Transcriptional Regulatory Complex Research Articles

Abstract 4705: Therapeutic activity of bivalent BRD4 inhibitor AZD5153 in hematological cancers

Abstract The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Across a number of tumor models pharmacological targeting of BRD4 bromodomains by small-molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor. AZD5153 is a candidate drug that possesses an unprecedented bivalent binding mode among reported BET inhibitors, which allows AZD5153 to ligate the tandem bromodomains in BRD4. The avidity resulted from the bivalent binding interaction translates into markedly enhanced cellular potency. Although AZD5153 demonstrates broad activity across a cancer cell line panel comprising solid and hematologic subtypes, there is enriched antitumor activity against hematologic cell lines, including acute myeloid leukemia (AML), multiple myeloma (MM), and diffuse large B-cell lymphoma (DLBCL). The activity of AZD5153 in hematologic tumors was further confirmed in five selected xenograft models of AML, MM, and DLBCL where AZD5153 treatment led to tumor stasis or regression, accompanied by concomitant modulation of BRD4 pharmacodynamic markers, such as MYC and HEXIM1. In order to characterize the transcriptional consequences elicited by AZD5153, we carried out transcriptional profiling of 11 hematologic tumor lines and identified the robust modulation of MYC, and E2F transcriptional programs. Moreover, our transcriptional data was used to identify candidate clinical PD biomarkers. The suitability and dynamic range of the top two candidate biomarkers for AZD5153 was confirmed using human whole blood from normal healthy volunteers. To identify protein biomarkers associated with sensitivity to AZD5153 treatment, we deployed reverse-phase protein array (RPPA) technology to quantitatively examine the level of 182 proteins following AZD5153 treatment. Our findings indicate that cell lines sensitive to AZD5153 uniquely exhibit a marked decrease in the level of mTOR-pathway associated proteins following AZD5153 treatment. Conversely, MYC modulation was observed in both sensitive and resistant groups. Thus, these data suggest that in hematologic malignancies, mTOR pathway downregulation may serve as an appropriate biomarker of sensitivity to BRD4 inhibitors such as AZD5153. Our study establishes AZD5153 as a novel and potent BRD4/BET inhibitor possessing a unique bivalent binding property. We have characterized the pharmacological consequences of BRD4/BET inhibition by AZD5153 via unbiased transcriptional and proteome profiling. These efforts are the first to identify mTOR modulation as a putative biomarker of sensitivity to BET bromodomain inhibition in hematologic tumors and may help to inform future clinical evaluation of AZD5153 and other BET bromodomain inhibitors. Citation Format: Huawei (Ray) Chen, Maureen Hattersley, Garrett Rhyasen, Austin Dulak, Wendy Wang, Phil Petteruti, Ian Dale, Tony Cheung, Shenghua Wen, Lilian Castriotta, Deborah Lawson, Mike Collins, Miika Ahdesmaki, Graeme Walker, Al Rabow, Jonathan Dry, Corinne Reimer, Paul Lyne, Steve Fawell, MIke Waring, Mike Zinda, Ed Clark, Ed Clark. Therapeutic activity of bivalent BRD4 inhibitor AZD5153 in hematological cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4705.

Mutant p53 and ETS2, a Tale of Reciprocity.

TP53 is one of the most frequently inactivated tumor suppressor genes in human cancer. However, unlike other tumor suppressor genes whose expression is lost, TP53 is usually inactivated as a result of a single nucleotide change within the coding region. Typically, these single nucleotide mutations result in a codon change that creates an amino acid substitution. Thus, unlike other tumor suppressor genes whose expression is lost due to genetic or epigenetic changes, the p53 gene primarily suffers missense mutations, and therefore, the cells retain and express a mutant form of the p53 protein (mtp53). It is now well established that mtp53 contributes to tumor development through its gain-of-function (GOF) activities. These GOF activities can arise from novel protein–protein interactions that can either disable other tumor suppressors (e.g., p63 and p73) or enable oncogenes such as ETS2, an ETS family member. In this review, I will focus on the identification of the mtp53/ETS2 complex and outline the diverse activities that this transcriptional regulatory complex controls to promote cancer.

Abstract B21: Suppression of adaptive resistance mechanisms to trametinib by inhibition of BET bromodomains in TNBC

Abstract The RAF-MEK-ERK signaling pathway regulates the proliferation of Triple Negative Breast Cancer (TNBC). Although MEK inhibition by trametinib in TNBC cell lines and mouse models inhibits growth, the response is not durable and the tumor develops resistance. The lack of durability of targeted kinase inhibitors is a common problem in many tumor types due to induction of alternative bypass signaling pathways that lead to resistance. We have profiled the response of TNBC to MEK inhibition using trametinib in order to identify adaptive signaling pathways which drive resistance. We used a chemical proteomics method [multiplexed inhibitor beads coupled to mass spectrometry (MIB/MS)], to quantify global changes in kinase activity coupled with transcriptional changes in response to trametinib using RNAseq. Our studies revealed that adaptive resistance to trametinib occurs through increased expression and activation of multiple receptor tyrosine kinases, leading to reactivation of ERK. These adaptive kinases are heterogeneous across TNBC including PDGFRβ, VEGFR2, DDR1, AXL, KIT and FGFR2. There is no single targeted kinase inhibitor able to block all the adaptive kinases induced and activated, making the design of combination targeted kinase inhibitor therapies impractical. We therefore hypothesized an alternative therapeutic approach was required to inhibit adaptive reprogramming. Our goal was to block the initial transcriptional response to trametinib which upregulates adaptive kinase expression. Several studies in the lab determined that we could block adaptive kinase expression by inhibiting BET family bromodomain proteins that interact with acetylated histones and facilitate the formation of transcriptional regulatory complexes. Our work demonstrated that BET family bromodomain inhibitors (JQ1 and iBET151), synergize with trametinib to prevent TNBC growth in short-term (4 days) and long-term (4 weeks) growth assays. Using MIB/MS we found that combination trametinib/BET bromodomain inhibitor treatment blocks upregulation of adaptive kinase expression and activation. RNAseq demonstrated that BET bromodomain inhibitors synergize with trametinib to block the induction of kinases expressed as part of the adaptive bypass response. ChIP-seq of the BET bromodomain protein BRD4 and H3K27ac, a marker of active enhancers, showed BRD4 is recruited to super-enhancer regions proximal to adaptive response kinases as early as 4hr following trametinib treatment. Furthermore, the recruitment of BRD4 to enhancer regions is maintained in cells made resistant to trametinib by constant culture in low-dose drug. In sum, these results suggest that BRD4 recruitment to enhancer regions, following trametinib treatment, leads to the formation of transcriptional complexes which maintain adaptive gene expression. Ongoing studies seek to understand the mechanism by which BRD4 is recruited to super-enhancers following trametinib treatment in TNBC. Mouse xenograft studies also show promising evidence of enhanced tumor growth suppression in vivo using combination trametinib/iBET151 treatment. The results presented provide a novel treatment strategy to block adaptive resistance and have the potential to lead to durable clinical responses of TNBC. Citation Format: Samantha M. Miller, Jon S. Zawistowski, Timothy J. Stuhlmiller, Noah Sciaky, Charlene M. Santos, David B. Darr, Gary L. Johnson. Suppression of adaptive resistance mechanisms to trametinib by inhibition of BET bromodomains in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B21.

Smad3-mSin3A-HDAC1 Complex is Required for TGF-β1-Induced Transcriptional Inhibition of PPARγ in Mouse Cardiac Fibroblasts

Background: We have recently demonstrated that activated transforming growth factor-β (TGF-β) signaling suppresses myocardial peroxisome proliferator-activated receptor γ (PPARγ) expression in the pressure overloaded heart. In this study, we aim to further define the molecular mechanisms that underlie TGF-β-induced PPARγ transcriptional inhibition. Methods: Adult mouse cardiac fibroblasts were isolated and cultured. PPARγ promoter activity was measured by the dual-Luciferase reporter assay. Interactions between transcription factors and the target gene were identified. Results: In cultured cardiac fibroblasts transfected with a plasmid containing a human PPARγ promoter, co-transfection of Smad3 and Smad4, but not Smad2, plasmids significantly enhanced TGF-β1-induced inhibition of PPARγ promoter activity. Promoter deletion analysis and site-directed mutagenesis assays defined two Smad binding elements on the promoter of the PPARγ gene. Utilizing chromatin immunoprecipitation analysis and DNA-affinity precipitation methods, we demonstrated that the transcriptional regulatory complex consisting of Smad3, mSin3A and HDAC1 bound to the promoter of the PPARγ gene in cardiac fibroblasts in response to TGF-β1 stimulation. Either silencing endogenous mSin3A expression by Lentivirus-mediated transduction of mSin3A shRNA or pretreatment with the specific HDAC1 inhibitor MS-275 effectively attenuated TGF-β-induced transcriptional suppression of PPARγ. Conclusion: These results suggest that TGF-β1-induced inhibition of PPARγ transcription depends on formation of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the PPARγ promoter.

Evidence for a non-canonical role of HDAC5 in regulation of the cardiac Ncx1 and Bnp genes.

Class IIa histone deacetylases (HDACs) are very important for tissue specific gene regulation in development and pathology. Because class IIa HDAC catalytic activity is low, their exact molecular roles have not been fully elucidated. Studies have suggested that class IIa HDACs may serve as a scaffold to recruit the catalytically active class I HDAC complexes to their substrate. Here we directly address whether the class IIa HDAC, HDAC5 may function as a scaffold to recruit co-repressor complexes to promoters. We examined two well-characterized cardiac promoters, the sodium calcium exchanger (Ncx1) and the brain natriuretic peptide (Bnp) whose hypertrophic upregulation is mediated by both class I and IIa HDACs. Selective inhibition of class IIa HDACs did not prevent adrenergic stimulated Ncx1 upregulation, however HDAC5 knockout prevented pressure overload induced Ncx1 upregulation. Using the HDAC5(-/-) mouse we show that HDAC5 is required for the interaction of the HDAC1/2/Sin3a co-repressor complexes with the Nkx2.5 and YY1 transcription factors and critical for recruitment of the HDAC1/Sin3a co-repressor complex to either the Ncx1 or Bnp promoter. Our novel findings support a non-canonical role of class IIa HDACs in the scaffolding of transcriptional regulatory complexes, which may be relevant for therapeutic intervention for pathologies.

Regulatory principles governing Salmonella and Yersinia virulence.

Enteric pathogens such as Salmonella and Yersinia evolved numerous strategies to survive and proliferate in different environmental reservoirs and mammalian hosts. Deciphering common and pathogen-specific principles for how these bacteria adjust and coordinate spatiotemporal expression of virulence determinants, stress adaptation, and metabolic functions is fundamental to understand microbial pathogenesis. In order to manage sudden environmental changes, attacks by the host immune systems and microbial competition, the pathogens employ a plethora of transcriptional and post-transcriptional control elements, including transcription factors, sensory and regulatory RNAs, RNAses, and proteases, to fine-tune and control complex gene regulatory networks. Many of the contributing global regulators and the molecular mechanisms of regulation are frequently conserved between Yersinia and Salmonella. However, the interplay, arrangement, and composition of the control elements vary between these closely related enteric pathogens, which generate phenotypic differences leading to distinct pathogenic properties. In this overview we present common and different regulatory networks used by Salmonella and Yersinia to coordinate the expression of crucial motility, cell adhesion and invasion determinants, immune defense strategies, and metabolic adaptation processes. We highlight evolutionary changes of the gene regulatory circuits that result in different properties of the regulatory elements and how this influences the overall outcome of the infection process.

Downregulation of Toll-Like Receptor 9 Expression by Beta Human Papillomavirus 38 and Implications for Cell Cycle Control.

Innate immunity is the first line of host defense against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus 38 (HPV38) E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor Toll-like receptor 9 (TLR9) in human foreskin keratinocytes (HFK), a key mediator of the antiviral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA downregulation by promoting accumulation of ΔNp73α, an antagonist of p53 and p73. Inhibition of ΔNp73α expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of ΔNp73α in this event. Chromatin immunoprecipitation experiments showed that ΔNp73α is part of a negative transcriptional regulatory complex with IκB kinase beta (IKKβ) that binds to a NF-κB responsive element within the TLR9 promoter. In addition, the Polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex, leading to histone 3 trimethylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21(WAF1) and p27(Kip1), decreased CDK2-associated kinase activity, and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can downregulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation. The mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the host immune system in order to guarantee the persistence of the infection. Their capability to evade the immune system is as important as their ability to promote cellular transformation. Therefore, understanding the viral mechanisms involved in viral persistence is a valid tool to evaluate their potential role in human carcinogenesis. Here, we show that E6 and E7 oncoproteins from the cutaneous HPV38 downregulate the expression of the double-stranded DNA sensor TLR9 of innate immunity. We also present evidence that the HPV38-mediated downregulation of TLR9 expression, in addition to its potential impact on the innate immune response, is linked to cell cycle deregulation.

Histone Core Phosphorylation Regulates DNA Accessibility

Nucleosome unwrapping dynamics provide transient access to the complexes involved in DNA transcription, repair, and replication, whereas regulation of nucleosome unwrapping modulates occupancy of these complexes. Histone H3 is phosphorylated at tyrosine 41 (H3Y41ph) and threonine 45 (H3T45ph). H3Y41ph is implicated in regulating transcription, whereas H3T45ph is involved in DNA replication and apoptosis. These modifications are located in the DNA-histone interface near where the DNA exits the nucleosome, and are thus poised to disrupt DNA-histone interactions. However, the impact of histone phosphorylation on nucleosome unwrapping and accessibility is unknown. We find that the phosphorylation mimics H3Y41E and H3T45E, and the chemically correct modification, H3Y41ph, significantly increase nucleosome unwrapping. This enhances DNA accessibility to protein binding by 3-fold. H3K56 acetylation (H3K56ac) is also located in the same DNA-histone interface and increases DNA unwrapping. H3K56ac is implicated in transcription regulation, suggesting that H3Y41ph and H3K56ac could function together. We find that the combination of H3Y41ph with H3K56ac increases DNA accessibility by over an order of magnitude. These results suggest that phosphorylation within the nucleosome DNA entry-exit region increases access to DNA binding complexes and that the combination of phosphorylation with acetylation has the potential to significantly influence DNA accessibility to transcription regulatory complexes.

Abstract 2199: Evaluation of critical residues in the C-terminal binding protein (CtBP) dehydrogenase domain contributing to substrate binding, catalysis, and oncogenic activity

Abstract C-terminal binding proteins (CtBP) 1 and 2 are transcriptional coregulators that are upregulated in several cancers, including a majority of studied breast, colorectal, and ovarian tumor samples. CtBPs drive many cellular oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes, such as E-cadherin, PTEN, BRCA1, and p16. CtBPs encode an intrinsic dehydrogenase activity regulated by both intracellular NADH concentration and the putative substrate 4-methylthio-2-oxobutyric acid (MTOB). NADH binding induces CtBP dimerization, which regulates the recruitment of transcriptional regulatory complexes. High levels of MTOB appear to inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. However, the function of the substrate-binding domain has yet to be examined with regard to CtBP's oncogenic activity. To this end, we have created several point mutations in the substrate-binding pocket of CtBP and have determined key residues for CtBP's enzymatic activity using an in vitro dehydrogenase assay. We have found that a conserved tryptophan in the catalytic domain, and specifically its aromatic moiety, is imperative for enzyme activity. This tryptophan is unique to CtBP family proteins and distinguishes the CtBP substrate-binding domain from that of all other families of dehydrogenases. Additionally, we found arginine and histidine residues lining the substrate pocket that are necessary for CtBP dehydrogenase function. Knowledge of these residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Moreover, to investigate the importance of the catalytic domain on CtBP's oncogenic potential, we have transfected human cancer cell lines with several CtBP substrate-binding domain mutants and observed the effects on cellular processes, including growth, survival, migration, and recruitment of key components of the transcriptional repressor complex. From this work we have investigated the utility of CtBP, and specifically the CtBP substrate-binding domain, as a target for cancer therapeutics. We also provide preliminary insights into the function of this domain in cellular models of cancer. Citation Format: Benjamin L. Morris, Priyadarshan Damle, Zaid Nawaz, Steven R. Grossman. Evaluation of critical residues in the C-terminal binding protein (CtBP) dehydrogenase domain contributing to substrate binding, catalysis, and oncogenic activity. [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 2199. doi:10.1158/1538-7445.AM2015-2199

A role for OVATE FAMILY PROTEIN1 (OFP1) and OFP4 in a BLH6-KNAT7 multi-protein complex regulating secondary cell wall formation in Arabidopsis thaliana

Formation of secondary walls is a complex process that requires the coordinated and developmentally regulated expression of secondary wall biosynthetic genes. In Arabidopsis thaliana, a transcriptional network orchestrates the biosynthesis and deposition of the main SCW components in xylem and fiber cells. It was recently reported that interacting TALE homeodomain proteins BEL-LIKE HOMEODOMAIN6 (BLH6) and KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) negatively regulate secondary cell wall formation in the interfascicular fibers of Arabidopsis inflorescence stems. Members of the Arabidopsis OVATE FAMILY PROTEIN (OFP) family of transcriptional regulators have been shown to physically interact in yeast with various KNAT and BLH proteins, forming a proposed TALE-OFP protein interaction network. This study presents molecular and genetic data indicating that OFP1 and OFP4, previously reported to interact with TALE homeodomain proteins, enhance the repression activity of BLH6, supporting a role for these OFPs as components of a putative multi-protein transcription regulatory complex containing BLH6 and KNAT7.

PML suppresses IL-6-induced STAT3 activation by interfering with STAT3 and HDAC3 interaction

The promyelocytic leukemia protein PML acts as a tumor suppressor by forming transcription–regulatory complexes with a variety of repressor proteins. In the present study, we found that endogenous PML suppresses interleukin (IL)-6-induced gene expression as well as phosphorylation and transcriptional activation of STAT3 in hepatoma cells. We also found that PML-mediated suppression of IL-6-induced STAT3 activation by disrupting interactions between STAT3 and HDAC3. These results indicate that PML modulates IL-6-induced STAT3 activation and hepatoma cell growth by interacting with HDAC3.

Chromatin signatures of cancer.

Changes in the pattern of gene expression play an important role in allowing cancer cells to acquire their hallmark characteristics, while genomic instability enables cells to acquire genetic alterations that promote oncogenesis. Chromatin plays central roles in both transcriptional regulation and the maintenance of genomic stability. Studies by cancer genome consortiums have identified frequent mutations in genes encoding chromatin regulatory factors and histone proteins in human cancer, implicating them as major mediators in the pathogenesis of both hematological malignancies and solid tumors. Here, we review recent advances in our understanding of the role of chromatin in cancer, focusing on transcriptional regulatory complexes, enhancer-associated factors, histone point mutations, and alterations in heterochromatin-interacting factors.

Mechanistic Role of HEXIM1 Induction in BRD4-Antagonist Mediated Growth Inhibition, Differentiation and in Vivo Lethal Activity Against Human AML Blast Progenitor Cells

The BET (bromodomain and extra terminal) protein family, including BRD4 are chromatin reader proteins that bind to acetylated lysines on histone proteins and recruit transcriptional regulatory complexes to gene promoters, thereby coupling histone acetylation to RNA polymerase II (RNAP2)-mediated transcript elongation. Preferential binding of BRD4 to clustered enhancers or ‘super enhancers’ enables BRD4, through the mediator complex, to regulate the transcription of specific oncogenes, including c-MYC, BCL2 and CDK4/6 in AML blast progenitor cells (BPCs). Through its C-terminal domain (CTD), BRD4 recruits pTEFb (positive transcription elongation factor b), the heterodimer composed of cyclin T and CDK9, which phosphorylates the heptad repeats on the CTD of RNAP2 at serine 2. The kinase activity of CDK9 in pTEFb is also required for the phosphorylation of DSIF and NELF, which abrogates their inhibitory hold on the pause-release of RNAP2 and productive transcript elongation of specific oncogenes and their target genes. pTEFb exists in two forms, the catalytically active form, which is associated with BRD4 and RNAP2, and a catalytically inactive form associated with 7SK snRNP (small nuclear ribonucleoprotein) and the inhibitory protein HEXIM1 (hexamethylene bis-acetamide or HMBA-inducible protein 1). The transition of pTEFb between the inactive and active complex is dynamic and regulated by cell growth and stress signaling. HEXIM1 binds to and sequesters pTEFb, thereby inhibiting its kinase activity for RNAP II. We recently reported (Mol Cancer Ther, 2014) that the BRD4 antagonist JQ1 and I-BET151 alone, and synergistically with the pan-histone deacetylase inhibitor panobinostat, inhibits the growth and induces apoptosis in cultured (OCI-AML3 and MOLM13) and patient-derived primary AML (pAML) BPCs. JQ1 treatment also improved the survival of the immune-depleted mice engrafted with MOLM13 or pAML BPCs. Notably, the lethal activity of JQ1 was associated with marked induction of HEXIM1. Therefore, we hypothesized that HEXIM1 induction, by sequestering pTEFb and inhibiting RNAP2, contributes to the growth inhibitory and lethal effects of JQ1. We additionally hypothesized that co-treatment with those agents that further augment HEXIM1 induction would synergize with JQ1 against AML BPCs. To validate these hypotheses, we achieved a stable lentivirus mediated knockdown (KD) of the mRNA and protein expressions of HEXIM1 by approximately 90% in OCI-AML3 and MOLM13 cells, as well as transient KD in pAML BPCs. Compared to the parental control cells, OCI-AML3/HKD and MOLM13/HKD cells showing HEXIM1 knockdown (HKD) exhibited reduced binding of HEXIM1 with cyclin T (as revealed by cyclin T pull-down and immunofluorescence microscopy), but showed augmented suspension culture growth and increased protein levels (by Western blot) of cyclin T, pRNAP2 and cMYC levels. However, in contrast to the parental control cells, JQ1 (1000 nM) treatment-mediated HEXIM1 induction and binding to cyclin T was markedly inhibited in the HKD AML BPCs, which was associated with significant inhibition of JQ1-induced pRNAP2, as well as differentiation and apoptosis of HKD BPCs (p < 0.01). Notably, whereas treatment with JQ1 alone improved the median survival of NOD/SCID mice engrafted with MOLM13 BPCs, this survival improvement was significantly impaired in the mice engrafted with MOLM13/HKD AML BPCs (p < 0.02). This indicated that HEXIM1-induction mechanistically contributes to JQ1-induced growth inhibition, differentiation and apoptosis of AML BPCs. Further, the synergistic apoptosis observed in MOLM13, OCI-AML3 and primary AML cells induced by co-treatment with JQ1 (250 to 1000 nM) and PS (5 to 10 nM) or HMBA (1 to 2 mM) was also associated with greater induction of HEXIM1. However, this synergy was impaired in the cultured and pAML BPCs with HEXIM1 knockdown. These findings confirm that HEXIM1 induction is a biomarker and plays a mechanistic role in mediating growth inhibition, differentiation and apoptosis induced by BRD4 antagonist in human AML BPCs. These findings support, and we have embarked upon, studies involving future pre-clinical development of those agents that in combination significantly augment BRD4 antagonist-induced HEXIM1 in human AML BPCs. DisclosuresNo relevant conflicts of interest to declare.

Short call abstracts.

Short call abstracts.

Abstract SY37-04: Bcl-2 family proteins as a paradigm for protein:protein interactions as chemotherapy targets

Abstract Many aspects of cell signaling are regulated by protein:protein interactions including growth factor receptor signal transduction pathways, transcriptional regulatory complexes and programmed cell death. The Bcl-2 family proteins regulate programmed cell death via a series of protein:protein interactions that occur in the cytoplasm and in membranes. These interactions are regulated by the amount of the proteins, their locations and by post-translational modifications. The multi-domain pro-apoptotic Bcl-2 family members Bax and Bak directly regulate mitochondrial outer membrane permeabilization (MOMP), an event widely accepted as committing most cells to apoptosis. MOMP results from an ordered series of steps beginning with activation of one or more Bcl-2 homology 3 proteins (BH3-proteins). Once activated, BH3-proteins bind to mitochondria, directly recruit and activate Bax and the constitutively membrane bound Bak. In some cases ‘activation’ involves releasing a previously activated Bax or Bak from inhibition by an anti-apoptotic protein of the Bcl-2 family. Oligomerization of integral membrane Bax and/or Bak culminates in membrane permeabilization. The embedded together model describes these interactions as a series of dynamic equilibria that change when the proteins interact with membranes. Because of their role regulating apoptosis in cancer cells and in response to chemotherapy, Bcl-2 family proteins are attractive targets for the development of small molecule inhibitors. The rational development of small molecules requires understanding in detail the molecular interactions that regulate activity. However, studying protein:protein interactions for membrane bound proteins is fraught with difficulties. I will describe some of the challenges of studying this family of proteins and the successes and surprises that resulted from our analyzing and perturbing their interactions for a variety of Bcl-2 family proteins in live cells. I will highlight recent data that suggests new avenues to targeting Bcl-2 family protein:protein interactions therapeutically. Citation Format: David W. Andrews. Bcl-2 family proteins as a paradigm for protein:protein interactions as chemotherapy targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY37-04. doi:10.1158/1538-7445.AM2014-SY37-04

Chromatin reader L(3)mbt requires the Myb-MuvB/DREAM transcriptional regulatory complex for chromosomal recruitment.

Lethal malignant brain tumors (lmbt) result from the loss of the conserved transcriptional repressor l(3)mbt, in Drosophila melanogaster. Similar mutations in the human homolog L3MBTL1 correlate with some cancers. The protein's C-terminal MBT repeats bind mono and dimethylated histones in vitro, which could influence recruitment of L3MBTL1 to its target sites. The L(3)mbt chromatin targeting mechanism, however, is controversial and several studies suggest insufficiency or a minor role for histone methylation in determining the site specificity for recruitment. We report that L(3)mbt colocalizes with core members of the Myb-MuvB/DREAM (MMB/DREAM) transcriptional regulatory complex genome-wide, and that L(3)mbt-mediated repression requires this complex in salivary glands and larval brains. Loss of l(3)mbt or of MMB components through mutation cause similar spurious expression of genes, including the transposon regulatory gene piwi, in terminally differentiated cells. The DNA-binding MMB core component Mip120 (Lin54) is required for L(3)mbt recruitment to chromosomes, whereas Mip130 (Lin9) (an MMB core protein) and E2f2 (an MMB transcriptional repressor) are not, but are essential for repression. Cytolocalization experiments suggest the presence of site-specific differential composition of MMB in polytene chromosomes where some loci were bound by a Myb-containing or alternatively, an E2f2 and L(3)mbt form of the complex.

Ubiquitin‑specific protease 22‑induced autophagy is correlated with poor prognosis of pancreatic cancer.

Ubiquitin‑specific protease 22 (USP22) is a component of the transcription regulatory histone acetylation complex SAGA, which broadly regulates gene transcription and correlates with cancer progression, metastasis and prognosis. Autophagy is a cell pathway with dual functions that promotes cell survival or death. However, it is not known whether USP22 can regulate autophagy in pancreatic cancer. In the present study, we first identified that USP22 was overexpressed in a large number of pancreatic cancer patient samples, concomitant with the increased expression of LC3, a marker of autophagy. Statistical analysis revealed that the increase in USP22 and autophagy was positively correlated with poor prognosis of pancreatic cancer patients. Further investigation using a human pancreatic cancer cell (Panc‑1) identified that the overexpression of USP22 increased the processing of LC3 into the active form LC3‑II and the number of autophagosomes, thus leading to enhanced autophagy. Activation of ERK1/2 kinase rather than AKT1 by USP22 was found to be one of the mechanisms promoting LC3 processing. USP22‑induced autophagy was also found to enhance cell proliferation and resistance to starvation and chemotherapeutic drugs in Panc‑1 cells, therefore expressing an overall effect that promotes cell survival. Collectively, the present study demonstrated a new function of USP22 that induces autophagy, thus leading to the poor prognosis of pancreatic cancer.

The Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6 target a subclass of 'VQ-motif'-containing proteins to regulate immune responses.

Mitogen-activated protein kinase (MAPK) cascades play key roles in plant immune signalling, and elucidating their regulatory functions requires the identification of the pathway-specific substrates. We used yeast two-hybrid interaction screens, in vitro kinase assays and mass spectrometry-based phosphosite mapping to study a family of MAPK substrates. Site-directed mutagenesis and promoter-reporter fusion studies were performed to evaluate the impact of substrate phosphorylation on downstream signalling. A subset of the Arabidopsis thaliana VQ-motif-containing proteins (VQPs) were phosphorylated by the MAPKs MPK3 and MPK6, and renamed MPK3/6-targeted VQPs (MVQs). When plant protoplasts (expressing these MVQs) were treated with the flagellin-derived peptide flg22, several MVQs were destabilized in vivo. The MVQs interact with specific WRKY transcription factors. Detailed analysis of a representative member of the MVQ subset, MVQ1, indicated a negative role in WRKY-mediated defence gene expression - with mutation of the VQ-motif abrogating WRKY binding and causing mis-regulation of defence gene expression. We postulate the existence of a variety of WRKY-VQP-containing transcriptional regulatory protein complexes that depend on spatio-temporal VQP and WRKY expression patterns. Defence gene transcription can be modulated by changing the composition of these complexes - in part - through MAPK-mediated VQP degradation.

Hic-5 is a transcription coregulator that acts before and/or after glucocorticoid receptor genome occupancy in a gene-selective manner

Ligand activation and DNA-binding dictate the outcome of glucocorticoid receptor (GR)-mediated transcriptional regulation by inducing diverse receptor conformations that interact differentially with coregulators. GR recruits many coregulators via the well-characterized AF2 interaction surface in the GR ligand-binding domain, but Lin11, Isl-1, Mec-3 (LIM) domain coregulator Hic-5 (TGFB1I1) binds to the relatively uncharacterized tau2 activation domain in the hinge region of GR. Requirement of hydrogen peroxide-inducible clone-5 (Hic-5) for glucocorticoid-regulated gene expression was defined by Hic-5 depletion and global gene-expression analysis. Hic-5 depletion selectively affected both activation and repression of GR target genes, and Hic-5 served as an on/off switch for glucocorticoid regulation of many genes. For some hormone-induced genes, Hic-5 facilitated recruitment of Mediator complex. In contrast, many genes were not regulated by glucocorticoid until Hic-5 was depleted. On these genes Hic-5 prevented GR occupancy and chromatin remodeling and thereby inhibited their hormone-dependent regulation. Transcription factor binding to genomic sites is highly variable among different cell types; Hic-5 represents an alternative mechanism for regulating transcription factor-binding site selection that could apply both within a given cell type and among different cell types. Thus, Hic-5 is a versatile coregulator that acts by multiple gene-specific mechanisms that influence genomic occupancy of GR as well transcription complex assembly.

MED18 interaction with distinct transcription factors regulates multiple plant functions

Mediator is an evolutionarily conserved transcriptional regulatory complex. Mechanisms of Mediator function are poorly understood. Here we show that Arabidopsis MED18 is a multifunctional protein regulating plant immunity, flowering time and responses to hormones through interactions with distinct transcription factors. MED18 interacts with YIN YANG1 to suppress disease susceptibility genes glutaredoxins GRX480, GRXS13 and thioredoxin TRX-h5. Consequently, yy1 and med18 mutants exhibit deregulated expression of these genes and enhanced susceptibility to fungal infection. In addition, MED18 interacts with ABA INSENSITIVE 4 and SUPPRESSOR OF FRIGIDA4 to regulate abscisic acid responses and flowering time, respectively. MED18 associates with the promoter, coding and terminator regions of target genes suggesting its function in transcription initiation, elongation and termination. Notably, RNA polymerase II occupancy and histone H3 lysine tri-methylation of target genes are affected in the med18 mutant, reinforcing MED18 function in different mechanisms of transcriptional control. Overall, MED18 conveys distinct cues to engender transcription underpinning plant responses.