Inhibition Of P-TEFb Research Articles

Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.

XRT-06. RAPID PTEFB-DEPENDENT TRANSCRIPTIONAL REORGANIZATION UNDERPINS THE GLIOMA ADAPTIVE RESPONSE TO RADIOTHERAPY

Abstract Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using a combination of ChIP-, ATAC-, and RNA-seq in model systems of diffuse intrinsic pontine glioma and other pediatric high-grade gliomas, we observe a rapid genome-wide reorganization of active chromatin within hours of exposure to therapeutic ionizing radiation. This redistribution facilitates PTEFb-mediated pause-release of Pol II to drive nascent transcriptional induction of canonical DNA damage response programs. Concurrent inhibition of PTEFb imparts a transcription elongation defect, disrupting this chromatin reorganization and blunting transcriptional induction. Functionally, concurrent PTEFb inhibition abrogates key adaptive programs such as DNA repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

VIP152 is a selective CDK9 inhibitor with pre-clinical in vitro and in vivo efficacy in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is effectively treated with targeted therapies including Bruton tyrosine kinase inhibitors and BCL2 antagonists. When these become ineffective, treatment options are limited. Positive transcription elongation factor complex (P-TEFb), a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, functions to regulate short half-life transcripts by phosphorylation of RNA Polymerase II (POLII). These transcripts are frequently dysregulated in hematologic malignancies; however, therapies targeting inhibition of P-TEFb have not yet achieved approval for cancer treatment. VIP152 kinome profiling revealed CDK9 as the main enzyme inhibited at 100 nM, with over a 10-fold increase in potency compared with other inhibitors currently in development for this target. VIP152 induced cell death in CLL cell lines and primary patient samples. Transcriptome analysis revealed inhibition of RNA degradation through the AU-Rich Element (ARE) dysregulation. Mechanistically, VIP152 inhibits the assembly of P-TEFb onto the transcription machinery and disturbs binding partners. Finally, immune competent mice engrafted with CLL-like cells of Eµ-MTCP1 over-expressing mice and treated with VIP152 demonstrated reduced disease burden and improvement in overall survival compared to vehicle-treated mice. These data suggest that VIP152 is a highly selective inhibitor of CDK9 that represents an attractive new therapy for CLL.

HGG-12. Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy

BACKGROUND: Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated promoter proximal pause-release of Pol II is a conserved regulatory mechanism for synchronous transcriptional induction best described in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. DESIGN/METHOD: In order to examine the dynamics of chromatin reorganization following radiotherapy, we performed a combination of ChIP-, ATAC-, and RNA-seq in model systems of diffuse intrinsic pontine glioma (DIPG) and other pediatric high-grade gliomas (pHGG) following IR exposure. We interrogated IR-induced gene expression in the presence or absence of PTEFb blockade, including both mechanistic and functional consequences of concurrent inhibition or genetic depletion. We utilized culture models with live cell imaging to assess the therapeutic synergy of PTEFb inhibition with IR, as well as the therapeutic index of this intervention relative to normal controls. Finally, we employed orthotopic models of pHGG treated with conformal radiotherapy and CNS-penetrant PTEFb inhibitors in order to assess tolerability and anti-tumor effect in vivo. RESULTS: Rapid genome-wide redistribution of active chromatin features and PTEFb facilitates Pol II pause-release to drive nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb imparts a transcription elongation defect, abrogating canonical adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. CONCLUSION: These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

Abstract 3022: BAY 1143572, a first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, shows convincing anti-tumor activity in preclinical models of acute myeloid leukemia (AML)

Abstract PTEFb/CDK9 mediated transcription of short-lived anti-apoptotic survival proteins like Mcl-1 and Myc, plays a critical role in cancer cell growth and survival in various tumor entities including AML. In addition, these survival proteins exhibit important functions in the development of resistance to chemotherapy. In contrast to pan-CDK inhibitors, to our knowledge PTEFb selective inhibitors have not been explored for clinical utility. We report the preclinical activity of BAY 1143572, a novel selective PTEFb/CDK9 inhibitor (AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT02-02) currently being investigated in Phase I clinical trials in advanced cancer (NCT01938638) and acute leukemia (NCT02345382) in various in vitro, ex vivo and in vivo models of AML. BAY 1143572 inhibited the proliferation of 7 MLL-rearrangements positive and negative AML cell lines with a median IC50 of 385 nM (range 230-1100 nM) and induced apoptosis. Furthermore, BAY 1143572 showed potent in vitro activity in 8 out of 10 non-MLL-rearranged patient derived AML samples incl. NPM1 mutant and Flt3-ITD positive samples derived from intermediate and high risk patients. Moreover, we elucidated the dynamic changes of the cellular proteome/phosphoproteome upon pharmacological and genetic PTEFb inhibition and identified PTEFb interaction partners in various AML in vitro models. These analyses uncover the oncogenic PTEFb-dependent signaling networks and substantiate the molecular rationale for the use of PTEFb inhibitors in this indication. When applied in vivo, BAY 1143572 exhibited single agent efficacy at tolerated doses in 4 out of 5 AML xenograft tumor models in mice and in 2 out of 2 AML xenograft tumor models in rats upon once daily oral administration. Of note, partial or even complete remissions could be achieved in several models. Furthermore, intermittent dosing schedules with up to 4 days treatment pauses were feasible in terms of efficacy and tolerability. Using MOLM-13 xenografts in mice and rats to address the in vivo MoA of BAY 1143572, a transient inhibition of RNA polymerase II phosphorylation, MYC mRNA and protein levels, MCL-1 mRNA and protein levels, and an induction of apoptosis was documented. In conclusion, our data provide the rationale for the initiation of clinical development of BAY 1143572 as a potent and highly selective inhibitor of PTEFb/CDK9 with first-in-class potential for the treatment of AML patients. A phase I clinical trial to determine the safety, tolerability and recommended Phase 2 dose in this indication is ongoing (NCT02345382). Citation Format: Arne Scholz, Thomas Oellerich, Akhtar Hussain, Sarah Lindner, Ulrich Luecking, Annette O. Walter, Peter Ellinghaus, Ray Valencia, Franz von Nussbaum, Dominik Mumberg, Michael Brands, Stuart Ince, Hubert Serve, Karl Ziegelbauer. BAY 1143572, a first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, shows convincing anti-tumor activity in preclinical models of acute myeloid leukemia (AML). [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 3022.

T-bet Activates Th1 Genes through Mediator and the Super Elongation Complex.

SummaryThe transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates.

Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis

Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.

Abstract 2828: Rapid identification of potent and highly selective, oral PTEFb Inhibitor BAY 1143572 with first in class potential

Abstract PTEFb (positive transcription elongation factor b) is a heterodimer of the transcriptional control kinase CDK9 (Cyclin-dependent kinase 9) and Cyclin T. PTEFb phosphorylates and activates RNA polymerase II. PTEFb inhibition causes rapid depletion of short-lived mRNA transcripts and their associated protein products involved in proliferation and survival like Myc, or Mcl-1 which results in cell death of addicted tumor cells. We previously disclosed the profile of the lead compound PTEFb BAY1, a nanomolar PTEFb inhibitor with 50-fold selectivity within the CDK family and cellular potency of about 1 μM in proliferation assays on various human tumor cell lines [1]. PTEFb BAY1 also revealed in vivo efficacy in a human acute myeloid leukemia (AML) xenograft model in nude mice. However, the lead compound also displayed certain limitations in ADME properties like low aqueous solubility and a strong recognition by efflux transporters in the Caco2 assay. Based on these findings, extensive lead optimisation efforts led to the rapid identification of BAY 1143572 which is a more potent and highly selective, orally available PTEFb inhibitor with first-in-class potential. BAY 1143572 has a high aqueous solubility, reduced drug efflux and a moderate oral bioavailability across species that allows daily as well as intermittent dosing schedules in animal models. BAY 1143572 revealed strong in vitro and in vivo anti-tumor efficacy with various cell-lines. BAY 1143572 is currently being evaluated in a Phase I study to determine the safety, tolerability, pharmacokinetics and initial pharmacodynamic biomarker response in patients with advanced cancer. This presentation will highlight the key learnings from our PTEFb lead optimization program. [1]: AACR, April 5-9, 2014, San Diego, Poster Presentation, Abstract 4538, Cancer Res October 1, 2014, 74:4538; doi:10.1158/1538-7445.AM2014-4538 Citation Format: Ulrich TJ Luecking, Arne Scholz, Philip Lienau, Gerhard Siemeister, Dirk Kosemund, Rolf Bohlmann, Knut Eis, Mark Gnoth, Ildiko Terebesi, Kirstin Meyer, Katja Prelle, Ray Valencia, Stuart Ince, Franz von Nussbaum, Dominik Mumberg, Karl Ziegelbauer, Bert Klebl, Axel Choidas, Peter Nussbaumer, Matthias Baumann, Carsten Schultz-Fademrecht, Gerd Ruehter, Jan Eickhoff, Michael Brands. Rapid identification of potent and highly selective, oral PTEFb Inhibitor BAY 1143572 with first in class potential. [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 2828. doi:10.1158/1538-7445.AM2015-2828

Abstract 4538: BAY 1112054, a highly selective, potent and orally available inhibitor of PTEFb/CDK9, shows convincing anti-tumor activity

Abstract The family of cyclin-dependent kinase (CDK) proteins consists of multiple cell cycle regulating CDK members as well as members involved in the regulation of gene transcription like CDK9/PTEFb (positive transcription elongation factor b). Inhibition of PTEFb and its direct downstream target RNA polymerase II is thought to cause rapid depletion of short-lived mRNA transcripts of important survival proteins like c-myc and Mcl-1 and thereby to induce growth delay and apoptosis in addicted tumor cells. In contrast to pan-CDK inhibitiors which are currently evaluated in Phase I and II clinical trials, PTEFb selective inhibitors have not been explored for clinical utility. BAY 1112054 is a potent and highly selective PTEFb-kinase inhibitor with low nanomolar activity against PTEFb/CDK9 and an at least 50-fold selectivity against other CDKs in enzymatic assays. Furthermore, BAY 1112054 shows a favourable selectivity against non-CDK kinases in vitro. The compound exhibits broad anti-proliferative activity against a panel of tumor cell lines with sub-micromolar IC-50 values. In line with the proposed mode of action, a concentration-dependent inhibition of the phosphorylation of the RNA polymerase II was observed in A549 tumor cells. This inhibition was accompanied by a reduction of intracellular Mcl-1 protein levels. Furthermore, BAY 1112054 increased DNA fragmentation in synchronized HeLa cells upon compound treatment for 24 hours. BAY 1112054 showed convincing in vivo efficacy at tolerated doses in two xenograft models in mice. Once daily oral treatment led to complete tumor stasis in established MOLM-13 AML xenografts. Pharmacokinetic analysis revealed that unbound plasma levels were 8 to 12 hours above the cellular IC50 in this model. In vivo efficacy and tolerability of the once daily po schedule of BAY 1112054 was confirmed in NCI-H82 SCLC xenografts. Xenografted tumors of this model showed lower levels of RNA polymerase II phosphorylation and Mcl-1 upon treatment with BAY 1112054. In conclusion, our data provides in vitro and in vivo proof of concept for BAY 1112054, a potent and highly selective inhibitor of PTEFb/CDK9 with first-in-class potential, and warrant further clinical evaluation of PTEFb selective inhibitors for the treatment of cancers addicted to the transcription of short-lived anti-apoptotic survival proteins. Citation Format: Arne Scholz, Ulrich Lücking, Gerhard Siemeister, Philip Lienau, Knut Eis, Antje Wengner, Kirstin Petersen, Ulf Bömer, Peter Nussbaumer, Axel Choidas, Gerd Rühter, Jan Eickhoff, Carsten Schultz-Fademrecht, Bert Klebl, Stuart Ince, Franz von Nussbaum, Dominik Mumberg, Michael Brands, Karl Ziegelbauer. BAY 1112054, a highly selective, potent and orally available inhibitor of PTEFb/CDK9, shows convincing anti-tumor activity. [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 4538. doi:10.1158/1538-7445.AM2014-4538

Brd4 activates P-TEFb for RNA polymerase II CTD phosphorylation.

The bromodomain protein Brd4 regulates the transcription of signal-inducible genes. This is achieved by recruiting the positive transcription elongation factor P-TEFb to promoters by its P-TEFb interaction domain (PID). Here we show that Brd4 stimulates the kinase activity of P-TEFb for phosphorylation of the C-terminal domain (CTD) of RNA polymerase II over basal levels. The CTD phosphorylation saturation levels, the preferences for pre-phosphorylated substrates, and the phosphorylation specificity for Ser5 of the CTD however remain unchanged. Inhibition of P-TEFb by Hexim1 is relieved by Brd4, although no mutual displacement with the Cyclin T-binding domain of Hexim1 was observed. Brd4 PID shows a surprising sequence motif similarity to the trans-activating Tat protein from HIV-1, which includes a core RxL motif, a polybasic cluster known as arginine-rich motif, and a C-terminal leucine motif. Mutation of these motifs to alanine significantly diminished the stimulatory effect of Brd4 and fully abrogated its activation potential in presence of Hexim1. Yet the protein was not found to bind Cyclin T1 as Tat, but only P-TEFb with a dissociation constant of 0.5 μM. Our data suggest a model where Brd4 acts on the kinase subunit of P-TEFb to relieve inhibition and stimulate substrate recognition.

Abstract C77: Sangivamycin-like molecule 6 (SLM6) exhibits potent and selective anti-multiple myeloma activity through inhibition of P-TEFb.

Abstract We previously reported the anti-tumor and anti-angiogenic activity of a new class of sangivamycin-like molecules (SLMs). In this study we aimed to further the preclinical development of these compounds by identifying tumor types with the highest sensitivity to their cytotoxic effects. Of 35 tumor cell lines from 8 different tissues of origin that we screened, we found that multiple myeloma (MM) cells were approximately 3–10 times more sensitive to SLMs than other tumor types. SLM3 as well as SLM5, SLM6, and SLM7 reduced viability and induced apoptosis of MM cells at sub-micromolar concentrations in vitro. By contrast, SLMs induced negligible amounts of apoptosis in other tumor and normal cell lines. SLM6 exhibited the most potent activity in vivo where it significantly inhibited the growth of xenografted MM tumors. We found that SLM6 inhibited cyclin dependent kinase 9 (CDK9) resulting in hypophosphorylation of RNA polymerase II (Ser2) and transcriptional repression of oncogenes that are known to drive MM progression and resistance to therapy (c-Maf, cyclin D1, c-Myc, L-Myc, and Mcl-1). Furthermore, we found that SLM6 increased the cytotoxic activity of therapies that are currently approved for MM treatment (bortezomib, lenalidomide, dexamethasone, and doxorubicin). These findings suggest that CDK9 inhibition is a particularly effective targeted strategy for MM, and SLM6 is a promising CDK9-targeted agent, both as a single agent and in combination with currently approved therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C77.

7SK snRNA-mediated, gene-specific cooperativity of HMGA1 and P-TEFb

7SK small-nuclear RNA has been shown to negatively regulate P-TEFb transcription elongation on the one hand and control HMGA1 transcription initiation and chromatin remodeling on the other. The non-coding 7SK RNA thereby directly interacts with both factors through different regions. While the loop 2 of the RNA specifically binds to the first HMGA1 A/T hook, thereby competing with DNA binding to the same domain, loops 1, 3 and 4 are involved in P-TEFb interaction. This raises the question of whether HMGA1 and P-TEFb cooperate during gene transcription. Using transcriptome profiling, we have identifed genes that are oppositely regulated by 7SK RNA over-expression versus shRNA mediated knock-down. Inhibition of P-TEFb by competitive expression of a dominant-negative Cdk9 protein leads to highly similar changes in global gene expression as the over-expression of 7SK RNA, confirming the importance of P-TEFb inhibition by 7SK RNA. Furthermore, we have similarly assembled genes affected concomitantly by HMGA1 over-expression. HMGA1 and P-TEFb, in the case of select target genes, show strong cooperation in transcriptional activation. Finally, we provide evidence for 7SK RNA complexes containing simultaneously HMGA1 and P-TEFb. 7SK RNA thus establishes gene-dependent plasticity between HMGA1 chromatin remodeling and transcription initiation and P-TEFb transcription elongation.

The angiogenic properties of Kaposi's sarcoma-associated herpesvirus encoded G-protein coupled receptor are reduced by flavopiridol, an inhibitor of cyclin-dependent kinase 9

Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8) has been identified as the etiologic agent of Kaposi’s sarcoma (KS), a multifocal highly vascularized neoplasm that is the most common malignancy associated with AIDS. Although highly active antiretroviral therapy has decreased the incidence of KS, it remains an incurable tumor for which there is no established treatment. Due to the vascular nature of KS, an anti-angiogenic therapeutic approach is attractive. The KSHV-encoded G-protein-coupled receptor (vGPCR) is required and sufficient to initiate angiogenesis and tumorigenesis. Recent evidence suggests that inhibition of P-TEFb, a transcriptional elongation factor composed of cyclin dependent kinase 9 (CDK9) and its regulatory partner cyclin T, is anti-angiogenic. We hypothesized that flavopiridol, a novel inhibitor of CDK9, would inhibit vGPCR-induced angiogenesis by downregulating expression of angiogenic growth factors and/or Bcl-2. To test this hypothesis, in vitro and in vivo angiogenesis assays were carried out using primary human umbilical vein endothelial cells (HUVECs) transduced with either a control or a vGPCR-expressing retroviral vector and then treated with flavopiridol. Our results show that CDK9 activity is increased in vGPCR-expressing HUVECs and that pretreatment with 50 nM flavopiridol inhibited vGPCR-induced migration and capillary tubule formation. These results correlated with a significant decrease in expression of genes encoding the angiogenic factors VEGF-A and VEGF-C and the pro-survival factor Bcl-2. Initial studies to determine the molecular mechanisms by which CDK9 affects angiogenesis demonstrated that HUVECs treated with bFGF and VEGF had increased CDK9 activity as well as increased expression of the major isoform of Cdk942 and cyclin T. Although Cdk9 was described in the literature as a general transcription factor, we have observed that inhibition of CDK9 by flavopiridol decreased the expression of Bcl-2 but not p21 in HUVEC. Together these results suggest that CDK9 plays a role in mediating transcriptional regulation of vGPCR responsive genes and implicate CDK9 as a potential target to reduce vGPCR-enhanced endothelial cell survival, angiogenesis, and tumorigenesis. Experiments are currently under way to determine whether CDK9 is directly activated upon vGPCR expression and whether inhibition of CDK9 activity suppresses KSHV-enhanced angiogenesis and tumorigenesis in vivo.

Calpain Regulation of Megakaryopoiesis through a Positive Regulatory Loop Involving P-TEFb, GATA-1, and RUNX1.

Abstract 566GATA-1 and RUNX1 cooperate in programming megakaryocytic development through the critical intermediation of the active P-TEFb kinase complex (Cdk9/cyclin T1). RUNX1 on its own traps P-TEFb in inactive chromatin loops and causes RNA pol II (RNAP II) stalling. GATA-1 by contrast remodels chromatin loops and promotes RNAP II elongation. Thus, P-TEFb most likely integrates and resolves conflicting signals from RUNX1 and GATA-1 to coordinate orderly activation of megakaryocytic target genes during development. P-TEFb activity is tightly regulated by a large network of interacting factors including Cdk9, cyclin T1, HEXIM1 and 7SK snRNA, RNA processing factors, and transcriptional regulators. During megakaryocytic differentiation, global activation of P-TEFb involves dissociation of HEXIM1 and recruitment of GATA-1, in a manner dependent on Cdk9 activity. The current studies address factors that regulate this dramatic reconfiguration of the P-TEFb complex during initiation of megakaryocytic differentiation. Candidate factors were identified based on two criteria: participation in the P-TEFb complex and specific upregulation in megakaryocytic differentiation. Notably, analysis of a P-TEFb interactome database identified the protease calpain 2 and its cofactor calpain S1 as participants in this complex (Jeronimo CD. Et. al. Mol Cell 2007). We confirmed a physical interaction by coimmunoprecipitation of endogenous calpain 2 and cyclin T1. Analysis of gene expression databases revealed three striking features of calpain 2: 1) strong upregulation early in megakaryocytic differentiation, 2) defective upregulation in GATA-1-deficient megakaryocytes, and 3) defective upregulation in megakaryocytes expressing GATA-1s, a mutant form associated with Down syndrome-associated megakaryocytic disorders (DS-TMD and DS-AMKL). The role of calpain in megakaryocytic differentiation of primary human CD34+ progenitors was assessed by shRNA knockdown (kd) of calpain S1, a required cofactor for calpains 1 and 2, as well as by treatment of cells with the calpain inhibitors calpeptin and Calpain Inhibitor III. All three approaches blocked cellular enlargement, CD41 upregulation, and polyploidization, indicating a critical role for calpain activity in early steps of megakaryocytic differentiation. We next addressed the hypothesis that calpain contributed to megakaryocytic differentiation through positive regulation of P-TEFb activity. In support of this hypothesis, calpain inhibition prevented the P-TEFb-driven processes of RNAP II hyperphosphorylation and HEXIM1 upregulation, both normally seen in megakaryocytic differentiation. In addition, calpain inhibition blocked the transcriptional cooperation of RUNX1 and GATA-1, which we have previously shown to be dependent on P-TEFb activity. How calpain activity regulates P-TEFb remains unclear, but in vitro and in vivo assays identified cyclin T1 and RNAP II as highly sensitive targets of calpain 2/S1 protease activity. Because P-TEFb remodeling in megakaryopoiesis requires Cdk9 kinase activity, we examined the possibility that calpain itself might be regulated by Cdk9, a notion supported by multiple experiments. In particular, Cdk9 inhibition by shRNA kd or flavopiridol treatment prevented the calpain-dependent cleavage of cyclin T1 and RNAP II normally seen in megakaryocytic differentiation. Furthermore, using purified, recombinant factors, in vitro calpain 2 cleavage of the RNAP II carboxy terminal domain (CTD) showed an absolute requirement for active P-TEFb complex. We thus postulate the existence of a novel regulatory circuit in which P-TEFb and calpain control the activity of one another during megakaryocytic differentiation. The participation of RUNX1 and GATA-1 in this circuit is suggested by the requirements for P-TEFb and calpain activity for their transcriptional cooperation. In addition, a murine strain with megakaryocytic GATA-1 deficiency, the GATA-1Lo strain, showed drops in platelet counts when treated with the calpain inhibitor E64, in contrast to to wild type counterparts, which responded with increased platelet counts. This novel regulatory circuit most likely has clinical relevance for at least two reasons: 1) P-TEFb inhibition in GATA-1Lo mice has been shown to elicit a disorder resembling the DS-TMD, and 2) megakaryocytes expressing GATA-1s display defective upregulation of calpains 2 and S1. Disclosures:No relevant conflicts of interest to declare.

Identification of Flavopiridol Analogues that Selectively Inhibit Positive Transcription Elongation Factor (P‐TEFb) and Block HIV‐1 Replication

The positive transcription elongation factor (P-TEFb; CDK9/cyclin T1) regulates RNA polymerase II-dependent transcription of cellular and integrated viral genes. It is an essential cofactor for HIV-1 Tat transactivation, and selective inhibition of P-TEFb blocks HIV-1 replication without affecting cellular transcription; this indicates that P-TEFb could be a potential target for developing anti-HIV-1 therapeutics. Flavopiridol, a small molecule CDK inhibitor, blocks HIV-1 Tat transactivation and viral replication by inhibiting P-TEFb kinase activity, but it is highly cytotoxic. In the search for selective and less cytotoxic P-TEFb inhibitors, we prepared a series of flavopiridol analogues and evaluated their kinase inhibitory activity against P-TEFb and CDK2/cyclin A, and tested their cellular antiviral potency and cytotoxicity. We identified several analogues that selectively inhibit P-TEFb kinase activity in vitro and show antiviral potency comparable to that of flavopiridol, but with significantly reduced cytotoxicity. These compounds are valuable molecular probes for understanding P-TEFb-regulated cellular and HIV-1 gene transcription and provide potential anti-HIV-1 therapeutics.

A Murine Model for the Transient Myeloproliferative Disorder of Down Syndrome: Phenotypic Influence of p53 on Reversible Megakaryoblastic Disorder

Infants with Down syndrome (DS) display a high incidence of a reversible megakaryoblastic proliferation known as transient myeloproliferative disorder (DS-TMD). The clinical features of DS-TMD include marrow and liver infiltration by abnormal megakaryoctic precursors. These cells show a high propensity for spontaneous death, leading to liver damage and occasionally tumor lysis syndromes. Rapid disease onset is followed by gradual spontaneous remission over weeks to months. 10–20% of patients experience disease recurrence, which manifests as irreversible acute megakaryoblastic leukemia (DS-AMKL). DS-TMD pathogenesis requires trisomy 21 combined with acquired mutations of GATA-1, a transcription factor that programs megakaryocytic and erythroid maturation. The mutant sGATA-1 consists of an 84 amino acid amino-terminal truncation with diminished transcriptional activation. Evolution of DS-TMD to DS-AMKL may involve acquisition of p53 mutations, according to a recent clinical study in which such mutations occurred in 0/7 DS-TMD and in 2/3 DS-AMKL cases. Our lab has recently developed a murine model for DS-TMD based on GATA-1 cross-talk with the P-TEFb kinase complex promoting megakaryocytic maturation (see Elagib et al., Blood, prepublication 2008). In this model, Flavopiridol inhibition of P-TEFb in GATA-1Lo mice, which have megakaryocytic deficiency of GATA-1, induces a rapid onset megakaryoblastic proliferative disorder with many features of DS-TMD:high rate of spontaneous cell death within megakaryoblasts,collateral damage to normal cells in involved tissues (marrow and spleen),defective megakaryoblastic polyploidization,aberrant coexpression of erythroid antigens on megakaryoblasts,reversibility of disease upon withdrawal of P-TEFb inhibitor,requirement for defective GATA-1 in megakaryocytes.To determine the influence of p53 signaling on disease phenotype, the GATA-1Lo mutation was bred onto a TP53−/− background, followed by in vivo P-TEFb inhibition with low-dose Flavopiridol (5 mg/kg/day for 9 days). The resultant megakaryoblastic disorder in the GATA-1Lo::TP53−/− compound mutants showed several features distinct from findings in GATA-1Lo::TP53+/+ mice. In the peripheral blood, the GATA-1Lo::TP53−/− mice showed no significant decline in platelet counts: 2/5 mice had decreases, each <20%. By contrast, the majority (11/13) of GATA-1Lo::TP53+/+ mice showed marked declines (>50%) in platelet counts with P-TEFb inhibition. On necropsy, the GATA-1Lo::TP53−/− mice showed splenomegaly of ~2-fold, while GATA-1Lo::TP53+/+ mice showed splenic shrinkage. Light microscopy revealed extensive splenic infiltration by sheets of megakaryoblasts with minimal evidence of cell death in GATA-1Lo::TP53−/− mice. Marrows from these mice also showed infiltration by megakaryoblasts, but with relative preservation of tissue architecture and bystander cellular elements. Flow cytometry on these marrows confirmed the presence of an abnornal population of megakaryocytic cells with erythroid antigen coexpression and highlighted the lack of intramedullary cell death, distinct from the extensive cell death seen in the GATA-1Lo::TP53+/+ marrows. Another histologic feature unique to the GATA-1Lo::TP53−/− mice consisted of hepatic infiltration by megakaryoblasts, without evidence of hepatocellular damage. Withdrawal of Flavopiridol for 14 days lead to clearance of megakaryoblasts from all involved organs, as seen with GATA-1Lo::TP53+/+ mice. Thus, p53 clearly modulates the phenotype of the megakaryoblastic disease seen in GATA-1Lo mice undergoing P-TEFb inhibition. In the absence of p53 signaling, this disease shows more extensive proliferation, as indicated by the splenomegaly and liver infiltration, combined with markedly decreased cell death. This decrease in cell death is accompanied by a decrease in collateral damage of bystander cells/tissues and by an ability to maintain platelet counts at pre-treatment levels. These findings provide in vivo validation that P-TEFb inhibition can activate p53 (see Gomes et al., Genes Dev. , 20:601, 2006) and suggest that the cell death, tissue damage, and spontaneous regression seen in human DS-TMD could be p53-driven. Loss of p53 function may promote transformation to irreversible leukemia, but P-TEFb under such circumstances retains the potential to induce disease regression.

Bromodomain Protein Brd4 Regulates Human Immunodeficiency Virus Transcription through Phosphorylation of CDK9 at Threonine 29

Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, the bromodomain protein Brd4 has been shown to interact with the low-molecular-weight, active P-TEFb complex and recruit P-TEFb to the HIV type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions +14 and +36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.

P-TEFb Is Critical for the Maturation of RNA Polymerase II into Productive Elongation In Vivo

Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. Here, we demonstrate that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.

HEXIM1 is a promiscuous double-stranded RNA-binding protein and interacts with RNAs in addition to 7SK in cultured cells

P-TEFb regulates eukaryotic gene expression at the level of transcription elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein HEXIM1 or HEXIM2. In an effort to determine the minimal region of 7SK needed to interact with HEXIM1 in vitro, we found that an oligo comprised of nucleotides 10–48 sufficed. A bid to further narrow down the minimal region of 7SK led to a surprising finding that HEXIM1 binds to double-stranded RNA in a sequence-independent manner. Both dsRNA and 7SK (10–48), but not dsDNA, competed efficiently with full-length 7SK for HEXIM1 binding in vitro. Upon binding dsRNA, a large conformational change was observed in HEXIM1 that allowed the recruitment and inhibition of P-TEFb. Both subcellular fractionation and immunofluorescence demonstrated that, while most HEXIM1 is found in the nucleus, a significant fraction is found in the cytoplasm. Immunoprecipitation experiments demonstrated that both nuclear and cytoplasmic HEXIM1 is associated with RNA. Interestingly, the one microRNA examined (mir-16) was found in HEXIM1 immunoprecipitates, while the small nuclear RNAs, U6 and U2, were not. Our study illuminates novel properties of HEXIM1 both in vitro and in vivo, and suggests that HEXIM1 may be involved in other nuclear and cytoplasmic processes besides controlling P-TEFb.

Regulation of Two Key Nuclear Enzymatic Activities by the 7SK Small Nuclear RNA

7SK is a highly conserved small nuclear RNA (snRNA) in vertebrates. Since its discovery in 1968, little had been known about its function until recently, when 7SK was found to associate with the general transcription elongation factor P-TEFb. Together with the HEXIM1 protein, 7SK sequesters P-TEFb into a kinase-inactive complex, where it mediates HEXIM1's inhibition of P-TEFb. This helps maintain P-TEFb in a functional equilibrium to control transcription, cell growth, and differentiation. Although highly abundant, only a small fraction of 7SK is P-TEFb-bound. Using affinity purification, we have identified APOBEC3C as another 7SK-associated protein. As a member of the APOBEC family that functions in diverse processes through deaminating cytosine in DNA, it is unclear how APOBEC3C's activity is controlled to prevent its mutations of genomic DNA. We show that most of APOBEC3C interact with about half of nuclear 7SK, which suppresses APOBEC3C's deaminase activity and sequesters APOBEC3C in the nucleolus where it could be at a safe distance from most genomic sequences. Because the DNA substrate-binding site in APOBEC3C differs from the region for 7SK binding, 7SK does not act as a substrate competitor in inhibiting APOBEC3C. The demonstration of 7SK's suppression of yet another enzyme besides P-TEFb suggests a general role for this RNA in regulating key nuclear functions.