Core Regulatory Circuitry Research Articles

Long non-coding RNA JPX correlates with poor prognosis and tumor progression in non-small-cell lung cancer by interacting with miR-145-5p and CCND2

Emerging studies have shown that the aberrant expression and function of long non-coding RNAs (lncRNAs) are involved in carcinogenesis and the development of various cancers. The long noncoding RNA JPX (lncRNA JPX) on the X chromosome is an activator of X-inactive-specific transcript (XIST) and is a molecular switch for X-chromosome inactivation. However, the exact mechanism by which JPX acts in non-small-cell lung cancer (NSCLC) is not well studied. Here, through integrating clinical data and a series of functional experiments, we found that lncRNA JPX expression is significantly upregulated in NSCLC tissues compared with that in paired adjacent normal tissues from two independent datasets and significantly associated with a poor survival and other malignant phenotypes (tumor stage, tumor volume) of NSCLC. Furthermore, we elucidated that JPX functions as an oncogene in NSCLC-promoting cell proliferation and cell migration by affecting cell-cycle progression. Mechanistically, JPX upregulates cyclin D2 (CCND2) expression in a competing endogenous RNA mechanism by interacting with miR-145-5p, thus provoking the development and progression of NSCLC. These findings reveal the mechanism of X-chromosome lncRNA JPX and its core regulatory circuitry JPX/miR-145-5p/CCND2 in the development and progression of NSCLC, which bring us closer to an understanding of the molecular drivers of NSCLC.

A NOTCH feed-forward loop drives reprogramming from adrenergic to mesenchymal state in neuroblastoma

Transition between differentiation states in development occurs swift but the mechanisms leading to epigenetic and transcriptional reprogramming are poorly understood. The pediatric cancer neuroblastoma includes adrenergic (ADRN) and mesenchymal (MES) tumor cell types, which differ in phenotype, super-enhancers (SEs) and core regulatory circuitries. These cell types can spontaneously interconvert, but the mechanism remains largely unknown. Here, we unravel how a NOTCH3 intracellular domain reprogrammed the ADRN transcriptional landscape towards a MES state. A transcriptional feed-forward circuitry of NOTCH-family transcription factors amplifies the NOTCH signaling levels, explaining the swift transition between two semi-stable cellular states. This transition induces genome-wide remodeling of the H3K27ac landscape and a switch from ADRN SEs to MES SEs. Once established, the NOTCH feed-forward loop maintains the induced MES state. In vivo reprogramming of ADRN cells shows that MES and ADRN cells are equally oncogenic. Our results elucidate a swift transdifferentiation between two semi-stable epigenetic cellular states.

DREAM target reactivation by core transcriptional regulators supports neuroblastoma growth

ABSTRACTChromosome 17q gains are a common alteration in high-risk neuroblastomas with unknown functional significance. We identified a 17q super-enhancer regulated T-box Transcription Factor 2 (TBX2) as constituent of a core regulatory circuitry driving proliferation through enhancing V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived (avian) (MYCN)/Forkhead box protein M1(FOXM1) reactivation of dimerization partner, RB-like, E2F and multi-vulval class B (DREAM) targets, which can be affected synergistically by combined cyclin-dependent kinase 7 and Bromo-domain inhibition.

Enhancer Architecture and Essential Core Regulatory Circuitry of Chronic Lymphocytic Leukemia

Enhancer profiling is a powerful approach for discovering cis-regulatory elements that define the core transcriptional regulatory circuits of normal and malignant cells. Gene control through enhancer activity is often dominated by a subset of lineage-specific transcription factors. By integrating measures of chromatin accessibility and enrichment for H3K27 acetylation, we have generated regulatory landscapes of chronic lymphocytic leukemia (CLL) samples and representative cell lines. With super enhancer-based modeling of regulatory circuits and assessments of transcription factor dependencies, we discover that the essential super enhancer factor PAX5 dominates CLL regulatory nodes and is essential for CLL cell survival. Targeting enhancer signaling via BET bromodomain inhibition disrupts super enhancer-dependent gene expression with selective effects on CLL core regulatory circuitry, conferring potent anti-tumor activity.

Chromatin Accessibility Profiling Reveals Cis-Regulatory Heterogeneity and Novel Transcription Factor Dependencies in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by clinical and genomic heterogeneity. Recurrent IgH translocations, copy number abnormalities and somatic mutations have been reported to participate in myelomagenesis; however no universal driver of the disease has been identified. Here, we hypothesize that transcriptional deregulation is critical for MM pathogenesis and the maintenance of the MM cell state. In order to capture signatures of transcription factor engagement with the myeloma epigenome, we performed the assay for transposase-accessible chromatin sequencing (ATAC sequencing), deep RNA sequencing in 23 primary myeloma samples and 5 normal plasma cell samples (NPC) from healthy donors along with whole genome sequencing and H3K27ac ChIP-seq in a cohort of these primary MM samples. We identified 22,603 variable accessible loci between MM and NPC and correlated impact of these on expression of associated genes using RNA-seq data. Together with robust differential analysis of open chromatin regions and nuclease-accessibility footprints to identify discrete transcription factor binding events, we have discerned the myeloma-specific open chromatin landscape, identified transcription factor dependencies and potential new myeloma drivers. In our dataset we observe a vast number of loci with heterogeneous chromatin states across the sample cohort, and the majority of the open chromatin sites identified are unique to a single sample. However, distinct variable chromatin accessibility signatures indicative of the MM chromatin state when compared to normal plasma cells were observed. Remarkably, we observed more frequent recurrent loss of variable accessible loci compared to gains. In addition, specific open chromatin profiles evident in hyperdiploid and non-hyperdiploid MM were also identified. Accessibility footprinting revealed MM-specific enrichment for transcription factors known to be essential for MM cell survival including Interferon Regulatory Factors (IRFs), Nuclear Factor Kappa B (NFkB), Ikaros, and Sp1. Interestingly, we also identify the myocyte enhancer factor 2 (MEF2) family of transcription factors as being specifically enriched in open chromatin regions in MM cells. Using a CRISPR-Cas9 knockout system, we identify the MEF2 family member MEF2C as essential for MM cell proliferation and survival. MEF2C is significantly overexpressed at the RNA level in our study as well as in several independent cohorts and is a central enhancer-localized transcription factor in MM core regulatory circuitry as determined by H3K27ac ChIP-sequencing profiles of primary MM samples. In order to evaluate MEF2C as a therapeutic target, we used small molecule inhibitors targeting MEF2C activity via inhibition of MEF2C phosphorylation using inhibitors of salt-induced kinases (SIK) and microtubule-associated protein/microtubule affinity regulating kinases (MARK). SIK/MARK have been described to specifically activate MEF2C. SIK and MARK inhibition resulted in both dose- and time-dependent inhibition of MM cell growth and survival in a panel of 12 MM cell lines with various genotypic and phenotypic characteristics, revealing a potential approach to targeting the dysregulated gene regulatory state of myeloma. To conclude, here we identify here an altered chromatin accessibility landscape in multiple myeloma that likely contributes to oncogenic transcription states through the activity of transcription factors such as MEF2C, representing a new MM dependency and potential therapeutic target. DisclosuresAnderson:Millennium Takeda: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder. Young:Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Munshi:OncoPep: Other: Board of director.

TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract B20: Deciphering heterogeneity and plasticity in neuroblastoma

Abstract Through the analysis of neuroblastoma super-enhancer landscape, we could recently identy the core regulatory circuitries (CRC) that drive neuroblastoma identity, revealing two main types of identity: a noradrenergic identity defined by a CRC module including PHOX2B, HAND2, and GATA3, and a neural crest cell (NCC)-like identity driven by a CRC module containing AP-1 transcription factors (Boeva et al., Nature Genet 2017). We observed that the heterogeneous SK-N-SH cell line exhibited a mixed identity whereas the SH-SY5Y and SH-EP cell lines, subcloned from the parental SK-N-SH cell line, presented with a noradrenergic and NCC-like identity, respectively. We documented that treatment of SK-N-SH cells with chemotherapeutic agents resulted in enrichment of NCC-like cells. Here, we used several neuroblastoma cell lines, chemotherapeutic treatment, FACS sorting, and single-cell analysis using Chromium Single Cell 3 Solution (10X Genomics) to further investigate cell identity and plasticity in neuroblastoma. We showed that treatment of the noradrenergic SH-SY5Y cell line with chemotherapy resulted in a high proportion of cells exhibiting a mesenchymal phenotype, associated with a decreased expression of the noradrenergic module and increased expression of the NCC-like module. Interestingly, cells reacquired their neuroblastic morphology after treatment withdrawal. Differential analysis of RNA-seq data comparing group I (noradrenergic) and group II (NCC-like/mesenchymal) cell lines identified one membrane marker expressed in group II but not in group I cell lines. Interestingly, FACS analysis of the SK-N-SH cell line identified two distinct populations of negative and positive cells for this membrane marker, as well as few cells with a moderate expression. Further RNA-seq analysis indicated that sorted negative cells had highly similar expression profiles compared to noradrenergic SH-SY5Y cells, whereas sorted positive cells were close to the NCC-like/mesenchymal SH-EP cells, indicating that this membrane marker may be used as a surrogate marker of cell NCC-like/mesenchymal identity. Our recent data show that culturing SK-N-SH sorted cells presenting with high or low level of the surrogate marker for few weeks is sufficient to reconstitute cell heterogeneity, since FACS analysis after culture identifies again a heterogeneous population of cells with low, intermediate, or high level of expression of the surrogate marker. This observation is also true for the SH-SY5Y cell line presenting with a minor population of positive/NCC-like cells. However, in a subset of neuroblastoma cell lines our FACS analysis with the surrogate marker of NCC-like identified only a homogeneous population of cells, either negative or positive. These data highly suggest that some neuroblastoma cells exhibit plasticity and are able to shift between the NCC-like and noradrenergic identities. To further unravel cell identity heterogeneity in the SK-N-SH model, we performed single-cell RNA-seq analysis. We expect to be able to identify different populations of cells expressing specific gene signatures. The understanding of cell identity, heterogeneity, and plasticity in neuroblastoma is of upmost importance with respect to the development of new therapeutic strategies to eradicate tumor cells in neuroblastoma patients and improve treatment and outcome of this childhood cancer that continues to have very poor prognosis in its aggressive forms. Citation Format: Simon Durand, Valentina Boeva, Caroline Louis-Brennetot, Agathe Peltier, Cécile Pierre-Eugène, Sylvain Baulande, Olivier Delattre, Isabelle Janoueix-Lerosey. Deciphering heterogeneity and plasticity in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B20.

Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry.

Childhood high-risk neuroblastomas with MYCN gene amplification are difficult to treat effectively1. This has focused attention on tumor-specific gene dependencies that underlie tumorigenesis and thus provide valuable targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR-Cas9 approaches to detect genes involved in tumor cell growth and survival2–6, we identified 147 candidate gene dependencies selective for MYCN-amplified neuroblastoma cell lines, compared to over 300 other human cancer cell lines. We then used genome-wide ChIP-seq analysis to demonstrate that a small number of essential transcription factors: MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2, are members of the transcriptional core regulatory circuitry (CRC) that maintains cell state in MYCN-amplified neuroblastoma. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which act synergistically, in vitro and in vivo, with rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma that are essential for cell state and survival in this tumor.

Abstract 4131: Plasticity of transcriptional and epigenetic cellular states in neuroblastoma is driven by core lineage transcription factors

Abstract Background: Core Regulatory Circuitries (CRCs) of lineage transcription factors associate with super-enhancers and are drivers of cellular identity. We recently showed that most neuroblastomas include two types of tumor cells with divergent gene expression profiles. Undifferentiated mesenchymal (MES) cells and committed adrenergic (ADRN) tumor cells resemble cells from developmental stages of the adrenergic lineage. Results: We identified the unique super-enhancer (SE) landscapes of MES and ADRN-type cells. SEs associated with lineage transcription factors that identified the Core Regulatory Circuitries (CRC) for each cell type. MES cells were resistant to chemotherapy. Accordingly, MES cells were strongly enriched in post-treatment samples, suggesting that MES cells could play a role in resistance and relapse development. MES and ADRN cells of isogenic origin spontaneously interconverted, showing the transcriptional plasticity of CRCs. We studied the mechanism of MES and ADRN transdifferentiation. The MES TFs PRRX1 and NOTCH were expressed as single genes in ADRN cells. Each of the genes induced a step-wise reprogramming of the ADRN transcriptome towards a MES state. The transcriptional switch was accompanied by genome-wide remodeling of the epigenome to a MES enhancer state. MES TFs repressed super-enhancers of ADRN core TFs, leading to transcriptional downregulation of the ADRN CRC. Deregulated enhancers associated with Polycomb repression. The transdifferentiation was initially reversible, but became gradually stabilized. The transgenes activated an endogenous feed-forward loop including ligands, receptors and co-factors from the NOTCH signaling route. Ultimately, this endogenous NOTCH-cascade maintained a transgene-independent MES state. Super-enhancers in stable MES cell lines associated with NOTCH receptors and co-factors, consistent with NOTCH driving MES lineage identity. Both NOTCH-induced MES cells and ADRN cells were tumorigenic in vivo. Conclusions: Our results demonstrate that single TFs from the MES CRC impose transdifferentiation via remodeling of the epigenetic and transcriptional landscape of ADRN cells, mimicking spontaneous interconversion. Plasticity of CRCs and lineage identity may have profound implications for treatment strategies in neuroblastoma. Citation Format: Johan V. Nes, Tim V. Groningen, Jan Koster, Linda Valentijn, Danny Zwijnenburg, Ellen M. Westerhout, Mohamed Hamdi, Rogier Versteeg. Plasticity of transcriptional and epigenetic cellular states in neuroblastoma is driven by core lineage transcription factors [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 4131.

Abstract 2062: Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry

Abstract Childhood neuroblastomas with MYCN gene amplification form a particularly high-risk subset of this disease and are difficult to treat effectively. This has focused attention on tumor-specific gene dependencies that reflect important pathways in tumorigenesis, and thus could provide valuable targets for the development of novel therapeutics. Using genome-scale CRISPR-Cas9 approaches that allow unbiased detection of genes critically involved in tumor cell growth and survival, we identified 147 candidate genes associated with selective vulnerabilities in nine MYCN-amplified neuroblastoma cell lines, compared to findings in over 300 other human cancer cell lines representing multiple tumor cell types. We then used genome-wide ChIP-seq analysis to test the hypothesis that a small number of transcription factors - MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2, all represented in the selective dependency group - are members of the transcriptional core regulatory circuitry (CRC) that underlies cell state in MYCN-amplified neuroblastoma. We show that these transcription factors bind as dense clusters at defined epicentres within the enhancers of their own genes, as well as those of the other CRC transcription factor genes, creating a positive feed-forward autoregulatory loop that establishes and maintains high levels of gene expression. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which we postulated would act synergistically by targeting both transcriptional initiation and elongation required to synthesize regulatory transcription factors. MYCN-amplified neuroblastoma cells treated with both drugs were killed synergistically, in vitro and in vivo, and accompanied by rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma, a subset of which comprises the oncogenic transcriptional regulatory circuitry that underlies cell state and survival in this tumor. Citation Format: Adam D. Durbin, Mark W. Zimmerman, Neekesh V. Dharia, Brian J. Abraham, Brian J. Abraham, Amanda Balboni-Iniguez, Nina Weichert-Leahey, Shuning He, John M. Krill-Burger, David E. Root, Francisca Vazquez, Aviad Tsherniak, William C. Hahn, Todd R. Golub, Richard A. Young, A. Thomas Look, Kimberly Stegmaier. Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry [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 2062.

Abstract 2970: Multiple new susceptibility loci identified in genome-wide association study of Ewing sarcoma

Abstract Ewing sarcoma (EWS) is a rare pediatric tumor predominantly occurring in children of European ancestry and is characterized by the pathognomonic EWSR1-FLI1 fusion oncogene. To identify germline susceptibility loci associated with EWS risk, we performed a genome-wide association study (GWAS) meta-analysis of 749 EWS cases and 1,378 unaffected individuals of European ancestry from sample collections within the Institut Curie, National Cancer Institute and the Childhood Cancer Survivor Study. Our study replicated previously reported susceptibility loci at 1p36.22, 10q21.3 and 15q15.1 as well as identified novel loci at 6p25.1, 8q24.23, 20p11.22 and 20p11.23 (P-values<5×10-8). These seven EWS susceptibility loci discovered in only 749 cases make EWS one of the most productive GWAS studied cancers when considering a locus to case discovery ratio. All estimated effect estimates were high for cancer GWAS with odds ratios in excess of 1.7 observed. These high per allele effects among relatively common germline variants are striking in light of the rarity of EWS cases and lack of evidence of EWS as part of a familial cancer syndrome and therefore suggest a distinctive genetic architecture for EWS. Interestingly, in silico bioinformatics analysis identified that most EWS susceptibility loci reside near GGAA nucleotide repeat sequences where binding of the EWSR1-FLI1 fusion protein occurs. ChIP-seq analyses confirmed in vivo binding of EWSR1-FLI1, suggesting germline variation in these regions could alter EWSR1-FLI1 binding and potentially deregulate neighboring genes. To identify genes with allele specific expression differences, we carried out expression quantitative trait locus (eQTL) analyses at each identified EWS susceptibility locus. We identified eQTLs for plausible candidate genes at 6p25.1 with RREB1, a RAS-responsive element, and at 20p11.23 with KIZ, a centrosomal stabilization protein. We also noted the 20p11.22 locus is near NKX2-2, a highly overexpressed gene in EWS, although no eQTL was observed in our expression data. Furthermore, knockdown of EWSR1-FLI1 in EWS cell lines indicated a more than 2-fold difference in expression of RREB1 and NKX2-2, further supporting the role of specific regulation of these genes by EWSR1-FLI1 and suggesting RREB1 and NKX2-2 may be transcription factors involved in core regulatory circuitries of EWS. Overall, our study suggests a distinctive underlying genetic architecture for EWS in which moderate risk common germline variants interact with EWSR1-FLI1 binding to alter expression of nearby target genes. Citation Format: Mitchell J. Machiela, Thomas G. Grünewald, Didier Surdez, Stephanie Reynaud, Olivier Mirabeau, Eric Karlins, Rebeca Alba Rubio, Sakina Zaidi, Sandrine Grossetete-Lalami, Stelly Ballet, Eve Lapouble, Valérie Laurence, Jean Michon, Gaelle Pierron, Heinrich Kovar, Nathalie Gaspar, Udo Kontny, Anna González-Neira, Piero Picci, Javier Alonso, Ana Patino-Garcia, Nadège Corradini, Neal D. Freedman, Nathaniel Rothman, Casey L. Dagnall, Laurie Burdette, Kristine Jones, Michelle Manning, Kathleen Wyatt, Weiyin Zhou, Meredith Yeager, David G. Cox, Robert N. Hoover, Javed Khan, Gregory T. Armstrong, Wendy M. Leisenring, Smita Bhatia, Leslie L. Robison, Uta Dirksen, Markus Metzler, Wolfgang Hartmann, Konstantin Strauch, Thomas Kirchner, Andreas E. Kulozik, Lindsay M. Morton, Lisa Mirabello, Margaret A. Tucker, Franck Tirode, Stephen J. Chanock, Olivier Delattre. Multiple new susceptibility loci identified in genome-wide association study of Ewing sarcoma [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 2970.

Abstract 3358: FOXF1 defines the core-regulatory circuitry in gastrointestinal stromal tumor (GIST)

Abstract The cellular context that integrates upstream signaling and downstream nuclear response dictates the oncogenic behaviour and shapes treatment responses in distinct cancer types. Here, we uncover that in GIST, the forkhead family member, FOXF1, directly controls the transcription of two master regulators, KIT and ETV1, both required for GIST precursor-interstitial cells of Cajal (ICC) lineage-specification and GIST tumorigenesis. Further, FOXF1 co-localizes with ETV1 at enhancers and functions as a pioneer factor that regulates the ETV1-dependent GIST-lineage specific transcriptome through modulation of the local chromatin context, including chromatin accessibility, enhancer maintenance and ETV1 binding. Functionally, FOXF1 is required for human GIST cell growth in vitro and murine GIST tumor growth and maintenance in vivo. The simultaneous control of the upstream signaling and nuclear response sets up a unique regulatory paradigm and highlights the critical role of FOXF1 in enforcing the GIST cellular context for highly lineage-restricted clinical behaviour and treatment response. Citation Format: Leili Ran, Yuedan Chen, Wai Pung E. Wong, Dan Li, Zhen Cao, Shipra Shukla, Yuanyuan Xie, Deyou Zheng, Richard P. Koche, Cristina R. Antonescu, Yu Chen, Ping Chi. FOXF1 defines the core-regulatory circuitry in gastrointestinal stromal tumor (GIST) [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 3358.

MICMIC: identification of DNA methylation of distal regulatory regions with causal effects on tumorigenesis

Aberrant promoter methylation is a common mechanism for tumor suppressor inactivation in cancer. We develop a set of tools to identify genome-wide DNA methylation in distal regions with causal effect on tumorigenesis called MICMIC. Many predictions are directly validated by dCas9-based epigenetic editing to support the accuracy and efficiency of our tool. Oncogenic and lineage-specific transcription factors are shown to aberrantly shape the methylation landscape by modifying tumor-subtype core regulatory circuitry. Notably, the gene regulatory networks orchestrated by enhancer methylation across different cancer types are seen to converge on a common architecture. MICMIC is available on https://github.com/ZhangJlab/MICMIC.

FOXF1 Defines the Core-Regulatory Circuitry in Gastrointestinal Stromal Tumor.

The cellular context that integrates upstream signaling and downstream nuclear response dictates the oncogenic behavior and shapes treatment responses in distinct cancer types. Here, we uncover that in gastrointestinal stromal tumor (GIST), the forkhead family member FOXF1 directly controls the transcription of two master regulators, KIT and ETV1, both required for GIST precursor-interstitial cells of Cajal lineage specification and GIST tumorigenesis. Further, FOXF1 colocalizes with ETV1 at enhancers and functions as a pioneer factor that regulates the ETV1-dependent GIST lineage-specific transcriptome through modulation of the local chromatin context, including chromatin accessibility, enhancer maintenance, and ETV1 binding. Functionally, FOXF1 is required for human GIST cell growth in vitro and murine GIST tumor growth and maintenance in vivo The simultaneous control of the upstream signaling and nuclear response sets up a unique regulatory paradigm and highlights the critical role of FOXF1 in enforcing the GIST cellular context for highly lineage-restricted clinical behavior and treatment response.Significance: We uncover that FOXF1 defines the core-regulatory circuitry in GIST through both direct transcriptional regulation and pioneer factor function. The unique and simultaneous control of signaling and transcriptional circuitry by FOXF1 sets up an enforced transcriptional addiction to FOXF1 in GIST, which can be exploited diagnostically and therapeutically. Cancer Discov; 8(2); 234-51. ©2017 AACR.See related commentary by Lee and Duensing, p. 146This article is highlighted in the In This Issue feature, p. 127.

Mapping of the Multiple Myeloma Transcriptional Core Regulatory Circuitry Reveals TCF3 As a Novel Dependency and an Oncogenic Collaborator of MYC

Multiple Myeloma (MM) is a complex plasma cell malignancy driven by numerous genetic and epigenetic alterations that are acquired over time. The events controlling and modifying transcriptomic changes that drive MM cell growth and progression remains undefined. To reveal the epigenetic circuitry governing myeloma cells, we performed a comprehensive analysis integrating data obtained from Multiplexed Indexed T7 Chromatin IP (Mint-ChIP), Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), and RNA-seq in 10 primary MM cells as well as 3 MM cell lines to identify genome-wide the master transcription factors (TFs), the enhancer elements they occupy, and the genes they regulate. Using these data, we have identified myeloma-specific core regulatory circuitry which includes several well-established regulators of MM such as IKZF, E2F, MYC and IRF family of genes. For example, our data show elevated MYC at numerous tissue specific enhancers in myeloma cells, including those that regulate lineage specifying transcription factors such as IRF4 and TCF3 (aka E2A). When translocated to the immunoglobulin enhancer, MYC in turn is regulated by these lineage transcription factors thus integrating MYC into the interconnected transcriptional core regulatory circuitry of MM (Figure 1a,b).We propose that this oncogenic “re-wiring” accounts for the observed addiction of MM cells to lineage factors such as IRF4 and in this work, we implicate the B-cell factor TCF3 as a novel multiple myeloma dependency. Using myeloma cell lines and primary samples, we observed elevated enhancer activity at TCF3 in primary CD138+ cells from myeloma patients compared to normal plasma cells (NPCs) (Figure 1c). As a result, TCF3 expression is significantly upregulated in our large cohort of MM patients (n=370) compared to normal bone marrow plasma cells (n=18). As MYC proteins can only bind pre-established and acetylated regions of active chromatin, we hypothesize that enhancer specifying lineage transcription factors such as TCF3 may cooperate with MYC to alter tissue specific gene expression programs. We show that TCF3 is regulated by a large proximal enhancer that is bound by MYC, and is highly sensitive to chemical perturbation of enhancer co-activators such as BRD4. As a helix-loop-helix transcription factor that similar to MYC binds short (CANNTG) E-box sequences, we computationally predict co-occupancy of MYC and TCF3 at ~80% of all enhancers that form the multiple myeloma transcriptional core regulatory circuitry. To evaluate the functional role of TCF3 in myeloma cells, we established TCF3 knock down myeloma cell lines and followed the cell growth over time. Stable knockdown of TCF3 preferentially blocks proliferation of IgH MYC translocated cell lines (such as MM1.S cells) versus non-translocated lines (such as U266 cells). Finally, high expression of TCF3 correlates with poor clinical outcome in myeloma patients. Together these data suggest TCF3 acts as an oncogenic collaborator with deregulated MYC and implicates transcriptional control of lineage as a dependency in multiple myeloma.Figure 1: Transcriptional core regulatory circuitry of multiple myeloma: A) ChIP-Seq tracks of IRF4, MYC, BRD4, and H3K27ac occupancy at the IRF4, IgH enhancer, and TCF3 loci respectively. B) Schematic of transcription factor to enhancer connectivity of the partial multiple myeloma transcriptional core regulatory circuitry highlighting interactions between IRF4, MYC, and TCF3 (computationally predicted based on TCF3 motif data). C) ChIP-Seq tracks of H3K27ac occupancy at the TCF3 locus in patient multiple myeloma (top, n=3) or normal plasma cells (bottom, n=2). [Display omitted] DisclosuresBradner:Acetylon: Other: Scientific Founder; Novartis: Employment. Anderson:Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees.

Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.

Neuroblastoma is a tumor of the peripheral sympathetic nervous system, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.

Abstract LB-083: Core transcriptional regulatory circuitries in neuroblastoma

Abstract Background Neuroblastoma (NB) is a pediatric tumor derived from precursor cells of the sympathetic nervous system. NB accounts for 12% of all childhood cancer deaths with ~50% high-risk cases which frequently harbor amplified proto-oncogene MYCN. Evidence accumulates that epigenetic deregulation, including aberrant DNA methylation in high-risk disease or oncogene activation by enhancer hijacking, plays a prominent role in NB. The present study applies a comprehensive approach integrating chromatin modification data with genomic and expression data to elucidate NB subtype specific super-enhancer (SE) landscapes and core regulatory circuitries (CRCs) consisting of lineage-specific interconnected loops of SE-driven, auto-regulatory master transcription factors. Methods Chromatin immunoprecipitation sequencing (ChIP-seq) of histone 3 lysine 27 acetylation (H3K27ac) was used to identify active enhancer elements in 23 primary NBs. A validation cohort consisting of 16 NB cell lines and two human neural crest cell lines was used. ChIPmentation was applied to validate predicted transcription factor (TF) binding events. Circular chromatin conformation capture sequencing (4C-seq) was used to assay physical promoter-enhancer interactions. Results Unsupervised clustering of 23 primary NBs according to H3K27ac signal intensity at the most variable SEs (genome-wide) revealed two main subgroups, MYCN-amplified (n = 8) and MYCN single copy tumors (n = 15), with distinctive activity patterns. Calling of CRCs in the 23 primary NBs yielded a core set of NB master TFs (CRC TFs). Amongst the top ten of them are HAND2, PHOX2B and MYCN, all of which are implicated in NB biology and playing essential roles in the development of the sympathetic nervous system. In line with this, gene ontology analyses of the top 50 CRC TFs converge on biological processes like development of neural crest cells, sympathetic nervous system and peripheral nervous system neurons. ChIPmentation analyses of selected CRC TFs confirmed auto-binding to their assigned SEs and those of other CRC TFs in their respective network. Interactions between promoters and SEs of selected CRC TFs were verified via 4C-seq. Intriguingly, expression analysis of the top 50 CRC TFs in a cohort of 498 primary NBs revealed that less than 20% of the CRC TFs are up-regulated in MYCN-amplified tumors while the remaining 80% are down-regulated in that subgroup. This suggests a superordinate role of MYCN in differentially orchestrating NB master TFs. Conclusion The study identifies the core set of NB master transcription factors and assigns established NB regulators like HAND2, PHOX2B and MYCN to well-defined CRCs. It reveals an association of MYCN amplification with the global SE landscape of primary NBs and suggests a role for MYCN in differentially controlling subsets of CRC TFs and their networks. Specific targeting of the SE-dependent CRC networks may open a therapeutic window for epigenetic drugs, including BET inhibitors, CDK7 or EZH2 inhibition, in NB. Citation Format: Moritz Gartlgruber, Daniel Dreidax, Daria Doncevic, Sebastian Steinhauser, Stefan Gröschel, Kai Oliver Henrich, Young-Gyu Park, Carl Herrmann, Frank Westermann. Core transcriptional regulatory circuitries in neuroblastoma [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 LB-083. doi:10.1158/1538-7445.AM2017-LB-083

Abstract IA31: Phthalimide conjugation as a strategy for targeted protein degradation

Abstract Ligand-induced target protein destabilization is an efficacious therapeutic strategy in cancer, but discovery in this field has historically been serendipitous given the lack of rational design principles. Building on the molecular logic of immunomodulatory drugs (“IMiDs”), we innovated a facile chemical solution for targeted protein degradation using bifunctional small molecules capable of recruiting the E3 ligase cereblon (CRBN). These bifunctional molecules induce immediate, and complete proteasomal degradation of their target proteins and thus represent a novel class of small molecule therapeutics with an entirely uncharted and unique molecular pharmacology. Focusing our initial efforts on degraders of the BET family of proteins, we observed that they exhibit superior efficacy than BET inhibitors through unknown mechanisms. In order to dissect and mechanistically explain that differential molecular pharmacology, we used optimized small-molecule degraders and kinetic measures of chromatin structure and function to unveil an unrecognized, essential role for BRD4 in global control of transcriptional pause-release. Targeted BET degradation nucleates a collapse in the transcriptional core regulatory circuitry and thus enabled us to exploit transcriptional addictions in T-Cell acute lymphoblastic leukemia (T-ALL) that were intractable using competitive BET bromodomain inhibitors. Citation Format: Georg E. Winter, Dennis L. Buckley, James E. Bradner. Phthalimide conjugation as a strategy for targeted protein degradation. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA31.

Models of human core transcriptional regulatory circuitries.

A small set of core transcription factors (TFs) dominates control of the gene expression program in embryonic stem cells and other well-studied cellular models. These core TFs collectively regulate their own gene expression, thus forming an interconnected auto-regulatory loop that can be considered the core transcriptional regulatory circuitry (CRC) for that cell type. There is limited knowledge of core TFs, and thus models of core regulatory circuitry, for most cell types. We recently discovered that genes encoding known core TFs forming CRCs are driven by super-enhancers, which provides an opportunity to systematically predict CRCs in poorly studied cell types through super-enhancer mapping. Here, we use super-enhancer maps to generate CRC models for 75 human cell and tissue types. These core circuitry models should prove valuable for further investigating cell-type–specific transcriptional regulation in healthy and diseased cells.