Structural basis of Cdk7 activation by dual T-loop phosphorylation.

Cyclin-dependent kinase 7 (Cdk7) occupies a central position in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of the general transcription factor TFIIH. Cdk7 forms an active complex upon association with Cyclin H and Mat1, and its catalytic activity is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the fully activated human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates a set of basic residues conserved in other CDKs, pS164 nucleates an arginine network involving all three subunits that is unique to the ternary Cdk7 complex. We identify differential dependencies of kinase activity and substrate recognition on individual phosphorylations within the Cdk7 T loop. The CAK function of Cdk7 is not affected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by phosphorylation at T170. Moreover, dual T-loop phosphorylation at both T170 and S164 stimulates multi-site phosphorylation of transcriptional substrates-the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and the SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7-regulatory phosphorylation is a two-step process in which phosphorylation of S164 precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing Cdk7 processivity, while the canonical pT170 enhances kinase activity towards critical substrates involved in transcription.

A patent review of cyclin-dependent kinase 7 (CDK7) inhibitors (2018-2022).

INTRODUCTION: Cyclin-dependent kinase 7 (CDK7) is a member of the CDK family of serine/threonine protein kinases and participates in the regulation of the cell cycle and mRNA transcription. CDK7 is emerging as a possible drug target in oncology and six exciting drug candidates have already undergone early evaluation in clinical trials. AREAS COVERED: This review examines CDK7 inhibitors as anticancer drugs reported in patents published in the online databases of the World Intellectual Property Organization and European Patent Office in the 2018-2022 period. This review provides an overview of available inhibitors, including their chemical structures, biochemical profile and stage of development. EXPERT OPINION: Small-molecule CDK7 inhibitors represent attractive pharmacological modalities for the treatment of various cancer types. Highly potent and selective inhibitors have been discovered and many of them show promising results in several preclinical cancer models. Developed compounds act on the kinase by various mechanisms, including traditional ATP competition, irreversible binding to tractable cysteine 312 outside the active site of CDK7, and induced protein degradation by proteolysis targeting chimeras. Ongoing preclinical research and clinical trials should reveal which strategy will provide the highest benefits.

Analysis of radiation dose and influencing factors during interventional procedures for 94 patients with pediatric congenital heart disease / 中国辐射卫生

Objective To evaluate the radiation dose of interventional procedure for children with congenital heart disease, and to analyze the differences in radiation dose and influencing factors. Methods A total of 94 children who underwent interventional procedure for congenital heart disease at a grade A tertiary hospital in Jinan, Shandong Province, China from June 2021 to September 2022 were included in this study. The patients were divided into three groups according to the type of procedure: ventricular septal defect occlusion group (VSD, 48 cases), patent ductus arteriosus occlusion group (PDA, 29 cases), and atrial septal defect occlusion group (ASD, 17 cases). The basic information of patients and postoperative dose reports were recorded. A statistical analysis was performed using SPSS software. Results The median cumulative air kerma (CAK) of VSD, PDA, and ASD was 100.5, 43.7, and 12.1 mGy, respectively. The median air kerma area product (KAP) of VSD, PDA, and ASD was 3.309, 1.313, and 0.540 Gy·cm2, respectively. The median KAP·kg−1 of VSD, PDA, and ASD was 0.179, 0.088, and 0.031 Gy·cm2·kg−1, respectively. There were significant differences in fluoroscopy time, number of cine images, CAK, KAP, and KAP·kg−1 among the three types of interventional procedures (P<0.05). Compared with PDA and ASD, VSD showed significantly higher fluoroscopy time, number of cine images, CAK, KAP, and KAP·kg−1 (P<0.05). Multiple linear regression analysis found that age (B=52.445, P<0.05), weight (B=13.077, P<0.05), fluoroscopy time (B=0.425, P<0.05), tube current (B=0.872, P<0.05), and number of cine images (B=0.660, P<0.05) were positively correlated with KAP, while there was no significant association between height and KAP (P>0.05). Conclusion There are differences in radiation dose among the three types of procedures. Reducing fluoroscopy time, tube current, and number of cine images while meeting the procedure requirements is of great significance for reducing the radiation dose received by children.

Isc10, an inhibitor of the Smk1 MAPK, prevents activation loop autophosphorylation and substrate phosphorylation through separate mechanisms.

Many eukaryotic protein kinases are activated by the intramolecular autophosphorylation of activation loop residues. Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in yeast that autophosphorylates its activation loop tyrosine and thereby upregulates catalytic output. This reaction is controlled by an inhibitor, Isc10, that binds the MAPK during meiosis I and an activator, Ssp2, that binds Smk1/Isc10 during meiosis II. Upon completion of the meiotic divisions, Isc10 is degraded, and Smk1 undergoes autophosphorylation to generate the high activity form of the MAPK that controls spore formation. How Isc10 inhibits Smk1 is not clear. Here, we use a bacterial coexpression/reconstitution system to define a domain in the carboxy-terminal half of Isc10 that specifically inhibits Smk1 autophosphorylation. Nevertheless, Smk1 bound by this domain is able to phosphorylate other substrates, and it phosphorylates the amino-terminal half of Isc10 on serine 97. In turn, the phosphorylated motif in Isc10 inhibits the Smk1 active site. These data show that Isc10 inhibits autophosphorylation and the phosphorylation of substrates by separate mechanisms. Furthermore, we demonstrate Isc10 can inhibit the autophosphorylation of the mammalian intestinal cell kinase ICK1 (also known as CILK1), suggesting a conserved mechanism of action. These findings define a novel class of developmentally regulated molecules that prevent the self-activation of MAPKs and MAPK-like enzymes.

Three targets in one complex: A molecular perspective of TFIIH in cancer therapy.

The general transcription factor II H (TFIIH) plays an essential role in transcription and nucleotide excision DNA repair (NER). TFIIH is a complex 10 subunit containing molecular machine that harbors three enzymatic activities while the remaining subunits assume regulatory and/or structural functions. Intriguingly, the three enzymatic activities of the CDK7 kinase, the XPB translocase, and the XPD helicase exert different impacts on the overall activities of TFIIH. While the enzymatic function of the XPD helicase is exclusively required in NER, the CDK7 kinase is deeply involved in transcription, whereas XPB is essential to both processes. Recent structural and biochemical endeavors enabled unprecedented details towards the molecular basis of these different TFIIH functions and how the enzymatic activities are regulated within the entire complex. Due to its involvement in two fundamental processes, TFIIH has become increasingly important as a target in cancer therapy and two of the three enzymes have already been addressed successfully. Here we explore the possibilities of recent high resolution structures in the context of TFIIH druggability and shed light on the functional consequences of the different approaches towards TFIIH inhibition.

Mutant KRAS Promotes NKG2D+ T Cell Infiltration and CD155 Dependent Immune Evasion.

BACKGROUND/AIM: Roles for mutant (mt) KRAS in the innate immune microenvironment in colorectal cancer (CRC) were explored. MATERIALS AND METHODS: Human CRC HCT116-derived, mtKRAS-disrupted (HKe3) cells that express exogenous mtKRAS and allogenic cytokine-activated killer (CAK) cells were co-cultured in 3D floating (3DF) culture. The anti-CD155 antibody was used for function blocking and immuno histochemistry. RESULTS: Infiltration of CAK cells, including NKG2D+ T cells, into the deep layer of HKe3-mtKRAS spheroids, was observed. Surface expression of CD155 was found to be up-regulated by mtKRAS in 3DF culture and CRC tissues. Further, the number of CD3+ tumor-infiltrating cells in the invasion front that show substantial CD155 expression was significantly larger than the number showing weak expression in CRC tissues with mtKRAS. CD155 blockade decreased the growth of spheroids directly and indirectly through the release of CAK cells. CONCLUSION: CD155 blockade may be useful for therapies targeting tumors containing mtKRAS.

Expression and clinical significance of CAK complex in estrogen receptor-positive breast cancer / 国际肿瘤学杂志

Objective To evaluate the expression and clinical significance of cyclin dependent kinase (CDK)-activating kinase (CAK) complex including CDK7, cyclin H and accessory protein menage a trios 1 (MAT1) in estrogen receptor-positive breast cancer.Methods A total of 40 patients with estrogen receptor-positive breast cancer from Department of Galactophore, Baoji Maternal and Child Care Service Centre of Shaanxi Province were investigated in this study.Breast cancer tissues and adjacent normal tissues were obtained from patients undergoing surgery.The mRNA expressions of CDK7, cyclin H and MAT1 in two types of tissues were measured by real-time fluorescent quantitative (qRT)-PCR, and their correlations with clinicopathologic features of patients were analyzed.Results The expressions of CDK7, cyclin H and MAT1 in cancer tissues were 2.54±0.78, 2.21±0.56 and 2.46±0.58, while those in adjacent normal tissues were 1.26±0.30, 1.16±0.42 and 1.17±0.39, and there were significantly differences between different types of tissues (t=9.654, P<0.001;t=9.433, P<0.001;t=11.741, P<0.001).The higher expressions of cyclin H and MAT1 in patients′ cancer tissues had the lower clinical stage, with significant correlations (U=3.17, P=0.01;U=2.53, P=0.01).In addition, the tumors were smaller in patients with higher expression levels of MAT1 (χ2=14.16, P=0.01).Conclusion The expressions of CAK complex CDK7, cyclin H and MAT1 are elevated in estrogen receptor-positive breast cancer patients.Cyclin H and MAT1 are closely associated with clinical grade, and MAT1 is also significantly associated with tumor size.

Strontium concentrations and isotope ratios in a forest-river system in the South Qinling Mts., China.

The concentrations of dissolved strontium (Sr) and isotope ratios ((87)Sr/(86)Sr) in rainwater, river water, and water from forest soil are measured to investigate the contributions of these sources to a river during base flow conditions in the relatively pristine South Qinling Mountains, China. Dissolved Sr concentrations and (87)Sr/(86)Sr ratios vary significantly between different water types (p < 0.01) suggesting that it is suitable for differentiating sources. Dissolved Sr is also positively correlated with most ions and a range of physicochemical parameters (p < 0.01 and p < 0.05 respectively) in water samples including Ca(2+), Mg(2+), EC, and TDS (p < 0.001) indicating their similarities in the drivers of biogeochemical processes and common origins. The correlations between Sr isotopes and Ca/Na, Ca/K, and 1000/Sr ratios suggest that three end-members of atmospheric inputs, carbonate and silicate weathering control the Sr water chemistry in the river water. Using the three-source mixing model, atmospheric inputs, carbonate, and silicate weathering contribute 74%, 20%, and 6% respectively to the dissolved Sr in the river water. This research has provided new insights into the contribution of sources of Sr to a river system in a mountainous catchment.

A unified model for the molecular basis of Xeroderma pigmentosum-Cockayne Syndrome.

Nucleotide Excision Repair (NER) is a pathway that removes lesions distorting the DNA helix. The molecular basis of the rare diseases Xeroderma pigmentosum (XP) and Cockayne Syndrome (CS) are explained based on the defects happening in 2 NER branches: Global-Genome Repair and Transcription-Coupled Repair, respectively. Nevertheless, both afflictions sporadically occur together, giving rise to XP/CS; however, the molecular basis of XP/CS is not understood very well. Many efforts have been made to clarify why mutations in only 4 NER genes, namely XPB, XPD, XPF and XPG, are the basis of this disease. Effort has also been made to unravel why mutations within these genes lead to XP, XP/CS, or other pathologies. We have recently contributed to the disclosure of this puzzle by characterizing Rad3/XPD mutations in Saccharomyces cerevisiae and human cells. Based on our, and others', observations, we propose a model compatible with all XP/CS cases and the current bibliography.

Co-expressed Cyclin D variants cooperate to regulate proliferation of germline nuclei in a syncytium.

The role of the G1-phase Cyclin D-CDK 4/6 regulatory module in linking germline stem cell (GSC) proliferation to nutrition is evolutionarily variable. In invertebrate Drosophila and C. elegans GSC models, G1 is nearly absent and Cyclin E is expressed throughout the cell cycle, whereas vertebrate spermatogonial stem cells have a distinct G1 and Cyclin D1 plays an important role in GSC renewal. In the invertebrate, chordate, Oikopleura, where germline nuclei proliferate asynchronously in a syncytium, we show a distinct G1-phase in which 2 Cyclin D variants are co-expressed. Cyclin Dd, present in both somatic endocycling cells and the germline, localized to germline nuclei during G1 before declining at G1/S. Cyclin Db, restricted to the germline, remained cytoplasmic, co-localizing in foci with the Cyclin-dependent Kinase Inhibitor, CKIa. These foci showed a preferential spatial distribution adjacent to syncytial germline nuclei at G1/S. During nutrient-restricted growth arrest, upregulated CKIa accumulated in arrested somatic endoreduplicative nuclei but did not do so in germline nuclei. In the latter context, Cyclin Dd levels gradually decreased. In contrast, the Cyclin Dbß splice variant, lacking the Rb-interaction domain and phosphodegron, was specifically upregulated and the number of cytoplasmic foci containing this variant increased. This upregulation was dependent on stress response MAPK p38 signaling. We conclude that under favorable conditions, Cyclin Dbß-CDK6 sequesters CKIa in the cytoplasm to cooperate with Cyclin Dd-CDK6 in promoting germline nuclear proliferation. Under nutrient-restriction, this sequestration function is enhanced to permit continued, though reduced, cycling of the germline during somatic growth arrest.

The CDK4/CDK6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes.

CDK4 and CDK6 bound to D-type cyclins are master integrators of G1 phase cell cycle regulations by initiating the inactivating phosphorylation of the central oncosuppressor pRb. Because of their frequent deregulation in cancer, cyclin D-CDK4/6 complexes are emerging as especially promising therapeutic targets. The specific CDK4/6 inhibitor PD0332991 is currently tested in a growing number of phase II/III clinical trials against a variety of pRb-proficient chemotherapy-resistant cancers. We have previously shown that PD0332991 inhibits not only CDK4/6 activity but also the activation by phosphorylation of the bulk of cyclin D-CDK4 complexes stabilized by p21 binding. Here we show that PD0332991 has either a positive or a negative impact on the activation of cyclin D-CDK4/6 complexes, depending on their binding to p21. Indeed, whereas PD0332991 inhibits the phosphorylation and activity of p21-bound CDK4/6, it specifically stabilized activated cyclin D3-CDK4/6 complexes devoid of p21 and p27. After elimination of PD0332991, these activated cyclin D3-CDK4/6 complexes persisted for at least 24 h, resulting in paradoxical cell cycle entry in the absence of a mitogenic stimulation. This unsuspected positive effect of PD0332991 on cyclin D3-CDK4/6 activation should be carefully assessed in the clinical evaluation of PD0332991, which until now only involves discontinuous administration protocols.

NKG2D-directed cytokine-activated killer lymphocyte therapy combined with gemcitabine for patients with chemoresistant metastatic solid tumors.

Natural-killer group 2, member D (NKG2D) is an activating receptor found on activated natural killer cells and on activated T-cells, here termed cytokine-activated killer (CAK) cells. NKG2D ligands are expressed on various human cancer types. Gemcitabine is an anticancer drug which is a less immune-destructive agent than others. Herein, we investigated the clinical efficacy and the underlying mechanisms of a combination of CAK cell infusion therapy and gemcitabine. Twenty-three patients with disseminated carcinomas were treated with chemo-immunotherapy consisting of CAK cell infusion therapy following gemcitabine treatment. To investigate the underlying mechanisms by which CAK cells synergize with gemcitabine, we used enzyme-linked immunosorbent assay, Real-time reverse transcription polymerase chain reaction assay, calcein-release assay, and adherent target detachment assay. Using these assays we determined the NKG2D ligands such as major histocompatibility complex-class I-related chain (MIC)A/B expression in carcinoma cells and the level of cellular cytotoxicity generated by treatment with gemcitabine with/without CAK cells. The tumor responses differed among the patients (n=23). In vitro experiments revealed that MICA/B protein and mRNA expression were up-regulated in several carcinoma cell lines after gemcitabine treatment. Pre-treatment with gemcitabine and subsequent exposure to CAK cells induced greater cytotoxicity than either treatment alone. Inclusion of soluble MICB in CAK cell-mediated cytotoxicity assay significantly reduced cytotoxicity. Our clinical results of gemcitabine-CAK combinatorial therapy demonstrated long-term stable disease despite chemoresistance. In conclusion, the combination of gemcitabine and CAK cells may have clinical therapeutic significance for pancreatic, hepato-biliary tract, and urothelial tract cancer. Our study shows that combining CAK therapy with gemcitabine can lead to successful treatment of metastatic cancer.

Adaptors for disorders of the brain? The cancer signaling proteins NEDD9, CASS4, and PTK2B in Alzheimer's disease.

No treatment strategies effectively limit the progression of Alzheimer's disease (AD), a common and debilitating neurodegenerative disorder. The absence of viable treatment options reflects the fact that the pathophysiology and genotypic causes of the disease are not well understood. The advent of genome-wide association studies (GWAS) has made it possible to broadly investigate genotypic alterations driving phenotypic occurrences. Recent studies have associated single nucleotide polymorphisms (SNPs) in two paralogous scaffolding proteins, NEDD9 and CASS4, and the kinase PTK2B, with susceptibility to late-onset AD (LOAD). Intriguingly, NEDD9, CASS4, and PTK2B have been much studied as interacting partners regulating oncogenesis and metastasis, and all three are known to be active in the brain during development and in cancer. However, to date, the majority of studies of these proteins have emphasized their roles in the directly cancer relevant processes of migration and survival signaling. We here discuss evidence for roles of NEDD9, CASS4 and PTK2B in additional processes, including hypoxia, vascular changes, inflammation, microtubule stabilization and calcium signaling, as potentially relevant to the pathogenesis of LOAD. Reciprocally, these functions can better inform our understanding of the action of NEDD9, CASS4 and PTK2B in cancer.

ELL, a novel TFIIH partner, is involved in transcription restart after DNA repair.

DNA lesions that block transcription may cause cell death even when repaired, if transcription does not restart to reestablish cellular metabolism. However, transcription resumption after individual DNA-lesion repair remains poorly described in mechanistic terms and its players are largely unknown. The general transcription factor II H (TFIIH) is a major actor of both nucleotide excision repair subpathways of which transcription-coupled repair highlights the interplay between DNA repair and transcription. Using an unbiased proteomic approach, we have identified the protein eleven-nineteen lysine-rich leukemia (ELL) as a TFIIH partner. Here we show that ELL is recruited to UV-damaged chromatin in a Cdk7- dependent manner (a component of the cyclin-dependent activating kinase subcomplex of TFIIH). We demonstrate that depletion of ELL strongly hinders RNA polymerase II (RNA Pol II) transcription resumption after lesion removal and DNA gap filling. Lack of ELL was also observed to increase RNA Pol II retention to the chromatin during this process. Identifying ELL as an essential player for RNA Pol II restart during cellular DNA damage response opens the way to obtaining a mechanistic description of transcription resumption after DNA repair.

The lost intrinsic fragmentation of MAT1 protein during granulopoiesis promotes the growth and metastasis of leukemic myeloblasts.

MAT1, an assembly factor and targeting subunit of both cyclin-dependent kinase-activating kinase (CAK) and general transcription factor IIH (TFIIH) kinase, regulates cell cycle and transcription. Previous studies show that expression of intact MAT1 protein is associated with expansion of human hematopoietic stem cells (HSC), whereas intrinsically programmed or retinoic acid (RA)-induced MAT1 fragmentation accompanies granulocytic differentiation of HSC or leukemic myeloblasts. Here we determined that, in humanized mouse microenvironment, MAT1 overexpression resisted intrinsic MAT1 fragmentation to sustain hematopoietic CD34+ cell expansion while preventing granulopoiesis. Conversely, we mimicked MAT1 fragmentation in vitro and in a mouse model by overexpressing a fragmented 81-aa MAT1 polypeptide (pM9) that retains the domain for assembling CAK but cannot affix CAK to TFIIH-core. Our results showed that pM9 formed ΔCAK by competing with MAT1 for CAK assembly to mimic MAT1 fragmentation-depletion of CAK. This resulting ΔCAK acted as a dominant negative to inhibit the growth and metastasis of different leukemic myeloblasts, with or without RA resistance, by concurrently suppressing CAK and TFIIH kinase activities to inhibit cell cycle and gene transcription. These findings suggest that the intrinsically programmed MAT1 expression and fragmentation regulate granulopoiesis by inversely coordinating CAK and TFIIH activities, whereas pM9 shares a mechanistic resemblance with MAT1 fragmentation in suppressing myeloid leukemogenesis.

The CDK Network: Linking Cycles of Cell Division and Gene Expression.

Cyclin-dependent kinases (CDKs) play essential roles in cell proliferation and gene expression. Although distinct sets of CDKs work in cell division and transcription by RNA polymerase II (Pol II), they share a CDK-activating kinase (CAK), which is itself a CDK-Cdk7-in metazoans. Thus a unitary CDK network controls and may coordinate cycles of cell division and gene expression. Recent work reveals decisive roles for Cdk7 in both pathways. The CAK function of Cdk7 helps determine timing of activation and cyclin-binding preferences of different CDKs during the cell cycle. In the transcription cycle, Cdk7 is both an effector kinase, which phosphorylates Pol II and other proteins and helps establish promoter-proximal pausing; and a CAK for Cdk9 (P-TEFb), which releases Pol II from the pause. By governing the transition from initiation to elongation, Cdk7, Cdk9 and their substrates influence expression of genes important for developmental and cell-cycle decisions, and ensure co-transcriptional maturation of Pol II transcripts. Cdk7 engaged in transcription also appears to be regulated by phosphorylation within its own activation (T) loop. Here I review recent studies of CDK regulation in cell division and gene expression, and propose a model whereby mitogenic signals trigger a cascade of CDK T-loop phosphorylation that drives cells past the restriction (R) point, when continued cell-cycle progression becomes growth factor-independent. Because R-point control is frequently deregulated in cancer, the CAK-CDK pathway is an attractive target for chemical inhibition aimed at impeding the inappropriate commitment to cell division.