Enhancer Landscape Research Articles

In This Issue

In This Issue

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Abstract PR007: A local tumor microenvironment acquired super-enhancer induces an oncogenic driver for efficient growth under oxidative conditions in colorectal carcinoma

Abstract Tumors exhibit widespread enhancer landscape reprogramming compared to normal tissue. The etiology is believed to be largely cell-intrinsic in non-hormonal cancers, attributed to such genomic alterations as focal amplification of non-coding regions, aberrant activation of transcription factors, and non-coding mutations creating de novo transcription factor binding sites. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon epithelia, we find divergent epigenetic landscapes between primary CRC tumors and CRC cell lines. We identify a unique super-enhancer signature largely absent in cell culture. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over patient-matched normal epithelium suggesting novel insight into the etiology of enhancer reprogramming in CRC and its downstream relevance to tumor biology. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. CRISPR-dcas9-KRAB interference of this super-enhancer identifies PDZK1IP1 as its target gene, a gene previously observed to be highly up-regulated in CRC. However, the mechanism behind its transcriptional activation and contribution to tumor growth is not fully understood. We restored both the super-enhancer and PDZK1IP1 expression levels following treatment with cytokines or xenotransplantation into nude mice, which was also associated with high levels of cytokines, thus demonstrating its etiology via local tumor microenvironment acquisition. Deletion of inflammatory transcription factors RELA and STAT3 in human CRC cells inhibits PDZK1IP1 induction in xenografts. PDZK1IP1 appears to be critical for CRC growth in the setting of its super-enhancer induction in xenografts, but not in cell culture where the super-enhancer is absent and expression is largely silent. Building on its known role in glucose uptake via SGLT receptors, we demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity of CRC cancer cells via the pentose phosphate pathway using polar metabolomic profiling. We show this activation enables efficient growth under oxidative conditions both in vitro and in vivo, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the biologic significance of epigenomic profiling on patient-matched primary specimens and identify this microenvironment-acquired super-enhancer as an oncogenic driver in the setting of the inflamed tumor that is commonly upregulated in CRC. Citation Format: Royce Zhou, Ramon Parsons. A local tumor microenvironment acquired super-enhancer induces an oncogenic driver for efficient growth under oxidative conditions in colorectal carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR007.

OR16-4 Genomic Interrogation of the Phytoestrogen Genistein in Ovarian Cancer Cells

Abstract Although studies have shown beneficial effects of the phytoestrogen genistein on reducing ovarian cancer risk, little is known at the molecular level regarding how genistein inhibits ovarian cancer growth, with estrogen receptor beta (ERb), a hormone-regulated transcription factor, receiving the bulk of the attention. When endogenous and exogenous ligands engage ERb, the receptor binds to regulatory regions in the genome known as enhancers, where it associates with coregulatory proteins, increases histone modifications associated with active enhancers, promotes chromatin looping, stimulates enhancer transcription and the production of enhancer RNAs, and ultimately regulates target gene transcription. Since signaling pathways converge at the transcriptome level, an unbiased approach is needed to examine how genistein acts to suppress ovarian cancer growth beyond ERb. We have previously used global run-on sequencing (GRO-seq), a high-throughput method that identifies the location and orientation of all transcriptionally active RNA polymerases across the genome, to identify active enhancers based on enhancer transcription and enrichment of the aforementioned genomic features. To determine the genomic responses upon genistein treatment in ovarian cancer cells, we have used PRO-seq to characterize the enhancer landscape in OVCAR3 and OVCAR4 ovarian cancer cell lines treated with genistein. The advantage of this method is the lack of a priori assumptions needed to determine which signaling pathways are regulated by genistein, since PRO-seq allows pathway determination based on the universe of active enhancers. Ultimately, this approach will allow us to uncover signaling pathways beyond ERb and determine how genistein acts as a signaling ligand to change the transcriptome in ovarian cancer cells. Presentation: Sunday, June 12, 2022 11:45 a.m. - 12:00 p.m.

OR26-6 | LBMON298 The Role Of Nuclear Receptor Coregulator Grip1 In Homeostatic Programming Of Macrophages

Abstract Aberrant macrophage function is implicated in acute and chronic inflammatory settings, and central to diseases such as obesity, atherosclerosis and cancer. However, the transcriptional and epigenomic programs driving macrophage polarization toward distinct phenotypic states are not well understood. Macrophage phenotypes exist on a spectrum, with pro-inflammatory (M1-like) and homeostatic, tissue repair (M2-like) states viewed as polar opposites and relatively stable. The main driver of the M2-like program is a type-2 cytokine, IL4 which signals to STAT6 followed by the Krüppel-like factor 4 (KLF4), a master regulator for numerous M2-specific genes. Reportedly, glucocorticoid (GC) hormones also promote the M2-like state, although the extent to which the phenotypes elicited by such disparate signals overlap is unknown. Interestingly, we discovered that GRIP1, a well-known cofactor for nuclear receptors including GR, serves as a KLF4 coactivator, suggesting that GRIP1 could mediate transcriptional and phenotypic convergence of the IL4- and GC-driven pathways. Gene expression profiling on differentially polarized macrophages revealed a highly significant overlap between M2IL4 and M2GC transcriptomes which was mirrored by the H3K27ac de novo enhancer landscape. Notably, loss of GRIP1 blunted both up-regulation of M2- and downregulation of M1-associated genes irrespective of the signal driving M2 polarization. At the cellular level, polarization with either IL4 or GCs enhanced macrophage phagocytosis relative to non-polarized M0, and did so in a GRIP1-dependent manner. Finally, in the dextran sulphate sodium (DSS)-induced colitis model of intestinal inflammation and tissue damage, GRIP1-cKO mice lacking GRIP1 in myeloid cells, including macrophages, displayed more severe destruction of the colonic mucosa and greater immune cell infiltration into the mucosa and submucosa. Moreover, in total colon RNA, the abundance of many characteristic M2 transcripts was reduced in the cKO mice. Surprisingly, the expression of proinflammatory mediators such as TNF, IL1b and IL23 was unaffected by GRIP1 deletion, suggesting that the more severe disease in the cKO stems primarily from the failure of M2-like resolving macrophage to efficiently mitigate tissue destruction and repair the damage. Together, these findings point to GRIP1 as a crucial regulator of macrophage polarization to the homeostatic state in vitro and in vivo. Presentation: Monday, June 13, 2022 12:15 p.m. - 12:30 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Enhancer and super-enhancer landscape in polycystic kidney disease

Widespread aberrant gene expression is a pathological hallmark of polycystic kidney disease (PKD). Numerous pathogenic signaling cascades, including c-Myc, Fos, and Jun, are transactivated. However, the underlying epigenetic regulators are poorly defined. Here we show that H3K27ac, an acetylated modification of DNA packing protein histone H3 that marks active enhancers, is elevated in mouse and human samples of autosomal dominant PKD. Using comparative H3K27ac ChIP-Seq analysis, we mapped over 16000 active intronic and intergenic enhancer elements in Pkd1-mutant mouse kidneys. We found that the cystic kidney epigenetic landscape resembles that of a developing kidney, and over 90% of upregulated genes in Pkd1-mutant kidneys are co-housed with activated enhancers in the same topologically associated domains. Furthermore, we identified an evolutionarily conserved enhancer cluster downstream of the c-Myc gene and super-enhancers flanking both Jun and Fos loci in mouse and human models of autosomal dominant PKD. Deleting these regulatory elements reduced c-Myc, Jun, or Fos abundance and suppressed proliferation and 3D cyst growth of Pkd1-mutant cells. Finally, inhibiting glycolysis and glutaminolysis or activating Ppara in Pkd1-mutant cells lowerd global H3K27ac levels and its abundance on c-Myc enhancers. Thus, our work suggests that epigenetic rewiring mediates the transcriptomic dysregulation in PKD, and the regulatory elements can be targeted to slow cyst growth.

Genome-wide analysis of the FOXA1 transcriptional regulatory network identifies super enhancer associated LncRNAs in tamoxifen resistance.

Breast cancer is the leading cause of death in female cancers, and what’s worse, tamoxifen resistance occurs in almost 30% breast cancer patients and has seriously attenuated the therapeutic effect. It is widely studied that epigenetic regulation has played important role in the development of tamoxifen resistance. FOXA1 is a pioneer transcription factor that can translate epigenetic signature into transcription regulation and also drive genome-wide enhancer reprogramming in breast cancer. However, the chromatin super enhancer landscape orchestrated by FOXA1 and the key downstream targets of the FOXA1 oncogenic network in tamoxifen resistance remain elusive. Through analyzing the FOXA1 ChIP-seq data in tamoxifen sensitive MCF7 and tamoxifen resistant MCF7/TamR cells, we show that the FOXA1 chromatin occupancy is enhanced in both the promoter and enhancer regions, and the recruitment events may be E2 dependent in both MCF7 and MCF7/TamR cells. By integratively analyzing the FOXA1 ChIP-seq data and RNA-seq data of MCF7 and MCF7/TamR cells, we find that the enhanced or reduced FOXA1 chromatin binding densities may synchronize the transcriptional activity in tamoxifen resistance. Besides, we identify 1003 super enhancer associated protein coding genes and five super enhancer associated lncRNAs (ATP1A1−AS1, CASC11, CASC15, KCTD21−AS1, LINC00885) in tamoxifen resistance. By KM survival analysis, we find that high expression level of ATP1A1−AS1 and its sense transcript ATP1A1 indicates favorable clinical outcome among the luminal endocrine treated breast cancer patients. Further coexpression analysis indicates that ATP1A1-AS1 is significantly correlated with ATP1A1, and RT-qPCR results show that they both are downregulated in MCF7/TamR cells. Our study shows that the FOXA1 transcriptional regulatory network may promote the development of tamoxifen resistance, and identifies one super enhancer associated lncRNA ATP1A1-AS1 that may work as promising biomarker or drug target in tamoxifen resistance.

Single-cell sequencing reveals activation of core transcription factors in PRC2-deficient malignant peripheral nerve sheath tumor.

SUMMARYLoss-of-function mutations in the polycomb repressive complex 2 (PRC2) occur frequently in malignant peripheral nerve sheath tumor, an aggressive sarcoma that arises from NF1-deficient Schwann cells. To define the oncogenic mechanisms underlying PRC2 loss, we use engineered cells that dynamically reassemble a competent PRC2 coupled with single-cell sequencing from clinical samples. We discover a two-pronged oncogenic process: first, PRC2 loss leads to remodeling of the bivalent chromatin and enhancer landscape, causing the upregulation of developmentally regulated transcription factors that enforce a transcriptional circuit serving as the cell’s core vulnerability. Second, PRC2 loss reduces type I interferon signaling and antigen presentation as downstream consequences of hyperactivated Ras and its cross talk with STAT/IRF transcription factors. Mapping of the transcriptional program of these PRC2-deficient tumor cells onto a constructed developmental trajectory of normal Schwann cells reveals that changes induced by PRC2 loss enforce a cellular profile characteristic of a primitive mesenchymal neural crest stem cell.

Enhancer looping protein LDB1 regulates hepatocyte gene expression by cooperating with liver transcription factors.

Enhancers establish proximity with distant target genes to regulate temporospatial gene expression and specify cell identity. Lim domain binding protein 1 (LDB1) is a conserved and widely expressed protein that functions as an enhancer looping factor. Previous studies in erythroid cells and neuronal cells showed that LDB1 forms protein complexes with different transcription factors to regulate cell-specific gene expression. Here, we show that LDB1 regulates expression of liver genes by occupying enhancer elements and cooperating with hepatic transcription factors HNF4A, FOXA1, TCF7 and GATA4. Using the glucose transporter SLC2A2 gene, encoding GLUT2, as an example, we find that LDB1 regulates gene expression by mediating enhancer-promoter interactions. In vivo, we find that LDB1 deficiency in primary mouse hepatocytes dysregulates metabolic gene expression and changes the enhancer landscape. Conditional deletion of LDB1 in adult mouse liver induces glucose intolerance. However, Ldb1 knockout hepatocytes show improved liver pathology under high-fat diet conditions associated with increased expression of genes related to liver fatty acid metabolic processes. Thus, LDB1 is linked to liver metabolic functions under normal and obesogenic conditions.

KLF4 recruits SWI/SNF to increase chromatin accessibility and reprogram the endothelial enhancer landscape under laminar shear stress

Physiologic laminar shear stress (LSS) induces an endothelial gene expression profile that is vasculo-protective. In this report, we delineate how LSS mediates changes in the epigenetic landscape to promote this beneficial response. We show that under LSS, KLF4 interacts with the SWI/SNF nucleosome remodeling complex to increase accessibility at enhancer sites that promote the expression of homeostatic endothelial genes. By combining molecular and computational approaches we discover enhancers that loop to promoters of KLF4- and LSS-responsive genes that stabilize endothelial cells and suppress inflammation, such as BMPR2, SMAD5, and DUSP5. By linking enhancers to genes that they regulate under physiologic LSS, our work establishes a foundation for interpreting how non-coding DNA variants in these regions might disrupt protective gene expression to influence vascular disease.

Decoding YAP dependent transcription in the liver

The transcriptional coactivator YAP is emerging as a master regulator of cell growth. In the liver, YAP activity is linked to hepatomegaly, regeneration, dedifferentiation, and aggressive tumor growth. Here we present genomic studies to address how YAP may elicit such profound biological changes in murine models. YAP bound the genome in a TEAD-dependent manner, either at loci constitutively occupied by TEAD or by pioneering enhancers, which comprised a fraction of HNF4a/FOXA-bound embryonic enhancers active during embryonic development but silent in the adult. YAP triggered transcription on promoters by recruiting BRD4, enhancing H3K122 acetylation, and promoting RNApol2 loading and pause-release. YAP also repressed HNF4a target genes by binding to their promoters and enhancers, thus preventing RNApol2 pause-release. YAP activation led to the induction of hepatocyte proliferation, accompanied by tissue remodeling, characterized by polarized macrophages, exhausted T-lymphocytes and dedifferentiation of endothelial cells into proliferative progenitors. Overall, these analyses suggest that YAP is a master regulator of liver function that reshapes the enhancer landscape to control transcription of genes involved in metabolism, proliferation, and inflammation, subverts lineage specification programs by antagonizing HNF4a and modulating the immune infiltrate and the vascular architecture of the liver.

510 The role of histone demethylase UTX on epidermal homeostasis and carcinogenesis

Histone modifiers are among the most highly mutated genes in all forms of cancer, with histone demethylase UTX (KDM6A) being one of the most frequent. Found on the X chromosome, UTX is one of the few genes to escape X inactivation and functions as a major enhancer regulator. UTX establishes the active enhancer landscape through its histone demethylase activity as well as its ability to complex with other activating histone modifiers. UTX is implicated in many epithelial cancers and is commonly mutated or lost in cutaneous squamous cell carcinoma (cSCC). This is supported by loss of UTX expression in cSCC upon immunohistochemical staining. UTY, the Y-linked paralog of UTX, retains minimal catalytic function but can potentially compensate for UTX loss by other mechanisms. This may account for some of the sex specific differences that have been observed in the risk and severity of cSCC, given that mutations in UTX are typically heterozygous. Despite its demonstrated role in tumor suppression across cancers, there is virtually no understanding of how it functions during epidermal homeostasis or carcinogenesis. To address these questions, we generated mice with epidermal deletions of Utx. Intriguingly, we observed that homozygous Utx knockout female mice show regions of epidermal hyperplasia, an expanded keratin 14 basal layer and increased immune invasion in the skin when compared to controls. In contrast, male mice missing their one copy of Utx, as well as heterozygous female mice lacking only one copy of Utx, appear phenotypically normal. At the transcriptional level, we find that depletion of UTX leads to numerous alterations in the expression of both immune and metabolic genes, consistent with a recent discovery that UTX senses cellular oxygen. Taken together, these data offer new insights into how UTX may be critical for maintaining epidermal homeostasis and proper gene expression, and how when lost, UTX depletion may lead to increased cancer risk.

High enhancer activity is an epigenetic feature of HPV negative atypical head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with significant mortality and frequent recurrence. Prior efforts to transcriptionally classify HNSCC into groups of varying prognoses have identified four accepted molecular subtypes of the disease: Atypical (AT), Basal (BA), Classical (CL), and Mesenchymal (MS). Here, we investigate the active enhancer landscapes of these subtypes using representative HNSCC cell lines and identify samples belonging to the AT subtype as having increased enhancer activity compared to the other 3 HNSCC subtypes. Cell lines belonging to the AT subtype are more resistant to enhancer-blocking bromodomain inhibitors (BETi). Examination of nascent transcripts reveals that both AT TCGA tumors and cell lines express higher levels of enhancer RNA (eRNA) transcripts for enhancers controlling BETi resistance pathways, such as lipid metabolism and MAPK signaling. Additionally, investigation of higher-order chromatin structure suggests more enhancer-promoter (E-P) contacts in the AT subtype, including on genes identified in the eRNA analysis. Consistently, known BETi resistance pathways are upregulated upon exposure to these inhibitors. Together, our results identify that the AT subtype of HNSCC is associated with higher enhancer activity, resistance to enhancer blockade, and increased signaling through pathways that could serve as future targets for sensitizing HNSCC to BET inhibition.

Regulatory enhancer profiling of mesenchymal-type gastric cancer reveals subtype-specific epigenomic landscapes and targetable vulnerabilities

ObjectiveGastric cancer (GC) comprises multiple molecular subtypes. Recent studies have highlighted mesenchymal-subtype GC (Mes-GC) as a clinically aggressive subtype with few treatment options. Combining multiple studies, we derived and applied...

DBC1 is a key positive regulator of enhancer epigenomic writers KMT2D and p300

Histone modification is a key epigenetic mechanism for regulation of chromatin dynamics and gene expression. Deleted in breast cancer 1 (DBC1) has been shown to act as a negative regulator of epigenetic modifiers and as a co-activator for nuclear receptors and other transcription factors. However, little is known about the role of DBC1 in the regulation of histone modifications and chromatin landscapes. Here, we analyzed genome-wide profiles of active enhancer and promoter marks in colorectal cancer cells and report DBC1 as a critical positive regulator of histone epigenetic writers KMT2D (H3K4 methyltransferase) and p300 (histone acetyltransferase). DBC1 is required for establishing the landscape of active enhancers, for genome-wide chromatin binding and enhancer recruitment of KMT2D and p300, and for gene activation involved in colorectal cancer progression. DBC1 interacts directly with KMT2D and p300, and enhances KMT2D-mediated histone H3K4 methylation (H3K4me1/2/3) and p300-mediated H3 acetylation. Importantly, DBC1 contributes to super-enhancer formation and function by facilitating the recruitment of KMT2D and p300 and by enhancing their functional interaction and cooperative cross-talk. Our results highlight the critical role of DBC1 as a key positive regulator of KMT2D and p300, and provide insights into regulatory mechanisms underlying the interplay between the enhancer epigenomic writers in enhancer activation.

Mesp1 controls the chromatin and enhancer landscapes essential for spatiotemporal patterning of early cardiovascular progenitors.

The mammalian heart arises from various populations of Mesp1-expressing cardiovascular progenitors (CPs) that are specified during the early stages of gastrulation. Mesp1 is a transcription factor that acts as a master regulator of CP specification and differentiation. However, how Mesp1 regulates the chromatin landscape of nascent mesodermal cells to define the temporal and spatial patterning of the distinct populations of CPs remains unknown. Here, by combining ChIP-seq, RNA-seq and ATAC-seq during mouse pluripotent stem cell differentiation, we defined the dynamic remodelling of the chromatin landscape mediated by Mesp1. We identified different enhancers that are temporally regulated to erase the pluripotent state and specify the pools of CPs that mediate heart development. We identified Zic2 and Zic3 as essential cofactors that act with Mesp1 to regulate its transcription-factor activity at key mesodermal enhancers, thereby regulating the chromatin remodelling and gene expression associated with the specification of the different populations of CPs in vivo. Our study identifies the dynamics of the chromatin landscape and enhancer remodelling associated with temporal patterning of early mesodermal cells into the distinct populations of CPs that mediate heart development.

Epigenomic analysis reveals a dynamic and context-specific macrophage enhancer landscape associated with innate immune activation and tolerance

BackgroundChromatin states and enhancers associate gene expression, cell identity and disease. Here, we systematically delineate the acute innate immune response to endotoxin in terms of human macrophage enhancer activity and contrast with endotoxin tolerance, profiling the coding and non-coding transcriptome, chromatin accessibility and epigenetic modifications.ResultsWe describe the spectrum of enhancers under acute and tolerance conditions and the regulatory networks between these enhancers and biological processes including gene expression, splicing regulation, transcription factor binding and enhancer RNA signatures. We demonstrate that the vast majority of differentially regulated enhancers on acute stimulation are subject to tolerance and that expression quantitative trait loci, disease-risk variants and eRNAs are enriched in these regulatory regions and related to context-specific gene expression. We find enrichment for context-specific eQTL involving endotoxin response and specific infections and delineate specific differential regions informative for GWAS variants in inflammatory bowel disease and multiple sclerosis, together with a context-specific enhancer involving a bacterial infection eQTL for KLF4. We show enrichment in differential enhancers for tolerance involving transcription factors NFκB-p65, STATs and IRFs and prioritize putative causal genes directly linking genetic variants and disease risk enhancers. We further delineate similarities and differences in epigenetic landscape between stem cell-derived macrophages and primary cells and characterize the context-specific enhancer activities for key innate immune response genes KLF4, SLAMF1 and IL2RA.ConclusionsOur study demonstrates the importance of context-specific macrophage enhancers in gene regulation and utility for interpreting disease associations, providing a roadmap to link genetic variants with molecular and cellular functions.

Abstract 701: Loss of PRC2 enforces a mesenchymal neural crest stem cell phenotype in NF1-deficient cancer through activation of core transcription factors

Abstract Objective: Recurrent mutations in the polycomb repressive complex 2 (PRC2) occur in ~80% of malignant peripheral nerve sheath tumors (MPNST), which originate from NF1-deficient Schwann cells. In MPNST, PRC2 loss results in a diverse set of transcriptomic and phenotypic consequences, including gain of acetylated H3K27 (H3K27ac) accompanying the loss of trimethylated H3K27 (H3K27me3), hyperactivated Ras signaling, and tumor escape from immune surveillance. Mechanistic understanding of how PRC2 loss results in these diverse consequences remains unresolved. Methods: To delineate the oncogenic mechanisms mediated by PRC2 loss, we engineered MPNST cell lines to dynamically reassemble a functional PRC2 and evaluated the transcriptomic and epigenetic consequences of PRC2 restoration. We further extended these finding using single cell RNA sequencing (scRNAseq) from human MPNST. Results: Through integrative analysis of RNAseq and H3K27me3 ChIP-seq in PRC2-deficient MPNST cells, we identified 6134 H3K27me3 peaks gained when a functional PRC2 was restored. Of the 876 significantly altered genes, 699 genes were downregulated, and 177 genes were upregulated when a restored PRC2 caused genome-wide redistribution of H3K27me3 peaks. Detailed mechanistic dissection of these PRC2-regulated genes revealed a two-pronged oncogenic process mediated by PRC2 loss and they cooperatively contribute to the tumorigenesis of MPNST. First, PRC2 loss leads to the upregulation of a transcriptional circuit that remodels the enhancer landscape of MPNST cells and establishes an enhancer-driven transcription factor, FOXC1, as a master regulator and thus a core vulnerability of the cell. Second, PRC2 loss reduces type I interferon (IFN) signaling and antigen presentation as a downstream consequence of the upregulated Ras signaling. These data were further integrated with scRNAseq of human MPNST. Importantly, we discovered that the PRC2-deficient tumor cells have a corrupted transcriptional program characteristic of a mesenchymal precursor cell found during the trajectory of normal Schwann cell development. Interestingly, malignant cells expressing high levels of IFN and antigen presentation genes were missing in the metastatic MPNST, potentially allowing the tumor cell to escape the immune surveillance. The transcriptional circuit established by PRC2-regulated FOXC1 and its downstream targets sustains the malignant program in both primary and metastatic MPNST. Conclusion: In summary, we discovered the activation of a lineage specific oncogenic transcription program in PRC2-deficient MPNST, which is characteristic of a neural crest-derived mesenchymal stem cell. Our findings provide mechanistic understanding of the inherent metastatic potential, chemotherapy and radiotherapy resistance, and immune escape that are clinically characteristic of these tumors. Citation Format: Xiyuan Zhang, Hannah E. Lou, Vishaka Gopalan, Zhihui Liu, Hilda Jafarah, Haiyan Lei, Paige Jones, Carly M. Sayers, Marielle E. Yohe, Prashant Chittiboina, Brigitte C. Widemann, Carol J. Thiele, Michael C. Kelly, Sridhar Hannenhalli, Jack F. Shern. Loss of PRC2 enforces a mesenchymal neural crest stem cell phenotype in NF1-deficient cancer through activation of core transcription factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 701.

Abstract 3719: Loss of Kmt2c, of the COMPASS-like complex, cooperates with oncogenic signaling in cancer

Abstract In mammals, the histone methyltransferase KMT2C is one of the major regulators of gene expression and is one of the most commonly lost epigenetic regulators across all cancers. Studies in mice demonstrated that Kmt2c is a haploinsufficient tumor suppressor in AML and inoculation of mammary fat pads with breast cancer cells that lack KMT2C had significantly larger tumor growth and reduced survival compared to mice injected with isogenic wild-type cells. Analysis of cBioportal’s curated set of non-redundant cancer patient studies indicates that loss of function KMT2C mutations significantly co-occur with RAS gain of function mutations. Our lab previously discovered that cells expressing oncogenic RAS displayed altered activation of enhancer gene regulator elements. We will test how loss of KMT2C cooperates with oncogenic Ras by: 1) characterizing the impact of KMT2C loss, with or without expression of oncogenic RAS, on biological properties of cells and global chromatin architecture and 2) characterizing how KMT2C loss may cooperate with oncogenic RAS to alter hematopoiesis and the global enhancer landscape in animal models. MEFs expressing heterozygous loss of Kmt2c have a proliferative advantage relative to WT littermate controls. Interestingly, WT MEFs demonstrate enhanced migration in a wound assay, but heterozygotes demonstrate an advantage in a transwell membrane assay. Heterozygotes further display greater EdU incorporation, indicating a greater propensity for entering S-phase and undergoing cell-division. RNA-seq experiments have indicated an upregulation of MAPK & Ras signaling pathway genes in heterozygotes, whilst simultaneously downregulating gene pathways critical for proper extra-cellular matrix organization. Therefore, loss of Kmt2c disrupts biological cell phenotypes, in a manner relatable to the hallmarks of cancer, demonstrating how Kmt2c loss may promote malignancy. Further studies must now be performed to elucidate the molecular mechanisms altered by loss of KMT2C and how they cause changes in cellular phenotypes. Citation Format: Gabriel Prado, Richard L. Bennett, Jonathan D. Licht. Loss of Kmt2c, of the COMPASS-like complex, cooperates with oncogenic signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3719.

XIST loss impairs mammary stem cell differentiation and increases tumorigenicity through Mediator hyperactivation

X inactivation (XCI) is triggered by upregulation of XIST, which coats the chromosome in cis, promoting formation of a heterochromatic domain (Xi). XIST role beyond initiation of XCI is only beginning to be elucidated. Here, we demonstrate that XIST loss impairs differentiation of human mammary stem cells (MaSCs) and promotes emergence of highly tumorigenic and metastatic carcinomas. On the Xi, XIST deficiency triggers epigenetic changes and reactivation of genes overlapping Polycomb domains, including Mediator subunit MED14. MED14 overdosage results in increased Mediator levels and hyperactivation of the MaSC enhancer landscape and transcriptional program, making differentiation less favorable. We further demonstrate that loss of XIST and Xi transcriptional instability is common among human breast tumors of poor prognosis. We conclude that XIST is a gatekeeper of human mammary epithelium homeostasis, thus unveiling a paradigm in the control of somatic cell identity with potential consequences for our understanding of gender-specific malignancies.