Positive Transcription Elongation Factor Research Articles

The TAR binding dynamics and its implication in Tat degradation mechanism

Human immunodeficiency virus (HIV) is a retrovirus that progressively attacks the human immune system. It is known that the HIV viral protein Tat recruits the host elongation factor, positive transcription elongation factor b (P-TEFb), onto the nascent HIV viral transactivation response element (TAR) RNA to overcome the elongation pause for active transcription of the entire viral genome. Interestingly, there exists an amplifying feedback loop between Tat and TAR—a reduction in Tat increases the elongation pause, resulting in more TAR RNA fragments instead of the entire viral genome transcript, and the TAR fragments as a scaffold for PRC2 complex in turn promote Tat ubiquitination and degradation. In this study, the structural ensembles and binding dynamics of various interfaces in the Tat/TAR/P-TEFb complex are probed by all-atom accelerated sampling molecular dynamics simulations. The results show that a protein-binding inhibitor F07#13 targeting the Tat/P-TEFb interface initiates the above feedback loop and shuts down the active transcription. Another RNA binding inhibitor, JB181, targeting the Tat/TAR interface, can prevent TAR from pulling down the Tat from P-TEFb protein and further reducing Tat degradation. The detailed mechanism of the complex dynamics helps elucidate how Tat and TAR coordinate the regulation between HIV genome transcription versus possible HIV latency.

Transcriptional Reprogramming of Super-Enhancer Associated Oncogenes Following Inhibition of Cyclin-Dependent Kinase-9 (CDK9) in Aggressive Non-Hodgkin Lymphoma (NHL)

Introduction. CDKs are Ser/Thr kinases that associate with specific cyclins during the cell cycle to regulate its progression. We and others have shown that selective targeting of CDK9, a component of the positive transcription elongation factor b (pTEFb) complex which facilitates RNA transcription, is a promising approach in NHL leading to downmodulation of MYC and MCL1 (Hashiguchi et al, 2019). However, cells also exhibited resistance to CDK9i in vitro and in vivo. Here we studied the transcriptional and epigenetic effects of CDK9 inhibition using the novel selective CDK9 inhibitor AZD4573 which is under evaluation in clinical trials.Methods. A panel of NHL cell lines with variable sensitivity to CDK9i (OCI-LY3/19, SUDHL4/10/16, VAL, U2932) and primary NHL cells were employed. Response to CDK9i was characterized using RNA-Seq, ChIP-Seq and ATAC-Seq.Results. Treatment with AZD4573 potently inhibited proliferation of NHL cell lines and primary cells (IC 50 range 5-30 nM). RNA-Seq of cells treated with CDK9i demonstrated that up to 6,000 genes were significantly downregulated in NHL cells treated with CDK9i for 3 h, leading to downregulation of MAPK, PI3K, NFκB and TNFR signaling. However, following the initial nadir at 3 h, a subset of genes recovered transcription to at least baseline level (e.g., PIM1, MYC, IRF8, BCL6, BCL2L1, CXCR4 etc.), confirmed by RT-PCR and immunoblotting. Recovery was more prominent in less sensitive cells (OCI-LY3; by 6-8 h) and occurred despite continued p-RNAPII Ser2 blockade. ChIP-Seq analysis confirmed increased global RNAPII pausing at promoters in cells treated with AZD4573, including at recovery genes.Next, we conducted ChIP-Seq for H3K4me3 and H3K27ac in OCI-LY3 and VAL cells. We observed a global decrease in H3K4me3 signal across the promoters and gene bodies with AZD4573 treatment. Meanwhile, H3K27ac enhancer analysis showed a global decrease in VAL cells, but was stable/increased in OCI-LY3 cells, including at certain recovery genes (e.g., MYC and BCL2L1).BRD4 was previously shown to compensate for loss of CDK9 function to recover transcription of the MYC oncogene in HeLa cells, thereby compensating for loss of pTEFb activity. We observed increased interaction between BRD4 and RNAPII in OCI-LY3 cells treated with AZD4573 (by IP) and increased global BRD4 binding at transcription start sites (ChIP-Seq). Combination of BET bromodomain inhibitor JQ1 and CDK9i reduced expression of recovery genes including MYC and synergistically restricted proliferation of NHL cells.BRD4 inhibition leads to preferential loss of transcription at super enhancer (SE)-associated genes. Therefore we leveraged our H3K27ac ChIP-Seq data to elucidate the SE landscape in NHL cells. Enhancers were ranked by signal density and amplitude, followed by assignment of the closest gene. SE domains were identified near to genes known to contribute to lymphomagenesis. Notably the majority of the recovery genes had highly ranked SE association in the tested cell lines.Next, ATAC-Seq analysis of NHL cells revealed a significant number of genes with differential accessibility following CDK9i. Upon an 8 h treatment with AZD4573, 11121 and 4367 hyper-accessible chromatin regions were identified in VAL and OCI-LY3 cells, respectively, including MYC and BCL2L1 loci. E-box motif was found accessible across many genes at this timepoint.To investigate recovery genes as targets, we used PIM kinase inhibitors SGI1776 (PIM1) and AZD1208 (pan-PIM). Both drugs demonstrated synergy with AZD4573 in a panel of 4 cell lines and primary samples.Finally, to further elucidate genes and pathways which mediate susceptibility to CDK9i, we conducted a genome-wide loss of function CRISPR-Cas9 library screen. Cas9-expressing OCI-LY3 cells were transduced with a lentivirus encoding a CRISPR library comprised of ~5 unique sgRNA per gene. Knockout of genes in the PI3K/mTOR and DNA repair pathways sensitized cells to AZD4573. Confirming this result, combination treatment with AZD8835 (PI3K inhibitor) synergistically suppressed proliferation of the NHL cell lines and primary cells treated with AZD4573.Conclusions. CDK9i leads to transcriptional reprogramming including the upregulation of a subset of SE-associated oncogenes. Targeting recovery oncogenes may enhance sensitivity to CDK9i. DisclosuresDanilov: Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; TG Therapeutics: Consultancy, Research Funding; Takeda Oncology: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Rigel Pharm: Honoraria.

Pharmacologic targeting of the P-TEFb complex as a therapeutic strategy for chronic myeloid leukemia

BackgroundThe positive transcription elongation factor b (P-TEFb) kinase activity is involved in the process of transcription. Cyclin-dependent kinase 9 (CDK9), a core component of P-TEFb, regulates the process of transcription elongation, which is associated with differentiation and apoptosis in many cancer types. Wogonin, a natural CDK9 inhibitor isolated from Scutellaria baicalensis. This study aimed to investigate the involved molecular mechanisms of wogonin on anti- chronic myeloid leukemia (CML) cells.Materials and methodsmRNA and protein levels were analysed by RT-qPCR and western blot. Flow cytometry was used to assess cell differentiation and apoptosis. Cell transfection, immunofluorescence analysis and co-immunoprecipitation (co-IP) assays were applied to address the potential regulatory mechanism of wogonin. KU-812 cells xenograft NOD/SCID mice model was used to assess and verify the mechanism in vivo.ResultsWe reported that the anti-CML effects in K562, KU-812 and primary CML cells induced by wogonin were regulated by P-TEFb complex. We also confirmed the relationship between CDK9 and erythroid differentiation via knockdown the expression of CDK9. For further study the mechanism of erythroid differentiation induced by wogonin, co-IP experiments were used to demonstrate that wogonin increased the binding between GATA-1 and FOG-1 but decreased the binding between GATA-1 and RUNX1, which were depended on P-TEFb. Also, wogonin induced apoptosis and decreased the mRNA and protein levels of MCL-1 in KU-812 cells, which is the downstream of P-TEFb. In vivo studies showed wogonin had good anti-tumor effects in KU-812 xenografts NOD/ SCID mice model and decreased the proportion of human CD45+ cells in spleens of mice. We also verified that wogonin exhibited anti-CML effects through modulating P-TEFb activity in vivo.ConclusionsOur study indicated a special mechanism involving the regulation of P-TEFb kinase activity in CML cells, providing evidences for further application of wogonin in CML clinical treatment.8cwZMBLp1zasMdEPaNEt3gVideo

Development of 7SK snRNA Mimics That Inhibit HIV Transcription.

The 332-nucleotide small nuclear RNA (snRNA) 7SK is a highly conserved non-coding RNA that regulates transcriptional elongation. By binding with positive transcriptional elongation factor b (P-TEFb) via HEXIM1, 7SK snRNA decreases the kinase activity of P-TEFb and inhibits transcriptional elongation. Additionally, it is reported that 7SK inhibition results in the stimulation of human immunodeficiency virus (HIV)-specific transcription. These reports suggest that 7SK is a naturally occurring functional molecule as negative regulator of P-TEFb and HIV transcription. In this study, we developed functional oligonucleotides that mimic the function of 7SK (7SK mimics) as novel inhibitors of HIV replication. We defined the essential region of 7SK regarding its suppressive effects on transcriptional downregulation using an antisense strategy. Based on the results, we designed 7SK mimics containing the defined region. The inhibitory effects of 7SK mimics on HIV-1 long terminal repeat promoter specific transcription was drastic compared with those of the control mimic molecule. Notably, these effects were found to be more enhanced by co-transfection with Tat-expressing plasmids. From these results, it is indicated that 7SK mimics may have great therapeutic potential for HIV/AIDS treatment.

CPI-637 as a Potential Bifunctional Latency-Reversing Agent That Targets Both the BRD4 and TIP60 Proteins.

The failure of highly active antiretroviral therapy (HAART) has been largely responsible for the existence of latent human immunodeficiency virus type 1 (HIV-1) reservoirs. The “shock and kill” strategy was confirmed to reactivate HIV-1 latent reservoirs by latency-reversing agents (LRAs) for accelerated HIV-1 clearance. However, a single LRA might be insufficient to induce HIV-1 reactivation from latency due to the complexity of the multiple signaling regulatory pathways that establish the HIV-1 latent reservoir. Therefore, combinations of LRAs or dual-mechanism LRAs are urgently needed to purge the latent reservoirs. We demonstrate here for the first time that a dual-target inhibitor with a specific suppressive effect on both BRD4 and TIP60, CPI-637, could reactivate latent HIV-1 in vitro by permitting Tat to bind positive transcription elongation factor b (P-TEFb) and assembling Tat-super-elongation complex (SEC) formation. In addition, CPI-637-mediated TIP60 downregulation further stimulated BRD4 dissociation from the HIV-1 long terminal repeat (LTR) promoter, allowing Tat to more effectively bind P-TEFb compared to BRD4 inhibition alone. Much more importantly, CPI-637 exerted a potent synergistic effect but alleviated global T cell activation and blocked viral spread to uninfected bystander CD4+ T cells with minimal cytotoxicity. Our results indicate that CPI-637 opens up the prospect of novel dual-target inhibitors for antagonizing HIV-1 latency and deserves further investigation for development as a promising LRA with a “shock and kill” strategy.

Changing for the Better: Discovery of the Highly Potent and Selective CDK9 Inhibitor VIP152 Suitable for Once Weekly Intravenous Dosing for the Treatment of Cancer

Selective inhibition of exclusively transcription-regulating positive transcription elongation factor b/CDK9 is a promising new approach in cancer therapy. Starting from atuveciclib, the first selective CDK9 inhibitor to enter clinical development, lead optimization efforts aimed at identifying intravenously (iv) applicable CDK9 inhibitors with an improved therapeutic index led to the discovery of the highly potent and selective clinical candidate VIP152. The evaluation of various scaffold hops was instrumental in the identification of VIP152, which is characterized by the underexplored benzyl sulfoximine group. VIP152 exhibited the best preclinical overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats upon once weekly iv administration. VIP152 has entered clinical trials for the treatment of cancer with promising longterm, durable monotherapy activity in double-hit diffuse large B-cell lymphoma patients.

Allosteric transcription stimulation by RNA polymerase II super elongation complex

The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8Å resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that the stimulation of RNA elongation requires the dimerization module and the ELL2 linker that tethers the module to the RNA Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a closed conformation of the RNA Pol II active center cleft.

Abstract 1115: BET bromodomain inhibition synergizes with MEK inhibitors in uveal melanoma

Abstract Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Approximately 50% of the patients develop hepatic metastasis and the median survival rate is 12 months. Currently available drugs have shown limited clinical activity in patients with UM and there is an urgent need for new effective therapies. UM is characterized by activating mutations of G protein q/11, mutations in BAP1, and amplification of the oncogene MYC. Epigenetic dysregulation plays a critical role in UM pathogenesis, and we have demonstrated that Gnaq/11 mutant UM cells are addicted to BRD4 activity. BRD4 is a key regulator of transcriptional elongation by recruiting positive transcriptional elongation factor complex (PTEFb) to chromatin and activating RNA polymerase II-dependent transcription. While BET inhibitors have shown potent activity in pre-clinical studies, single-agent BET inhibitors exhibited limited efficacy in advanced UM clinical trials. MAPK signaling in constitutively active in Gnaq/11 mutant UM cells and previous studies have shown that the MAPK pathway mediates resistance to BET inhibition. Therefore, we sought to combine MEK and BET inhibitors in a panel of UM cells. We found synergistic activities with a variety of structurally distinct MEK inhibitors including trametinib and binimetinib in combination with BET inhibitors, such as I-BET and PLX2853. These combinations induced a greater reduction in cell viability and significant apoptosis compared to either monotherapy, in both parental and BET inhibitor-resistant cells. Western blot studies were performed to evaluate the mechanism of the combination treatment with BET and MEK inhibition and demonstrated a marked downregulation of p-ERK, MYC, Bcl-xL and upregulation of BIM with the combination therapy. Similarly, the combination treatment resulted in greater inhibition of tumor growth compared to either BET or MEK inhibition alone in a Gnaq mutant xenograft model. In conclusion, our studies suggest that co-targeting of MEK and BET proteins may be required to maximize the responses of UM cells to BET inhibitors. Clinical trials with the combination of BET and MEK inhibitors are warranted. Citation Format: Grazia Ambrosini, Elgilda Musi, Gary K. Schwartz. BET bromodomain inhibition synergizes with MEK inhibitors in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1115.

Epigenetic and Transcriptomic Profiling of MLL Rearranged Infant Acute Lymphoblastic Leukemia

[Background] Infant acute lymphoblastic leukemia (ALL) accounts for less than 5% of childhood leukemia, and is characterized by Mixed lineage leukemia (MLL also called KMT2A ) rearrangements found in approximately 80% of patients. Also found in 5-10% of ALL in older children and adults, MLL -rearrangement positivity is highly associated with poor outcome in ALL; there is an urgent need for molecular findings of potential therapeutic targets and/or risk stratification markers, which can be translated into the clinical practice. Recently, the mechanisms that MLL fusion proteins recruit epigenetic regulators, including histone H3 lysine 79 methyltransferase DOT1L and BET family histone readers, to activate the transcription of target genes have been reported, suggesting epigenetic dysregulation should play a central role in the leukemogenesis. However, DNA-level methylation profiles of MLL -rearranged leukemia and their associations with gene expression profiles are not fully characterized.[Materials and Methods] Our cohort included samples from the Infant Leukemia Sub-committee, Japan Children's Cancer Group. Both methylation array and RNA sequencing were performed on the diagnostic leukemia samples from 38 infants with ALL, consisting of 30 cases with MLL -rearrangements identified by fluorescence in situ hybridization or Southern blotting, and 8 MLL -germline cases. Methylation beta values were normalized with beta-mixture quantile normalization method. For RNA sequencing data analysis, we used publicly available Genomon Pipeline.[Results] RNA sequencing confirmed MLL -rearrangements in 30 cases, whose partner genes comprised AFF1 (n=15), MLLT1 (n=6), MLLT3 (n=3), MLLT10 (n=2), EPS15 (n=1), AFF2 (n=1), ABI1 (n=1), and SARNP (n=1). As expected, DNA methylation-based hierarchical clustering divided the cases into 2 clusters with highly distinctive methylation profiles: MLL -rearranged cluster (MLL -R cluster) and MLL -germline cluster (MLL -G cluster). Intriguingly, 3 out of 30 (10%) MLL -rearranged cases were classified into MLL -G cluster; these cases revealed uncommon partner genes, such as ABI1 , SARNP , and MLLT10, respectively. ABI1, an Eps8 SH3 domain-binding protein, and SARNP, a SAP domain containing ribonucleoprotein, are rare fusion partners of MLL. The MLL -germline leukemia-like methylation profiles suggested different leukemogenic mechanisms from common fusion partners, such as AFF1 and MLLT1, which directly or indirectly interact with DOT1L and positive transcription elongation factor b. Because DOT1L-binding domain of MLLT10 was retained in both of the 2 cases with MLL-MLLT10 in our cohort, additional mechanisms causing MLL-germline leukemia-like methylation in a single case may be present. As a result of inter-cluster comparison, MLL -R clusterexhibited global hypermethylation compared to MLL -G cluster (P -value < 2.2 × 10−16, Kolmogorov-Smirnov test). Methylation-based gene set enrichment analysis demonstrated significant promoter hypermethylation on cell development/differentiation-related gene sets and hypomethylation on DNA replication/cell proliferation-related gene sets (P -value < 0.01, hypergeometric test) in MLL -G cluster. Consistently, RNA sequencing-based gene expression analysis demonstrated overexpression of cell cycle regulators, such as cyclins/cyclin-dependent kinases, in MLL -R cluster, which may be associated with more aggressively proliferating diseases.[Conclusion] Corresponding to known aberrant histone modifications in MLL -rearranged leukemia, largely different DNA methylation profiles between MLL -R and MLL -G clusters have been demonstrated. Because DNA-level methylation on CpG dinucleotides interactively affects histone modifications, these results implicated aberrant DNA methylation in the pathogenesis of MLL -rearranged leukemia as a part of epigenetic dysregulation. We also identified a subset of MLL -rearranged cases with MLL -germline leukemia-like methylation/expression profiles, indicating that not only MLL -rearrangement positivity, but also the types of MLL -rearrangement partners may affect a biological feature of leukemic cells. These cases may possibly be therapeutically stratified as relatively lower risk group compared to other MLL -rearranged cases. Further detailed analyses using clinical information are planned. DisclosuresNo relevant conflicts of interest to declare.

The CDK7 Inhibitor THZ1 Alters RNA Polymerase Dynamics at the 5‘ and 3‘ Ends of Genes

The t(8;21) is one of the most frequent chromosomal translocations in acute myeloid leukemia (AML). This translocation encodes a fusion protein containing the DNA binding domain of RUNX1, a master regulator of hematopoiesis, linked to the majority of the myeloid translocation gene on chromosome 8 (MTG8, also known as eight-twenty-one or ETO; gene name RUNX1T1). This chimeric fusion protein binds to the RUNX1 DNA elements to control gene expression by recruiting transcriptional cofactors via the MTG8/ETO moiety. In addition, MTG/ETO family proteins co-purified in a complex that also contained “E proteins” and the cyclin dependent kinase CDK9, a component of the positive transcription elongation factor b (P-TEFb).P-TEFb contains Cyclin T1 and CDK9 and several potential CDK9 inhibitors have shown promise in the clinics for t(8;21) AML, such as flavopiridol (alvocidib). Transcription initiation, promoter-proximal pausing of RNA polymerase II (RNAPII), and transcription elongation are events that are highly regulated for genes involved in proliferation and differentiation such as MYC and BCL2 . CDK7 phosphorylates Ser5 of the C-terminal domain of RNAPII to trigger promoter-proximal pausing of RNA polymerase II, whereas CDK9 phosphorylates Ser2 of RNAPII and the negative elongation factors DSIF and NELF to release the polymerase into the body of the gene.In this study, we used inhibitors of CDK7 and CDK9 to probe for transcriptional vulnerabilities in t(8;21) AML. We used both broad spectrum (flavopiridol, dinacicilib) and a specific CDK9 inhibitor PHA767491 along with THZ1 (CDK7 inhibitor). Since CDK7 is also a CAK (CDK activating kinase) that activates cell-cycle CDKs and mutations in Cyclin D2 are common in t(8;21) AML, we also tested the highly selective CDK4/6 inhibitor palbociclib (PD-0332991). We observed that the t(8;21)-containing AML cell lines SKNO-1 and Kasumi-1, as well as primary AML patient cells, were extremely sensitive to all the CDK7/9 inhibitors, but not palbociclib. We used precision global run-on transcription sequencing (PROseq) to define the mechanism of action of these compounds. This technique creates high resolution genomic maps of the active RNA polymerases to define the effects on RNA polymerase dynamics. CDK9 inhibitors caused wide spread RNA Polymerase II promoter proximal pausing at nearly 80% of the expressed genes, as well as at enhancers where there was a general impairment of production of eRNAs and lncRNAs. Conversely, inhibition of CDK7, a critical component of TFIIH caused a loss of paused RNA polymerase II at promoters, which was associated with an increase of polymerases in the body of genes. While RUNX1 was reported to be regulated by CDK7 in T-ALL, there was no impact on the expression of RUNX1 or the fusion protein in t(8;21)-containing cells. Although enhancers were not affected, super enhancers were modestly affected by THZ1. Interestingly, within a subset of genes that showed increased RNA polymerase accumulation within the gene body, CDK7 inhibition altered 3' end transcription termination dynamics within 1 hr and this effect remained even at 2 hr. A time course of THZ1 treatment at 0, 15', 30', and 60' suggested that CDK7 inhibition altered RNA polymerase dynamics at 5'ends of the genes, and it's these effects that eventually affected transcription termination at the 3' ends of the genes. DisclosuresSavona:Amgen: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Takeda: Research Funding; Karyopharm: Consultancy, Equity Ownership; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Consultancy, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

CDK9 Inhibition with LDC526 Is an Effective Treatment for T-Cell Prolymphocytic Leukemia (T-PLL) in Vitro

▪IntroductionT-cell prolymphocytic leukemia (T-PLL) is a rare leukemia with an aggressive disease course, characterized by chemotherapy resistance and a median overall survival of less than two years. Apart from defects in DNA damage response genes like ATM and genes of JAK/STAT signaling, overexpression of MYC, often caused by gain of chromosomal material, is frequently observed in T-PLL. Intravenous application of the anti-CD52 antibody Alemtuzumab has been demonstrated to improve progression-free survival and is currently considered the standard of care. In younger patients remissions can be consolidated by allogeneic stem cell transplantation, which results in long-term remission in a small subgroup of patients. The prognosis in the majority of patients relapsing from their disease is dismal and thus novel treatment options are urgently needed. Cyclin-dependent kinase 9 (CDK9), a serine/ threonine kinase which is ubiquitously expressed, is the catalytic unit of the positive transcription elongation factor (P-TEFb). In the presence of cyclin T, p-TEFb stimulates transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNA Pol II). CDK9 inhibition by LDC526, a specific CDK9 inhibitor, leads to reduced phosphorylation of CTD-Ser2, Ser5 and Ser7 of RNA Pol II and subsequently to the downregulation of MYC and MCL-1.Methods</spanCD3+CD4+ tumor cells from cryopreserved peripheral blood samples of 11 T-PLL patients were enriched to a purity of >95% by fluorescence-activated cell sorting (FACS) or magnetic activated cell separation (MACS). RNA and DNA were isolated using the RNeasy Mini Kit® or the QIAamp DNA blood midi kit® (Qiagen, Hilden, Germany), respectively. To determine IC50 values, we performed cell viability assays for T-PLL cells of 11 patients treated with LDC526 and a panel of reference compounds compared to DMSO controls (CellTiter-Glo Luminescent Cell Viability Assay; Promega, Fitchburg, WI, USA). Gene expression profiling was performed in primary T-PLL cells treated with the CDK9 inhibitor LDC526 in a concentration of 10µM compared to DMSO (n=3) on Clariom S Pico Assays (Affymetrix, Santa Clara, CA, USA) and analyzed by gene set enrichment analysis (GSEA) and GeneTrail analysis. Gene expression measurement was performed with quantitative real-time PCR (qPCR) for MYC (Taqman, Thermo Fisher Scientific, Waltham, MA, USA). Protein expression was analyzed for pCTD Ser2, Ser5 and Ser7, MYC, p-STAT3 and MCL-1 by Western Blot (n=4) after LDC526-treatment in three different concentrations (10, 30 and 100 µM).ResultsLDC526 inhibited T-PLL cell survival in vitro in a dose-dependent manner. The median IC50 was 1.7 µM (range: 0.9 -3.5) which compared favourably to a panel of reference compounds including fludarabine, clofarabine, methotrexate, cytarabine and ruxolitinib (median IC50 values 6.4, 6.2, 29.9, 8.3, and 29.2, respectively). Similar experiments with BAY1143572, a structurally closely related CDK9 inhibitor, which is currently investigated in phase I studies of acute myeloid leukemia, revealed an even lower median IC50 of 0.9 µM (range: 0.5 - 1.7). Of note, the purine analogues fludarabine and clofarabine were only active in some of the patients tested, whereas both CDK9 inhibitors showed activity in all samples analyzed.Gene expression profiles of LDC526 treated T-PLL cells compared to DMSO controls revealed an enrichment of genes in gene sets associated with interferon and TNFA signatures, MYC targets as well as JAK/STAT genes, which were subsequently confirmed by qPCR experiments. At the protein level LDC526 dose-dependently reduced phosphorylation of CTD-Ser2, Ser7 and to a lesser extent of Ser5 compared to DMSO controls resulting in efficient down-regulation of MYC, MCL-1 and p-STAT3.ConclusionsOur experiments identify CDK9 as a novel therapeutic target in T-PLL which can be efficiently inhibited by LDC526 and BAY1143572 at clinically meaningful concentrations. CDK9 inhibition resulted in both reduced T-PLL cell survival and down-regulation of MYC and JAK/STAT signaling, which have a key role in the molecular pathogenesis of T-PLL. DisclosuresDürig:Lead Discovery Center: Research Funding.

DDB1 binds histone reader BRWD3 to activate the transcriptional cascade in adipogenesis and promote onset of obesity.

Obesity has become a global pandemic. Identification of key factors in adipogenesis helps to tackle obesity and related metabolic diseases. Here, we show that DDB1 binds the histone reader BRWD3 to promote adipogenesis and diet-induced obesity. Although typically recognized as a component of the CUL4-RING E3 ubiquitin ligase complex, DDB1 stimulates adipogenesis independently of CUL4. A DDB1 mutant that does not bind CUL4A or CUL4B fully restores adipogenesis in DDB1-deficient cells. Ddb1+/- mice show delayed postnatal development of white adipose tissues and are protected from diet-induced obesity. Mechanistically, by interacting with BRWD3, DDB1 is recruited to acetylated histones in the proximal promoters of ELK1 downstream immediate early response genes and facilitates the release of paused RNA polymerase II, thereby activating the transcriptional cascade in adipogenesis. Our findings have uncovered a CUL4-independent function of DDB1 in promoting the transcriptional cascade of adipogenesis, development of adipose tissues, and onset of obesity.

Latency Reversing Agents: Kick and Kill of HTLV-1?

Human T-cell leukemia virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), is a retrovirus, which integrates into the host genome and persistently infects CD4+ T-cells. Virus propagation is stimulated by (1) clonal expansion of infected cells and (2) de novo infection. Viral gene expression is induced by the transactivator protein Tax, which recruits host factors like positive transcription elongation factor b (P-TEFb) to the viral promoter. Since HTLV-1 gene expression is repressed in vivo by viral, cellular, and epigenetic mechanisms in late phases of infection, HTLV-1 avoids an efficient CD8+ cytotoxic T-cell (CTL) response directed against the immunodominant viral Tax antigen. Hence, therapeutic strategies using latency reversing agents (LRAs) sought to transiently activate viral gene expression and antigen presentation of Tax to enhance CTL responses towards HTLV-1, and thus, to expose the latent HTLV-1 reservoir to immune destruction. Here, we review strategies that aimed at enhancing Tax expression and Tax-specific CTL responses to interfere with HTLV-1 latency. Further, we provide an overview of LRAs including (1) histone deacetylase inhibitors (HDACi) and (2) activators of P-TEFb, that have mainly been studied in context of human immunodeficiency virus (HIV), but which may also be powerful in the context of HTLV-1.

Targeting CDK9 for Anti-Cancer Therapeutics.

Simple SummaryCDK9, in combination with Cyclin T1, is one of the major regulators of RNA Polymerase II mediated productive transcription of critical genes in any cell. The activity of CDK9 is significantly up-regulated in a wide variety of cancer entities, to aid in the overexpression of genes responsible for the regulation of functions, which are beneficial to the cancer cells, like proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Enhanced CDK9 activity, therefore, leads to poorer prognosis in many cancer types, offering the rationale to target it using small-molecule inhibitors. Several, increasingly specific inhibitors, have been developed, some of which are presently in clinical trials. Other approaches being tested involve combining inhibitors against CDK9 activity with those against CDK9’s upstream regulators like BRD4, SEC and HSP90; or downstream effectors like cMYC and MCL-1. The inhibition of CDK9’s activity holds the potential to be a highly effective anti-cancer therapeutic.Cyclin Dependent Kinase 9 (CDK9) is one of the most important transcription regulatory members of the CDK family. In conjunction with its main cyclin partner—Cyclin T1, it forms the Positive Transcription Elongation Factor b (P-TEFb) whose primary function in eukaryotic cells is to mediate the positive transcription elongation of nascent mRNA strands, by phosphorylating the S2 residues of the YSPTSPS tandem repeats at the C-terminus domain (CTD) of RNA Polymerase II (RNAP II). To aid in this process, P-TEFb also simultaneously phosphorylates and inactivates a number of negative transcription regulators like 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) Sensitivity-Inducing Factor (DSIF) and Negative Elongation Factor (NELF). Significantly enhanced activity of CDK9 is observed in multiple cancer types, which is universally associated with significantly shortened Overall Survival (OS) of the patients. In these cancer types, CDK9 regulates a plethora of cellular functions including proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Due to the extremely critical role of CDK9 in cancer cells, inhibiting its functions has been the subject of intense research, resulting the development of multiple, increasingly specific small-molecule inhibitors, some of which are presently in clinical trials. The search for newer generation CDK9 inhibitors with higher specificity and lower potential toxicities and suitable combination therapies continues. In fact, the Phase I clinical trials of the latest, highly specific CDK9 inhibitor BAY1251152, against different solid tumors have shown good anti-tumor and on-target activities and pharmacokinetics, combined with manageable safety profile while the phase I and II clinical trials of another inhibitor AT-7519 have been undertaken or are undergoing. To enhance the effectiveness and target diversity and reduce potential drug-resistance, the future of CDK9 inhibition would likely involve combining CDK9 inhibitors with inhibitors like those against BRD4, SEC, MYC, MCL-1 and HSP90.

The 7SK/P-TEFb snRNP controls ultraviolet radiation-induced transcriptional reprogramming.

Conversion of promoter-proximally paused RNA polymerase II (RNAPII) into elongating polymerase by the positive transcription elongation factor b (P-TEFb) is a central regulatory step of mRNA synthesis. The activity of P-TEFb is controlled mainly by the 7SK small nuclear ribonucleoprotein (snRNP), which sequesters active P-TEFb into inactive 7SK/P-TEFb snRNP. Here we demonstrate that under normal culture conditions, the lack of 7SK snRNP has only minor impacts on global RNAPII transcription without detectable consequences on cell proliferation. However, upon ultraviolet (UV)-light-induced DNA damage, cells lacking 7SK have a defective transcriptional response and reduced viability. Both UV-induced release of "lesion-scanning" polymerases and activation of key early-responsive genes are compromised in the absence of 7SK. Proper induction of 7SK-dependent UV-responsive genes requires P-TEFb activity directly mobilized from the nucleoplasmic 7SK/P-TEFb snRNP. Our data demonstrate that the primary function of the 7SK/P-TEFb snRNP is to orchestrate the proper transcriptional response to stress.

Npac Is A Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse Embryonic Stem Cells

Chromatin modification contributes to pluripotency maintenance in embryonic stem cells (ESCs). However, the related mechanisms remain obscure. Here, we show that Npac, a “reader” of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC (mESC) pluripotency since knockdown of Npac causes mESC differentiation. Depletion of Npac in mouse embryonic fibroblasts (MEFs) inhibits reprogramming efficiency. Furthermore, our chromatin immunoprecipitation followed by sequencing (ChIP-seq) results of Npac reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs. Interestingly, we find that Npac interacts with positive transcription elongation factor b (p-TEFb), Ser2-phosphorylated RNA Pol II (RNA Pol II Ser2P), and Ser5-phosphorylated RNA Pol II (RNA Pol II Ser5P). Furthermore, depletion of Npac disrupts transcriptional elongation of the pluripotency genes Nanog and Rif1. Taken together, we propose that Npac is essential for the transcriptional elongation of pluripotency genes by recruiting p-TEFb and interacting with RNA Pol II Ser2P and Ser5P.

Transcriptional programming drives Ibrutinib-resistance evolution in mantle cell lymphoma

SUMMARYIbrutinib, a bruton’s tyrosine kinase (BTK) inhibitor, provokes robust clinical responses in aggressive mantle cell lymphoma (MCL), yet many patients relapse with lethal Ibrutinib-resistant (IR) disease. Here, using genomic, chemical proteomic, and drug screen profiling, we report that enhancer remodeling-mediated transcriptional activation and adaptive signaling changes drive the aggressive phenotypes of IR. Accordingly, IR MCL cells are vulnerable to inhibitors of the transcriptional machinery and especially so to inhibitors of cyclin-dependent kinase 9 (CDK9), the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Further, CDK9 inhibition disables reprogrammed signaling circuits and prevents the emergence of IR in MCL. Finally, and importantly, we find that a robust and facile ex vivo image-based functional drug screening platform can predict clinical therapeutic responses of IR MCL and identify vulnerabilities that can be targeted to disable the evolution of IR.

Resveratrol Promotes HIV-1 Tat Accumulation via AKT/FOXO1 Signaling Axis and Potentiates Vorinostat to Antagonize HIV-1 Latency.

The latent reservoir of HIV-1 is a major barrier to achieving the eradication of HIV-1/AIDS. One strategy is termed "shock and kill", which aims to awaken the latent HIV-1 using latency reversing agents (LRAs) to replicate and produce HIV-1 particles. Subsequently, the host cells containing HIV-1 can be recognized and eliminated by the immune response and anti-retroviral therapy. Although many LRAs have been found and tested, their clinical trials were dissatisfactory. To aim of the study was to investigate how resveratrol reactivates silent HIV-1 transcription and assess if resveratrol could be a candidate drug for the "shock" phase in "shock and kill" strategy. We used established HIV-1 transcription cell models (HeLa-based NH1 and NH2 cells) and HIV-1 latent cell models (J-Lat A72 and Jurkat 2D10 cells). We performed resveratrol treatment on these cell lines and studied the mechanism of how resveratrol stimulates HIV-1 gene transcription. We also tested resveratrol's bioactivity on primary cells isolated from HIV-1 latent infected patients. Resveratrol promoted HIV-1 Tat protein levels, and resveratrol-induced Tat promotion was found to be dependent on the AKT/FOXO1 signaling axis. Resveratrol could partially dissociate P-TEFb (Positive Transcription Elongation Factor b) from 7SK snRNP (7SK small nuclear Ribonucleoprotein) and promote Tat-SEC (Super Elongation Complex) interaction. Preclinical studies showed that resveratrol potentiated Vorinostat to awaken HIV-1 latency in HIV-1 latent infected cells isolated from patients. We found a new mechanism of resveratrol stimulating the production of HIV-1. Resveratrol could be a promising candidate drug to eradicate HIV-1 reservoirs.

MBRS-67. ROLE OF CYCLIN DEPENDENT KINASE-9 IN Myc-ENHANCED MEDULLOBLASTOMA

Myc is highly expressed in group 3 medulloblastoma (Myc-MB) and influences cell growth, proliferation and oncogenesis by directly promoting the activity of RNA polymerases (RNA Pol). Myc driven RNA Pol II activity is mediated by Positive Transcription Elongation Factor b (pTEFb). pTEFb’s catalytic core consists of cyclin dependent kinase-9 (CDK9) and Cyclin T, that phosphorylate and release RNA Pol II into active elongation. CDK9 is over expressed in group 3 MB suggesting that MB may be vulnerable to inhibition of CDK9 (CDK9i). The exact mechanism is not completely known in MB. Genetic depletion of CDK9 suppressed Myc-MB cell clonogenicity in vitro and tumor growth in vivo. CDK9i by two clinically relevant inhibitors, Atuveciclib and AZD4573, suppressed clonogenicity and cell self-renewal of Myc-MB cell lines. CDK9i in Myc-MB cell lines downregulated Myc and RNA Pol II phosphorylation at Ser2 and Ser5, and, upregulated P21. Further, mice with orthotopic xenografts treated with CDK9 inhibitors survived significantly longer than control mice. RNA-Seq-based gene set enrichment analysis showed that CDK9i decreased c-Myc driven transcriptomic programs and enhanced differentiation networks. ChIP-Seq for Pol2 and Myc, demonstrated that the Myc-driven aberrant transcriptional input can be reversed via CDK9i. These findings highlight the role of CDK9 in Myc-driven pathogenesis and that its inhibition is critical to the treatment of Myc-MB.

Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors

RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6 to 22 μm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.