Convergent organization of aberrant MYB complex controls oncogenic gene expression in acute myeloid leukemia.

Dysregulated gene expression contributes to most prevalent features in human cancers. Here, we show that most subtypes of acute myeloid leukemia (AML) depend on the aberrant assembly of MYB transcriptional co-activator complex. By rapid and selective peptidomimetic interference with the binding of CBP/P300 to MYB, but not CREB or MLL1, we find that the leukemic functions of MYB are mediated by CBP/P300 co-activation of a distinct set of transcription factor complexes. These MYB complexes assemble aberrantly with LYL1, E2A, C/EBP family members, LMO2, and SATB1. They are organized convergently in genetically diverse subtypes of AML and are at least in part associated with inappropriate transcription factor co-expression. Peptidomimetic remodeling of oncogenic MYB complexes is accompanied by specific proteolysis and dynamic redistribution of CBP/P300 with alternative transcription factors such as RUNX1 to induce myeloid differentiation and apoptosis. Thus, aberrant assembly and sequestration of MYB:CBP/P300 complexes provide a unifying mechanism of oncogenic gene expression in AML. This work establishes a compelling strategy for their pharmacologic reprogramming and therapeutic targeting for diverse leukemias and possibly other human cancers caused by dysregulated gene control

Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond

MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here, we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Kmt2c cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Kmt2c restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network

GC-Rich Sequence Elements Recruit PRC2 in Mammalian ES Cells

Polycomb proteins are epigenetic regulators that localize to developmental loci in the early embryo where they mediate lineage-specific gene repression. In Drosophila, these repressors are recruited to sequence elements by DNA binding proteins associated with Polycomb repressive complex 2 (PRC2). However, the sequences that recruit PRC2 in mammalian cells have remained obscure. To address this, we integrated a series of engineered bacterial artificial chromosomes into embryonic stem (ES) cells and examined their chromatin. We found that a 44 kb region corresponding to the Zfpm2 locus initiates de novo recruitment of PRC2. We then pinpointed a CpG island within this locus as both necessary and sufficient for PRC2 recruitment. Based on this causal demonstration and prior genomic analyses, we hypothesized that large GC-rich elements depleted of activating transcription factor motifs mediate PRC2 recruitment in mammals. We validated this model in two ways. First, we showed that a constitutively active CpG island is able to recruit PRC2 after excision of a cluster of activating motifs. Second, we showed that two 1 kb sequence intervals from the Escherichia coli genome with GC-contents comparable to a mammalian CpG island are both capable of recruiting PRC2 when integrated into the ES cell genome. Our findings demonstrate a causal role for GC-rich sequences in PRC2 recruitment and implicate a specific subset of CpG islands depleted of activating motifs as instrumental for the initial localization of this key regulator in mammalian genomes.Burroughs Wellcome FundCharles E. Culpeper FoundationMassachusetts General HospitalBroad Institute of MIT and Harvar

Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in neuroblastoma

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.Open Access funding enabled and organized by Projekt DEAL. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748. A.G.H. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 398299703 Helmsauer is supported by Boehringer Ingelheim Fonds. This work was also supported by the TransTumVar project - PN01360

Analysis of the Global Methylation Profile of Accelerated and Blast Phase Myeloproliferative Neoplasms and Its Association with Response to Decitabine-Based Therapy

Methylation profiling in myeloid malignancies such as Acute Myeloid Leukemia (AML) and Chronic Myelomonocytic Leukemia (CMML) has demonstrated the ability to define distinct biological and clinical subgroups, including predicting which patients will respond to therapy with a hypomethylating agent (HMA). The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) carry an inherent risk of progression to an accelerated-phase disease (AP; 10-19% blasts in the peripheral blood or bone marrow), as well as to blast phase disease (BP; ≥ 20% blasts in the peripheral blood or bone marrow), which is associated with a poor prognosis. It is unknown whether the methylation profiles of MPN-AP/BP cases may further help identify distinct biological, genomic, and clinical subgroups, including identifying patients more likely to respond to HMA. We recently carried out a phase I/II study to test the safety and efficacy of combination therapy with the JAK1/2 inhibitor ruxolitinib (RUX) and the HMA Decitabine (DAC) in patients with MPN-AP/BP (MPD-RC 109 study; NCT02076191). A total of 46 patients were accrued to the phase I and II studies. 37 patients were evaluable for response. Complete response (CR) occurred in 10%, Complete Response with incomplete count recovery (CRi) in 24%, Partial Response in 24%. 42% of patients had no response to therapy. Using samples available from the MPD-RC 109 study, we sought to assess whether the baseline global methylation profile predicts for response to this regimen. Further, we sought to utilize this dataset to determine if IDH2 mutations (amongst the most common mutations in MPN-AP/BP) are associated with a distinct methylation profile, as has been demonstrated in de novo AML. We carried out a pilot study of 11 MPN-AP/BP patients from the MPD-109 phase I/II trial and performed Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) for DNA methylation quantification at ~3M CpG sites across the genome. Baseline DNA methylation profiles were compared between Responder (R) and Non-responder (NR) patients. Notably, unsupervised analysis using correspondence analysis (COA) demonstrated an almost complete separation of the two groups of patients (Fig 1A), while supervised analysis using a beta binomial model identified 134 differentially methylated regions (DMRs) (FDR25%) between the two groups at diagnosis (Fig 1B). Similar to our prior observation in CMML, response-associated DMRs were depleted from promoter regions (p<0.001) and enriched at enhancers (p<0.001), and were annotated to genes in pathways related to myeloid biology and metabolic processes (Fig 1C). We next carried out a pilot study to characterize the epigenetic abnormalities of IDH2-mutant MPN-AP/BP cases. For this purpose, we compared the genome-wide DNA methylation profiles of 12 IDH2-mutant to 7 IDH1/2 wild type MPN-AP/BP cases using ERRBS. Unsupervised analysis based on the DNA methylation profiles alone showed a strong trend to naturally segregate mutant from wild-type cases, indicating strong underlying epigenetic differences (Fig.1D). A supervised analysis using the beta binomial method identified 1,477 differentially methylated regions (DMRs) between the two groups (average absolute methylation difference ≥25% and FDR <10%) (Fig 1E). Eighty percent of these DMRs corresponded to sites that were hypermethylated in IDH2-mutant cases compared to wild type. These DMRs were strongly enriched in CpG shores and enhancer regions (p value < 0.001 for both) (Fig 1F). Our data demonstrate that the methylation profile of MPN-AP/BP may predict for response to HMA-based therapy. Such data could be used to guide therapeutic decisions and select patient for whom HMA has the highest likelihood of procuring a response. As well, these findings indicate that IDH2-mutant MPN-AP/BP are epigenetically distinct, and given the preferred targeting of regulatory elements, these epigenetic differences may play a functional role in disease biology. Further validation of these observations is required. Updated data, including analysis of further cases, and RNA-sequencing analysis of gene-expression and pathway enrichment of genes differentially methylated between responders and non-responders, and IDH2 mutated and wildtype cases will be presented at the conference. Disclosures Rampal: Constellation: Research Funding; Pharmaessentia: Consultancy; CTI Biopharma: Consultancy; Promedior: Consultancy; Celgene: Consultancy; Incyte: Consultancy, Research Funding; Abbvie: Consultancy; Galecto: Consultancy; Jazz Pharmaceuticals: Consultancy; Blueprint: Consultancy; Stemline: Consultancy, Research Funding. Mascarenhas:Celgene, Prelude, Galecto, Promedior, Geron, Constellation, and Incyte: Consultancy; Incyte, Kartos, Roche, Promedior, Merck, Merus, Arog, CTI Biopharma, Janssen, and PharmaEssentia: Other: Research funding (institution). Levine:Morphosys: Consultancy; Prelude Therapeutics: Research Funding; Novartis: Consultancy; Amgen: Honoraria; Lilly: Consultancy, Honoraria; Janssen: Consultancy; Astellas: Consultancy; Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria. Hoffman:Novartis: Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; Forbius: Consultancy; Dompe: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees

MPP8 is essential for sustaining self-renewal of ground-state pluripotent stem cells

Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. Nonetheless, the epigenetic regulation of ground-state mouse embryonic stem cells (mESCs) is not fully understood. Here, we identify the epigenetic protein MPP8 as being essential for ground-state pluripotency. Its depletion leads to cell cycle arrest and spontaneous differentiation. MPP8 has been suggested to repress LINE1 elements by recruiting the human silencing hub (HUSH) complex to H3K9me3-rich regions. Unexpectedly, we find that LINE1 elements are efficiently repressed by MPP8 lacking the chromodomain, while the unannotated C-terminus is essential for its function. Moreover, we show that SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE1 elements is maintained without retaining H3K9me3 levels. Taken together, our findings demonstrate that MPP8 protects the DNA-hypomethylated pluripotent ground state through its association with the HUSH core complex, however, independently of detectable chromatin binding and maintenance of H3K9me3

UDP-glucose pyrophosphorylase 2, a regulator of glycogen synthesis and glycosylation, is critical for pancreatic cancer growth.

UDP-glucose pyrophosphorylase 2 (UGP2), the enzyme that synthesizes uridine diphosphate (UDP)-glucose, rests at the convergence of multiple metabolic pathways, however, the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an important role for UGP2 in the maintenance of pancreatic ductal adenocarcinoma (PDAC) growth in both in vitro and in vivo tumor models. We found that transcription of UGP2 is directly regulated by the Yes-associated protein 1 (YAP)-TEA domain transcription factor (TEAD) complex, identifying UGP2 as a bona fide YAP target gene. Loss of UGP2 leads to decreased intracellular glycogen levels and defects in N-glycosylation targets that are important for the survival of PDACs, including the epidermal growth factor receptor (EGFR). These critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer insights into therapeutic options for otherwise intractable PDACs