Vivek S. Chopra

Stalled Hox promoters as chromosomal boundaries

Many developmental control genes contain stalled RNA Polymerase II (Pol II) in the early Drosophila embryo, including four of the eight Hox genes. Here, we present evidence that the stalled Hox promoters possess an intrinsic insulator activity. The enhancer-blocking activities of these promoters are dependent on general transcription factors that inhibit Pol II elongation, including components of the DSIF and NELF complexes. The activities of conventional insulators are also impaired in embryos containing reduced levels of DSIF and NELF. Thus, promoter-proximal stalling factors might help promote insulator-promoter interactions. We propose that stalled promoters help organize gene complexes within chromosomal loop domains.

Regulation of Hox gene activity by transcriptional elongation in Drosophila

Hox genes control the anterior-posterior patterning of most metazoan embryos. Their sequential expression is initially established by the segmentation gene cascade in the early Drosophila embryo [1]. The maintenance of these patterns depends on the Polycomb group (PcG) and trithorax group (trxG) complexes during the remainder of the life cycle [2]. We provide both genetic and molecular evidence that the Hox genes are subject to an additional tier of regulation, i.e., at the level of transcription elongation. Both Ultrabithorax (Ubx) and Abdominal-B (Abd-B) genes contain stalled or paused RNA polymerase II (Pol II) even when silent [3, 4]. The Pol II elongation factors Elongin-A and Cdk9 are essential for optimal Ubx and Abd-B expression. Mitotic recombination assays suggest that these elongation factors are also important for the regulation of Notch-, EGF-, and Dpp-signaling genes. Stalled Pol II persists in tissues where Ubx and Abd-B are silenced by the PcG complex. We propose that stalling fosters both the rapid induction and precise silencing of Hox gene expression during development.

CCAT1 is an enhancer-templated RNA that predicts BET sensitivity in colorectal cancer

Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we used a CRISPR loss-of-function screen and identified a number of essential genes, including the bromodomain and extraterminal (BET) protein BRD4. We found that BRD4 is critical for colon cancer proliferation, and its knockdown led to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct superenhancer in CIMP+ colon cancers that regulates cMYC transcription. We found that the long noncoding RNA colon cancer-associated transcript 1 (CCAT1) is transcribed from this superenhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose that CCAT1 is a clinically tractable biomarker for identifying patients who are likely to benefit from BET inhibitors.

Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer

Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we perform exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumours and 32 cell lines. Meta-analysis of exome data and previously published data sets reveals 24 significantly mutated genes in microsatellite stable (MSS) tumours and 16 in microsatellite instable (MSI) tumours. Over half the patients in our collection could potentially benefit from targeted therapies. We identify 55 splice site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumours. ZAK kinase isoform TV1 is preferentially upregulated in gastric tumours and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder and breast cancers. Overexpression of this particular isoform activates multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibits proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK.

An integrative analysis of colon cancer identifies an essential function for PRPF6 in tumor growth

The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.

Evolving enhancer-promoter interactions within the tinman complex of the flour beetle, Tribolium castaneum

Modifications of cis-regulatory DNAs, particularly enhancers, underlie changes in gene expression during animal evolution. Here, we present evidence for a distinct mechanism of regulatory evolution, whereby a novel pattern of gene expression arises from altered gene targeting of a conserved enhancer. The tinman gene complex (Tin-C) controls the patterning of dorsal mesodermal tissues, including the dorsal vessel or heart in Drosophila. Despite broad conservation of Tin-C gene expression patterns in the flour beetle (Tribolium castaneum), the honeybee (Apis mellifera) and the fruit fly (Drosophila melanogaster), the expression of a key pericardial determinant, ladybird, is absent from the dorsal mesoderm of Tribolium embryos. Evidence is presented that this loss in expression is replaced by expression of C15, the neighboring gene in the complex. This switch in expression from ladybird to C15 appears to arise from an inversion within the tinman complex, which redirects a conserved ladybird 3′ enhancer to regulate C15. In Drosophila, this enhancer fails to activate C15 expression owing to the activity of an insulator at the intervening ladybird early promoter. By contrast, a chromosomal inversion allows the cardiac enhancer to bypass the ladybird insulator in Tribolium. Given the high frequency of genome rearrangements in insects, it is possible that such enhancer switching might be widely used in the diversification of the arthropods.

Transcriptional repression via antilooping in the Drosophila embryo

Transcriptional repressors are thought to inhibit gene expression by interfering with the binding or function of RNA Polymerase II, perhaps by promoting local chromatin condensation. Here, we present evidence for a distinctive mechanism of repression, whereby sequence-specific repressors prevent the looping of distal enhancers to the promoter. Particular efforts focus on the Snail repressor, which plays a conserved role in promoting epithelial-mesenchyme transitions in both invertebrates and vertebrates, including mesoderm invagination in Drosophila, neural crest migration in vertebrates, and tumorigenesis in mammals. Chromosome conformation capture experiments were used to examine enhancer looping at Snail target genes in wild-type and mutant embryos. These studies suggest that the Snail repressor blocks the formation of fruitful enhancer–promoter interactions when bound to a distal enhancer. This higher-order mechanism of transcriptional repression has broad implications for the control of gene activity in metazoan development.

Combinatorial patterning mechanisms in the Drosophila embryo

The classical concept of the morphogen gradient proposes that small differences in the levels of a signalling molecule or transcription factor are responsible for producing a continuous spectrum of distinctive cellular identities across a naïve field of cells. In this review, we discuss how the Dorsal gradient controls the dorsal-ventral patterning of the early Drosophila embryo. This gradient extends from the ventral midline of the embryo into dorso-lateral regions, encompassing a cross-sectional field of approximately 20 cells. There is no evidence that these cells acquire distinctive identities due to subtle changes in the nuclear concentrations of the Dorsal protein. Rather, a variety of evidence suggests that the Dorsal gradient generates just three primary thresholds of gene activity. High levels activate gene expression in the presumptive mesoderm, while intermediate and low levels activate gene expression in the ventral and dorsal neurogenic ectoderm, respectively. We discuss how these primary readouts of the gradient establish localized domains of cell signalling, which work in a combinatorial manner with transcriptional networks to produce complex patterns of gene expression and tissue differentiation.

Abstract 5383: DOT1L inhibitor EPZ-5676 synergizes with cytarabine and azacitidine in preclinical models of MLL-rearranged leukemia

EPZ-5676 is a small molecule inhibitor of the histone methyltransferase DOT1L currently in clinical development and represents a first in class novel therapeutic agent for the treatment of MLL-rearranged (MLL-r) leukemia. In preclinical studies, EPZ-5676 selectively inhibited intracellular histone H3K79 methylation, downstream target gene expression and demonstrated complete tumor regression in a MLL-r leukemia xenograft model. We previously reported synergistic and durable anti-proliferative activity when EPZ-5676 was combined with current AML standard of care drugs, cytarabine and daunorubicin in MLL-r leukemia models MOLM-13 (MLL-AF9) and MV4-11 (MLL-AF4). Combination benefit was also observed when MLL-r cells were treated with cytarabine, prior to co-treatment with EPZ-5676. Additionally, both cytarabine and the DNA methyltransferase inhibitor azacitidine, displayed synergistic anti-leukemic activity in MLL-r rearranged cells in a 7 day co-treatment model (7 days of continuous treatment with EPZ-5676 and second agent; see Klaus et al, JPET, 2014). In this report we discuss results of investigating additional treatment schedules using EPZ-5676 in combination with azacitidine in MLL-r cells. Cells were pretreated with azacitidine at nanomolar concentrations known to reverse promoter DNA-hypermethylation and alter the chromatin state (Tsai et al., Cancer Cell, 2012). We found treating MV4-11 and MOLM-13 cells once daily for three consecutive days followed by sequential treatment with EPZ-5676 elicited a synergistic anti-proliferative effect using the Chou-Talalay method (Chou, Pharmacol Rev., 2006). Results of studies to investigate the mechanism of this synergistic cell killing, including evaluation of differentiation markers and Annexin V staining will be reported. To determine if combinations of EPZ-5676 with cytarabine or azacitidine were tolerable and efficacious in vivo, nude rats implanted subcutaneously with MV4-11 tumors were treated using a range of doses and schedules. Azacitidine and cytarabine were delivered by intraperitoneal injection once daily for 14 days at their respective maximum tolerated doses of 2 and 200 mg/kg. Dosing at the established MTD, these agents inhibited the subcutaneous MV4-11 tumor growth by 50% compared to vehicle controls. Efficacy results from the EPZ-5676 combination studies with cytarabine or azacitidine will be presented. In summary, our results indicate that EPZ-5676 in combination with cytarabine or azacitidine revealed a synergistic effect, regardless of the treatment schedule used in preclinical models of MLL-r leukemia. Tolerable in vivo rat combination doses for EPZ-5676 with both cytarabine and azacitidine have been determined in support of potential future assessment of these combinations in MLL-r leukemia patients. Citation Format: Christine R. Klaus, Scott R. Daigle, Vivek Chopra, Jeffrey A. Keats, Carly T. Campbell, Dorothy Iwanowicz, Edward J. Olhava, Margaret P. Scott, Roy M. Pollock, Robert A. Copeland, Jesse J. Smith, Jorge DiMartino, Stephen J. Blakemore, Alejandra Raimondi. DOT1L inhibitor EPZ-5676 synergizes with cytarabine and azacitidine in preclinical models of MLL-rearranged leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5383. doi:10.1158/1538-7445.AM2015-5383

Abstract 3203: The tri-snRNP spliceosome complex is required for MYC-dependent cancer growth.

Spliceosome coordinated RNA splicing is an essential cellular process that can generate an immensely diverse repertoire of RNA. While cancer cells have been known to hijack this process to generate splice forms with oncogenic function, the specific role of spliceosome components in this process is not well understood. In this study, we use an integrative genomic approach to identify PRPF6, a member of the tri-snRNP spliceosome complex as essential for colon cancer growth. We show that, in addition to PRPF6, other tri-snRNP components are coordinately overexpressed or amplified in cancer cells. Inhibition of the tri-snRNP complex, but not other spliceosome components, abrogated cancer cell growth only in dependent cancer cell lines in vitro and in vivo. High resolution transcriptome analysis reveals that the tri-snRNP complex binds and regulates the splicing of a relatively small number of genes, many of which are transcriptional targets of the c-MYC oncogene. Intriguingly, many components of the tri-snRNP complex are mutated in a genetic form of Retinitis Pigmentosa. This genetic corollary suggests that the tri-snRNP complex is necessary in specific cellular contexts that may depend on the proper transcription and splicing of a tri-snRNP regulated set of genes. Citation Format: Adam Adler, Zhaoshi Jiang, Mark McCleland, Elizabeth Blackwood, Sharon Yee, Benjamin Haley, Jean-Philippe Stephan, Sofia Hussain, Vivek Chopra, Ron Firestein. The tri-snRNP spliceosome complex is required for MYC-dependent cancer growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3203. doi:10.1158/1538-7445.AM2013-3203 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.