Positive Transcription Elongation Factor B Research Articles

First in Human (FIH) Study of GFH009, a Highly Selective Cyclin-Dependent Kinase 9 (CDK9) Inhibitor, in Patients with Relapsed/Refractory (r/r) Hematologic Malignancies

Background: The key roles of cyclin-dependent kinases in cell proliferation and various aspects of transcription have led to efforts to develop CDK inhibitors as cancer therapeutics. Positive transcription elongation factor b (PTEFb) consisting of Cyclin T and CDK9 is a key regulator of transcription in cancer cells. It is vital for transcription of short-lived gene products, such as MYC and MCL-1, that maintain cancer cell survival. GFH009 is a potent and highly selective CDK9 inhibitor. Here we report preliminary safety and efficacy data from the FIH study of GFH009 in patients with r/r hematologic malignancies (NCT04588922). Methods: This is a phase I, multicenter, dose-escalation trial of GFH009 enrolling patients with relapsed/refractory acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL)/ small lymphocytic lymphoma (SLL) or lymphoma. The Bayesian optimal interval (BOIN) design is employed in the dose escalation cohorts of 3-6 patients. In the first part of the trial, GFH009 was administered intravenously over 30-60 minutes twice per week. The primary endpoint was to evaluate the safety of GFH009 measured by adverse events using common terminology criteria for adverse event (CTCAE) v5.0 and dose limiting toxicities (DLTs). Disease responses were evaluated using Lugano (2014) for lymphoma, IWG criteria (2003) for AML, and iwCLL (2018) for CLL/SLL. Blood samples were collected and analyzed for pharmacokinetics (PK) and pharmacodynamics (PD). Results: As of 15 July 2022, a total of 46 patients were screened, and 30 received at least one dose of GFH009 as monotherapy, including 16 lymphoma patients in 2.5, 4.5, 9 and 15 mg dose cohorts and 14 AML patients in 9, 15, 22.5 and 30 mg dose cohorts. The median age was 52 (range 18-73), and 53% were male. Nine patients (30%) discontinued treatment due to disease progression, eight (27%) due to subjects’ decision and six (20%) due to lack of efficacy. No DLTs were observed to date. The most common drug related AEs were white blood cell decreased [20.0% (6/30), 6.7% (2/30) of which was ≥ G3], neutrophil count decreased [16.7% (5/30), 6.7% (2/30) ≥ G3] and anemia [13.3% (4/30), 3.3% (1/30) ≥ G3]. G3/G4 treatment emergent AEs recorded in >10% of patients included anemia, white blood cell decreased, platelet count decreased, neutrophil count decreased and pneumonia. The total of 12 serious adverse events (SAEs) occurred in 11 subjects, three of which were drug related. All the drug related SAEs were G3 pneumonia. One death was recorded due to disease progression of AML. Four lymphoma patients (3 in 4.5 mg and 1 in 9 mg) achieved stable disease (SD) including one peripheral T cell lymphoma (PTCL), one diffuse large B-cell lymphoma, one Hodgkin lymphoma and one mucosa-associated lymphoid tissue (MALT) lymphoma. The patient with PTCL at 9 mg dose had 62% decrease in sum of the product of the perpendicular diameters for multiple lesions (SPD) based on computed tomography (CT). The duration of treatment of the PTCL patient and MALT lymphoma patient was 12 weeks and 41 weeks respectively and they continue treatment at the cut-off date. No objective response was observed in AML patients, but two patients, one at 9 mg dose level and one at 15 mg dose level, had decrease of bone marrow blasts ≥ 50%. PK exposure increased in a dose-proportional manner as increasing dose from 2.5 mg to 22.5 mg. The mean half-life ranged from 14.9 h to 17.7 h. Pharmacodynamic assessment of MYC and MCL1 mRNA in whole blood showed post-dose reduction of short half-life mRNA transcripts at higher dose levels. Conclusion: GFH009 as monotherapy is well tolerated at the tested doses and has shown clinical activity in patients with r/r lymphoma. The dose escalation in patients with AML and lymphoma is ongoing. Different dosing regimens are currently being explored for potentially better inhibition on the target. GFH009 add-back in patients refractory to or relapsed while on venetoclax and hypomethylating agents is planned in AML patients. Key words: cyclin-dependent kinases, CDK9 inhibitor, MYC, MCL-1, AML, lymphoma, CLL, SLL, PTCL, MALT lymphoma

Identification of Novel Nucleocapsid Chimeric Proteins Inhibiting HIV-1 Replication

The positive transcription elongation factor b (P-TEFb) is an essential factor that induces transcription elongation and is also negatively regulated by the cellular factor HEXIM1. Previously, the chimeric protein HEXIM1-Tat (HT) was demonstrated to inhibit human immunodeficiency virus-1 (HIV)-1 transcription. In this study, we attempted to develop an improved antiviral protein that specifically binds viral RNA (vRNA) by fusing HT to HIV-1 nucleocapsid (NC). Thus, we synthesized NC-HEXIM1-Tat (NHT) and HEXIM1-Tat-NC (HTN). NHT and HTN inhibited virus proliferation more effectively than HT, and they did not attenuate the function of HT. Notably, NHT and HTN inhibited the infectivity of the progeny virus, whereas HT had no such effect. NHT and HTN selectively and effectively interacted with vRNA and inhibited the proper packaging of the HIV-1 genome. Taken together, our results illustrated that the novel NC-fused chimeric proteins NHT and HTN display novel mechanisms of anti-HIV effects by inhibiting both HIV-1 transcription and packaging.

Dynamic regulation of P-TEFb by 7SK snRNP is integral to the DNA damage response to regulate chemotherapy sensitivity

SummaryTesticular germ cell tumors and closely related embryonal stem cells are exquisitely sensitive to cisplatin, a feature thought to be linked to their pluripotent state and p53 status. It remains unclear whether and how cellular state is coordinated with p53 to confer cisplatin sensitivity. Here, we report that positive transcription elongation factor b (P-TEFb) determines cell fate upon DNA damage. We find that cisplatin rapidly activates P-TEFb by releasing it from inhibitory 7SK small nuclear ribonucleoprotein complex. P-TEFb directly phosphorylates pluripotency factor estrogen-related receptor beta (ESRRB), and induces its proteasomal degradation to enhance pro-survival glycolysis. On the other hand, P-TEFb is required for the transcription of a substantial portion of p53 target genes, triggering cell death during prolonged cisplatin treatment. These results reveal previously underappreciated roles of P-TEFb to coordinate the DNA damage response. We discuss the implications for using P-TEFb inhibitors to treat cancer and ameliorate cisplatin-induced ototoxicity.

Abstract 5471: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, is active in preclinical models of prostate cancer

Abstract CDK9 is a serine/threonine kinase belonging to the subclass of the transcription associated CDKs. CDK9 complexes with cyclin T and cyclin K, the positive transcription elongation factor b (P-TEFb), and phosphorylates Serine 2 of RNA polymerase II (pSer2 RNAPII) to activate transcription. Consequently, targeting CDK9 could effectively interfere with epigenetic and transcriptional reprogramming and prevent disease progression and treatment resistance in human cancers. Androgen receptor (AR)-dependence in prostate cancer is linked to CDK9 function. CDK9 stabilizes AR-associated proteins, and pharmacological inhibition of CDK9 can inhibit AR, AR variants, and their downstream transcription programs. We evaluated the novel CDK9 inhibitor, PRT2527, in prostate cancer models to evaluate its effects on cell proliferation, stem-like tumor cells, and tumor growth. PRT2527 is a potent inhibitor of CDK9/CyclinT1 complex, and when evaluated at concentration 200 times the biochemical IC50, PRT2527 was highly selective for CDK9 inhibition. We verified in biochemical assays the ability of PRT2527 to suppress pSer2 RNAPII and reduce expression of c-Myc, a common target of CDK9, in a concentration-dependent manner in multiple human prostate cancer cell lines. PRT2527 also inhibited c-Myc-dependent transcription in vitro in luciferase reporter assays. Furthermore, RNA sequencing showed altered expression of several genes with significant enrichment of c-Myc and E2F targets and RNAPII dependent transcription among downregulated genes in PRT2527-treated VCaP cells. In vitro, PRT2527 inhibited the proliferation of androgen-dependent and androgen-independent prostate cancer cell lines (IC50, ≤50 nM). PRT2527 was highly effective (IC50, ≤10 nM) in tumor-spheroid assays in blocking the growth of stem-like tumor cells and significantly suppressed the in vitro growth of tumor organoids from both human cell lines and patient-derived xenografts (PDXs). In mice, PRT2527 IV administration reduced pSer2 RNAPII in tumor xenografts and c-Myc-dependent transcriptional activity in a DU145 luminescence reporter model. PRT2527 administration in mice significantly reduced growth of PDX LuCaP 35 (castration-sensitive, adenocarcinoma) and LuCaP 145.2 (castration-resistant, neuroendocrine) along with the fraction of tumor-initiating stem-like cells in ex vivo assays. PRT2527 reduced pSer2 RNAPII in both PDXs, whereas c-Myc decreased in LuCaP 35 and Sox2 in LuCaP 145.2, relative to basal expression levels. Collectively, our data demonstrate that PRT2527 has potent pharmacodynamic and antitumor activity in multiple models of castration-sensitive and castration-resistant prostate cancer. PRT2527 is advancing into phase 1 studies in solid tumors. Citation Format: Elisa Federici, Gianluca Civenni, Aleksandra Kokanovic, Giada Sandrini, Luca Guarrera, Simone Mosole, Alessia Cacciatore, Valeria Uboldi, Manuel Lessi, Giovanni Papa, Domenico Albino, Elisa Storelli, Jessica Merulla, Andrea Rinaldi, Marco Bolis, Yang Zhang, Kris Vaddi, Peggy Scherle, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, is active in preclinical models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5471.

Abstract 3955: Targeting transcription elongation via CDK9 in mantle cell lymphoma patients with dual resistance to BTK inhibition and CAR T therapy

Abstract Introduction Clinical relapse following CD19 CAR T therapy after failure to BTK inhibitors (BTKi) is a novel and fast growing medical challenge in treating patients with mantle cell lymphoma (MCL). Thus, developing a novel therapy to overcome this BTKi-CAR T dual resistance (Dual-R) is an urgent need. Our unpublished data revealed MYC targets and cyclin-dependent kinase 9 (CDK9) are highly upregulated in the Dual-R compared to BTKi-R samples. CDK9 is a critical component of the positive transcription elongation factor b (P-TEFb) complex. Its inhibition induces acute decline of short-lived mRNA and proteins, especially MYC and MCL-1, in acute myeloid leukemia and diffuse large B-cell lymphoma. Therefore, we hypothesize that targeting CDK9 may turn off MYC-driven tumor survival and drug resistance. BAY-1251152 is a novel selective CDK9 inhibitor with nanomolar potency. However, whether it has the potential to overcome BTKi-CAR T dual resistance has not been assessed. Methods To validate the correlation of MYC and CDK9 with drug resistance and clinical outcome, we performed whole transcriptomic profiling using primary patient samples. To assess the in vitro efficacy of BAY-1251152, we performed cell viability assay and cell apoptosis assay using MCL cell lines and primary patient samples and followed up with functional studies. furthermore, we assessed its in vivo efficacy using patient-derived xenograft (PDX) models derived from MCL patients including one with Dual-R. Results The expression of MYC oncogene associates with ibrutinib resistance and Dual-R, and poor clinical outcome. CDK9 expression does not correlates with ibrutinib resistance but it does associate with Dual-R and poor clinical outcome in CAR T-relapsed patients. CDK9 inhibition by BAY-1251152 is highly potent in anti-MCL activity in MCL cell lines with low nanomolar range of IC50 (59.6-172.3 nM) by inducing robust cell apoptosis. BAY-1251152 induces dose and time-dependent CDK9 inhibition. A rapid decline of phosphorylation of RNA polymerase II, MYC, MCL-1, and Cyclin D1 can be observed as early as 2 hours. Furthermore, BAY-1251152 (10mg/kg, QW) significantly inhibited tumor growth (p = 0.000003) in a PDX model derived from a Dual-R patient without causing apparent toxicity in NSG mice. In addition, BAY-1251152 also significantly suppressed tumor growth in the PDX models derived from a BTKi-R patient (p=0.00015) and a BTKi-venetoclax dual-resistant patient (p=0.009). Conclusion Our findings showed that targeting CDK9 by its specific inhibitor BAY-1251152 led to potent in vitro anti-MCL activity. BAY-1251152 induces fast CDK9 inhibition and rapid decline of MYC, MCL-1 and Cyclin D1 to induce robust cell death. BAY-1251152 is also potent in inhibiting tumor growth in PDX models. These data support that CDK9 is a promising target to overcome BTKi-CAR T dual resistance in MCL, which is in urgent need. Citation Format: Vivian Changying Jiang, Lingzhi Li, Alexa Jordan, Yu Xue, Fangfang Yan, Joseph McIntosh, Yang Liu, Yuxuan Che, Yijing Li, Qingsong Cai, Angela Leeming, Lukas Simon, Zhongming Zhao, Jia Zhou, Michael Wang. Targeting transcription elongation via CDK9 in mantle cell lymphoma patients with dual resistance to BTK inhibition and CAR T therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3955.

DDB1 prepares brown adipocytes for cold-induced thermogenesis

Abstract Brown adipose tissue (BAT) plays a key role in thermogenesis during acute cold exposure. However, it remains unclear how BAT is prepared to rapidly turn on thermogenic genes. Here, we show that damage-specific DNA binding protein 1 (DDB1) mediates the rapid transcription of thermogenic genes upon acute cold exposure. Adipose- or BAT-specific Ddb1 knockout mice show severely whitened BAT and significantly decreased expression of thermogenic genes. These mice develop hypothermia when subjected to acute cold exposure at 4 °C and partial lipodystrophy on a high-fat diet due to deficiency in fatty acid oxidation. Mechanistically, DDB1 binds the promoters of Ucp1 and Ppargc1a and recruits positive transcriptional elongation factor b (P-TEFb) to release promoter-proximally paused RNA polymerase II (Pol II), thereby enabling rapid and synchronized transcription of thermogenic genes upon acute cold exposure. Our findings have thus provided a regulatory mechanism of how BAT is prepared to respond to acute cold challenge.

Measuring the precise position of transitions in transcription with PRO‐IP‐seq

Transcription is a fundamental process for all known organisms. Transcription by RNA Polymerase II (Pol II), the enzyme responsible for mRNA synthesis, is highly regulated by protein factors at every step. In multicellular eukaryotes, Pol II pauses 20‐60 bases downstream of the transcription start site in nearly all genes. This promoter‐proximal pausing is established as an important step in transcription regulation and is dependent on several pausing and elongation factors. The protein complexes DSIF (DRB Sensitivity Inducing Factor) and NELF (Negative Elongation Factor) bind to Pol II early in transcription to induce pausing. Pause release occurs upon phosphorylation of Pol II, DSIF and NELF by the Cdk9, Cyclin T1 heterodimer P‐TEFb (Positive Transcription Elongation Factor b). Although Pol II pausing is a key step in transcription regulation and has been extensively studied, the molecular mechanisms of pause and pause release are incompletely understood. Furthermore, recent studies have implicated controlled termination early in the gene as a mechanism to control transcription. A challenge to studying these key regulatory steps is that they occur in a narrow window: from initiation to Pol II pausing and release occurs in 20‐60 base range. Current approaches to study transcription factor interactions are unable to provide high enough resolution or sensitivity to address precisely where key transitions in transcription occur.To address this problem, I am developing an assay to measure the exact position of Pol II, when it is bound by a factor of interest. The technique couples our lab’s Precision Run‐on sequencing technique (PRO‐seq), which measures the position of active Pol II with single base‐pair resolution, and an immunoprecipitation enrichment step. Initial PRO‐IP‐seq (Precision Run‐on Immunoprecipitation sequencing) data shows that we can enrich for populations of Pol II bound by specific transcription factors. Utilizing this tool, we will be able to measure genomically where both stable and more transient transcription factors interact with Pol II at near single‐base resolution.

Investigating the Effect of PARP Mediated Compartments on Transcription Dynamics

Transcription of DNA into RNA is a fundamental, universal, and highly regulated process in all living organisms. RNA Polymerase II (Pol II), is the enzyme that catalyzes this reaction and it is highly regulated by the pausing factors DRB Sensitivity Inducing Factor (DSIF) and Negative Elongation Factor (NELF), and elongation factors, including Positive Transcription Elongation Factor b (P‐TEFb) and Polymerase Associated Factor 1 (PAF1). However, most previous studies of these factors have only provided snapshots of factor binding or snapshots of mRNA production following their depletion. While valuable, these studies provide limited information about the time frames in which Pol II and key transcription factors interact with each other and with the DNA. Using live‐cell imaging of amplified and interphase‐extended Drosophila polytene chromosomes with endogenously tagged transcription factors and Pol II, we can analyze the real time behavior of these molecules at single genetic loci and at high resolution. In particular, heat shock genes, like heat shock protein 70 (Hsp70), have been shown to be a robust system for studying transcription because of their inducible nature; they are able to transition quickly from a paused to a highly active state. Previous work from our lab utilizing this system suggested the formation of a transcription compartment during prolonged bouts of transcription. We propose that transcription compartments form around Hsp70 loci during an extended heat shock due to the activity of Poly ADP Ribose Polymerase (PARP) polymerizing large PAR chains that trap Pol II and other transcription factors, restricting their movement and preventing free diffusion with other molecules in the nucleus. We are tagging proteins of interest with photo‐activatable fluorescent proteins to minimize background by selectively activating a population of molecules only in the small 3‐D volume of specific loci. Utilizing this system we have begun measuring the dynamics of Pol II at specific loci and are working toward measuring the dynamics of key transcription regulating factors and further characterizing PARP‐mediated transcription compartments.

Structural basis of RNA conformational switching in the transcriptional regulator 7SK RNP

7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is hijacked by HIV-1 for viral transcription and replication. Methylphosphate capping enzyme (MePCE) and La-related protein 7 (Larp7) constitutively associate with 7SK to form a core RNP, while P-TEFb and other proteins dynamically assemble to form different complexes. Here, we present the cryo-EM structures of 7SK core RNP formed with two 7SK conformations, circular and linear, and uncover a common RNA-dependent MePCE-Larp7 complex. Together with NMR, biochemical, and cellular data, these structures reveal the mechanism of MePCE catalytic inactivation in the core RNP, unexpected interactions between Larp7 and RNA that facilitate a role as an RNP chaperone, and that MePCE-7SK-Larp7 core RNP serves as a scaffold for switching between different 7SK conformations essential for RNP assembly and regulation of P-TEFb sequestration and release.

The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription.

Inhibition of host cell apoptosis is crucial for survival and replication of several intracellular bacterial pathogens. To interfere with apoptotic pathways, some pathogens use specialized secretion systems to inject bacterial effector proteins into the host cell cytosol. One of these pathogens is the obligate intracellular bacterium Coxiella burnetii, the etiological agent of the zoonotic disease Q fever. In this study, we analyzed the molecular activity of the anti-apoptotic T4SS effector protein AnkG (CBU0781) to understand how C. burnetii manipulates host cell viability. We demonstrate by co- and RNA-immunoprecipitation that AnkG binds to the host cell DExD box RNA helicase 21 (DDX21) as well as to the host cell 7SK small nuclear ribonucleoprotein (7SK snRNP) complex, an important regulator of the positive transcription elongation factor b (P-TEFb). The co-immunoprecipitation of AnkG with DDX21 is probably mediated by salt bridges and is independent of AnkG-7SK snRNP binding, and vice versa. It is known that DDX21 facilitates the release of P-TEFb from the 7SK snRNP complex. Consistent with the documented function of released P-TEFb in RNA Pol II pause release, RNA sequencing experiments confirmed AnkG-mediated transcriptional reprogramming and showed that expression of genes involved in apoptosis, trafficking, and transcription are influenced by AnkG. Importantly, DDX21 and P-TEFb are both essential for AnkG-mediated inhibition of host cell apoptosis, emphasizing the significance of the interaction of AnkG with both, the DDX21 protein and the 7SK RNA. In line with a critical function of AnkG in pathogenesis, the AnkG deletion C. burnetii strain was severely affected in its ability to inhibit host cell apoptosis and to generate a replicative C. burnetii-containing vacuole. In conclusion, the interference with the activity of regulatory host cell RNAs mediated by a bacterial effector protein represent a novel mechanism through which C. burnetii modulates host cell transcription, thereby enhancing permissiveness to bacterial infection.

CAMP-Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease.

Formation of biomolecular condensates by phase separation has recently emerged as a new principle for regulating gene expression in response to extracellular signaling. However, the molecular mechanisms underlying the coupling of signal transduction and gene activation through condensate formation, and how dysregulation of these mechanisms contributes to disease progression, remain elusive. Here, the authors report that CREB‐regulated transcription coactivator 2 (CRTC2) translocates to the nucleus and forms phase‐separated condensates upon activation of cAMP signaling. They show that intranuclear CRTC2 interacts with positive transcription elongation factor b (P‐TEFb) and activates P‐TEFb by disrupting the inhibitory 7SK snRNP complex. Aberrantly elevated cAMP signaling plays central roles in the development of autosomal dominant polycystic kidney disease (ADPKD). They find that CRTC2 localizes to the nucleus and forms condensates in cystic epithelial cells of both mouse and human ADPKD kidneys. Genetic depletion of CRTC2 suppresses cyst growth in an orthologous ADPKD mouse model. Using integrative transcriptomic and cistromic analyses, they identify CRTC2‐regulated cystogenesis‐associated genes, whose activation depends on CRTC2 condensate‐facilitated P‐TEFb recruitment and the release of paused RNA polymerase II. Together, their findings elucidate a mechanism by which CRTC2 nuclear condensation conveys cAMP signaling to transcription elongation activation and thereby promotes cystogenesis in ADPKD.

A synthetic resveratrol analog termed Q205 reactivates latent HIV-1 through activation of P-TEFb

The persistence of HIV-1 latent reservoir creates the major obstacle toward an HIV-1 cure. The “shock and kill” strategy aims to reverse HIV-1 proviral latency using latency-reversing agents (LRAs), thus boosting immune recognition and clearance to residual infected cells. Unfortunately, to date, none of these tested LRA candidates has been demonstrated effectiveness and/or safety in reactivation HIV-1 latency. The discovery and development of effective, safe and affordable LRA candidates are urgently needed for creating an HIV-1 functional cure. Here, we designed and synthesized a series of small-molecule phenoxyacetic acid derivatives based on the resveratrol scaffold and found one of them, named 5, 7-dimethoxy-2-(5-(methoxymethyl) furan-2-yl) quinazolin-4(3H)-one (Q205), effectively reactivated latent HIV-1 in latent HIV-1-infected cells without a corresponding increase in induction of potentially damaging cytokines. The molecular mechanism of Q205 is shown to increase the phosphorylation of the CDK9 T-loop at position Thr186, dissociate positive transcription elongation factor b (P-TEFb) from BRD4, and promote the Tat-mediated HIV-1 transcription and RNA polymerase II (RNAPII) C-terminal domain (CTD) on Ser (CTD-Ser2P) to bind to the HIV-1 promoter. This study provides a unique insight into resveratrol modified derivatives as promising leads for preclinical LRAs, which in turn may help toward inhibitor design and chemical optimization for improving HIV-1 shock-and kill-based efforts.

Mechanistic Insights into a CDK9 Inhibitor Via Orthogonal Proteomics Methods.

Cyclin-dependent-kinases (CDKs) are members of the serine/threonine kinase family and are highly regulated by cyclins, a family of regulatory subunits that bind to CDKs. CDK9 represents one of the most studied examples of these transcriptional CDKs. CDK9 forms a heterodimeric complex with its regulatory subunit cyclins T1, T2 and K to form the positive transcription elongation factor b (P-TEFb). This complex regulates transcription via the phosphorylation of RNA polymerase II (RNAPolII) on Ser-2, facilitating promoter clearance and transcription elongation and thus remains an attractive therapeutic target. Herein, we have utilized classical affinity purification chemical proteomics, kinobeads assay, compressed CEllular Thermal Shift Assay (CETSA)-MS and Limited Proteolysis (LiP) to study the selectivity, target engagement and downstream mechanistic insights of a CDK9 tool compound. The above experiments highlight the value of quantitative mass spectrometry approaches to drug discovery, specifically proteome wide target identification and selectivity profiling. The approaches utilized in this study unanimously indicated that the CDK family of kinases are the main target of the compound of interest, with CDK9, showing the highest target affinity with remarkable consistency across approaches. We aim to provide guidance to the scientific community on the available chemical biology/proteomic tools to study advanced lead molecules and to highlight pros and cons of each technology while describing our findings in the context of the CDKs biology.

Reversible phosphorylation of cyclin T1 promotes assembly and stability of P-TEFb.

The positive transcription elongation factor b (P-TEFb) is a critical coactivator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC-mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells.

Downregulation of Dihydrotestosterone and Estradiol Levels by HEXIM1.

We have previously reported that hexamethylene bis-acetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound estrogen receptor α (ERα) and the androgen receptor (AR) by disrupting the interaction between these receptors and positive transcriptional elongation factor b (P-TEFb) and attenuating RNA polymerase II (RNAPII) phosphorylation at serine 2. Functional consequences of the inhibition of transcriptional activity of ERα and AR by HEXIM1 include the inhibition of ERα- and AR-dependent gene expression, respectively, and the resulting attenuation of breast cancer (BCa) and prostate cancer (PCa) cell proliferation and growth. In our present study, we determined that HEXIM1 inhibited AKR1C3 expression in BCa and PCa cells. AKR1C3, also known as 17β-hydroxysteroid dehydrogenase (17β-HSD) type 5, is a key enzyme involved in the synthesis of 17β-estradiol (E2) and 5-dihydrotestosterone (DHT). Downregulation of AKR1C3 by HEXIM1 influenced E2 and DHT production, estrogen- and androgen-dependent gene expression, and cell proliferation. Our studies indicate that HEXIM1 has the unique ability to inhibit both the transcriptional activity of the ER and AR and the synthesis of the endogenous ligands of these receptors.

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.

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.

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.