mutations, similar to gain-of-function ones, involve regulatory noncoding alterations (80). This deletion destroys different types of structural, copy number, and point mutations the nodal CTCF binding site that forms the boundary of (Figure 2). A recent paper by Li and colleagues identified and two independent, insulated genomic neighborhoods. As a validated more than two hundred tumor suppressive or oncogenic consequence, this structural variation allows the hijacking of enhancers recurrently mutated in hematopoietic malignancies an active enhancer from one neighborhood by the PRLR using enhancer CRISPR/dCas9 perturbation (activation or promoter located in the other neighborhood, leading to PRLR repression) (88). Interestingly, some of these reside in proximity upregulation and JAK2 signaling activation. In the same paper, to nuclear receptor–binding genomic regions, contributing to the authors also report a recurrent intronic point mutation that aberrant nuclear receptor signaling in blood malignancies. One activates TAL1 oncogene transcription, a frequently mutated example is two noncoding variants within the PER2 enhancer that oncogene in T-ALL (81, 82). This point mutation creates a have tumor suppressive properties in AML and are potentially new active enhancer characterized by a de novo recognition controlled by the nuclear receptor program. PER2 controls motif for YY1. Interestingly, this transcription factor has circadian rhythm and it has been suggested as a tumor structural properties similar to CTCF in bridging promoter– suppressor gene (89–91). This work elegantly addresses the enhancer chromatin interactions (50), in addition to previous major challenge of prioritizing noncoding mutations and studies that show that ectopic expression of TAL1 can be also due assigning functional relevance to them. Approaches like this will to CTCF binding alterations that cause loss of TAD boundary be needed to translate the knowledge about the noncoding insulation and subsequently enhancer hijacking (83, 84). genome in blood cancer into patient benefits. Functional validation will be needed to better understand the complex interplay between noncoding genetic mutations, transactivation and three-dimensional chromatin organization to promote malignant transformation. FUTURE PERSPECTIVES Noncoding copy number variation and point mutations are not exclusive to T-ALL. For instance, Cornish and colleagues Even though compelling evidence for the contribution of identified recurrent mutations in B-cell lymphoma at cis- noncoding mutations and epimutations to oncogenesis exists, Frontiers in Immunology | www.frontiersin.org 5 October 2020 | Volume 11 | Article 592087 Rovirosa et al. Noncoding Mutations in Blood Cancer there is still a long way to go before we can translate them into as ChIA-PET (92) or HiChIP (93), is crucial. Nonetheless, all these new strategies to characterize, treat and monitor blood cancer. methods need millions of cells, which hinders the analysis of rare To achieve this translational challenge, many considerations cell populations such as hematopoietic stem cells or hematopoietic should be taken into account. precursors, which are the origin cells of the majority of leukemias. First, reliably identifying noncoding driver mutations from Methodological breakthroughs allowing lower inputs will be passenger ones remains a great endeavor due to sequencing and fundamental in the incipient era of noncoding driver mutations. mapping artifacts, poorly understood mutational processes, and In conclusion, the current methodological breakthroughs have inaccurate estimation of the mutational background. To face positioned the scientific community in a perfect situation to these impediments, adequate statistical methods, larger datasets, explore the noncoding genome in the context of cancer. Cancer higher sequence coverage, and longer and more accurate genomics is rapidly moving from a static, one-dimensional sequencing reads will be fundamental. In addition, the picture, to a time-dependent three-dimensional scenario to sequencing of normal tissues alongside malignant ones can provide biological relevance of noncoding mutations at help separate acquired from germline mutations and shed light regulatory elements. We anticipate a very exciting time ahead, in on differentiating driver from passenger mutations. which we will be fascinated by the power of noncoding mutations Second, given the vastness of the noncoding genome, we need and epimutations in malignant transformation and the new to restrict the search to relevant noncoding driver mutations. To clinical opportunities these genetic alterations will involve. this end, comparative genomic analysis, high-throughput in vitro reporter assays, and genome-wide histone modification profiling, coupled with chromatin accessibility and expression analysis, are indispensable. However, due to the highly dynamic, cell- and AUTHOR CONTRIBUTIONS stimulus-specific nature of regulatory regions, it is critical to All authors listed have made a substantial, direct, and intellectual identify the right cell-type and to extend this descriptive contribution to the work and approved it for publication. interrogation not only to the cells of origin but also to the transformed cells. Third, the unknown functional role of noncoding mutations imposes important limitations. To ascertain biological and FUNDING mechanistic relevance, it is essential to integrate genetic and epigenetic profiling with genome conformation data LR is funded by AGAUR project number 2019FI-B00017 of the and CRISPR-based functional validation. As previously Catalan Government (Generalitat de Catalunya). AR-M is described, many noncoding mutations map to enhancers, and funded by the José Carreras Leukämie-Stiftung (08R/2019). BJ these can exert their pathological function by altering the is funded by FEDER/Spanish Ministry of Science and Innovation expression level of their target genes. However, connecting (project number RTI2018-094788-A-I00), by La Caixa Banking enhancers and target genes is not trivial, and due to cell- Foundation Junior Leader project (LCF/BQ/PI19/11690001), by specificity and complexity, most of the associations remain the José Carreras Leukämie-Stiftung (08R/2019), and by the unknown. Quantitative associations between noncoding variants European Hematology Association Advance Research Grant. and gene expression, and regulatory biochemical properties The funder bodies were not involved in the study design, combined with sequencing-based chromatin conformation collection, analysis, interpretation of data, the writing of this capture methods, such as Hi-C (24), have started providing article or the decision to submit it for publication. some insights. However, reliable and reproducible identification of significant interactions between individual restriction fragments is not feasible unless Hi-C libraries are subjected to ultra-deep ACKNOWLEDGMENTS sequencing, which is not an economically viable solution for analyzing a comprehensive collection of cell-types or tumoral We apologize to the authors of many relevant studies for not samples. To overcome it, the development of sequence-specific citing their work due to space limitations. We thank the capturing approaches to enrich for promoter interactions and members of the Javierre laboratory for helpful discussion and mutations in Hi-C libraries (6, 11, 69, 85), or other methods such Christina Usher for her editing suggestions. 3. Weinhold N, Jacobsen A, Schultz N, Sander C, Lee W. Genome-wide analysis REFERENCES of noncoding regulatory mutations in cancer. Nat Genet (2014) 46(11):1160– 1. Maurano MT, Humbert R, Rynes E, Thurman RE, Haugen E, Wang H, 5. doi: 10.1038/ng.3101 et al. 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The use, distribution or reproduction in other forums is permitted, provided (1997) 91(7):1055–64. doi: 10.1016/S0092-8674(00)80495-X the original author(s) and the copyright owner(s) are credited and that the original 90. Gery S, Gombart AF, Yi WS, Koeffler C, Hofmann W-K, Koeffler HP. publication in this journal is cited, in accordance with accepted academic practice. No Transcription profiling of C/EBP targets identifies Per2 as a gene implicated use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 9 October 2020 | Volume 11 | Article 592087