Disruption of CDK7 signaling leads to catastrophic chromosomal instability coupled with a loss of condensin-mediated chromatin compaction.

Chromatin organization is highly dynamic and modulates DNA replication, transcription, and chromosome segregation. Condensin is essential for chromosome assembly during mitosis and meiosis, as well as maintenance of chromosome structure during interphase. While it is well established that sustained condensin expression is necessary to ensure chromosome stability, the mechanisms that control its expression are not yet known. Herein, we report that disruption of cyclin-dependent kinase 7 (CDK7), the core catalytic subunit of CDK-activating kinase, leads to reduced transcription of several condensin subunits, including structural maintenance of chromosomes 2 (SMC2). Live and static microscopy revealed that inhibiting CDK7 signaling prolongs mitosis and induces chromatin bridge formation, DNA double-strand breaks, and abnormal nuclear features, all of which are indicative of mitotic catastrophe and chromosome instability. Affirming the importance of condensin regulation by CDK7, genetic suppression of the expression of SMC2, a core subunit of this complex, phenocopies CDK7 inhibition. Moreover, analysis of genome-wide chromatin conformation using Hi-C revealed that sustained activity of CDK7 is necessary to maintain chromatin sublooping, a function that is ascribed to condensin. Notably, the regulation of condensin subunit gene expression is independent of superenhancers. Together, these studies reveal a new role for CDK7 in sustaining chromatin configuration by ensuring the expression of condensin genes, including SMC2.

A Phase I Dose-Escalation Study of LY3405105, a Covalent Inhibitor of Cyclin-Dependent Kinase 7, Administered to Patients With Advanced Solid Tumors.

BACKGROUND: This study aimed to evaluate the safety, pharmacokinetics (PKs), and preliminary activity of LY3405105, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), in patients with advanced solid tumors. MATERIALS AND METHODS: LY3405105 monotherapy was given once daily (QD; part A1) or thrice weekly (TIW; part A2) starting at 1 and 2 mg orally, respectively, and escalated per a Bayesian design in adult patients. The primary endpoint was safety, and secondary endpoints included PKs and antitumor activity. RESULTS: Fifty-four patients were enrolled: 43 in part A1 and 11 in part A2. Seven patients had dose-limiting toxicities, all in part A1 (45 mg: n = 3; 35 mg: n = 3; 25 mg: n = 1). Thirty-five patients (64.8%) reported at least one treatment-related adverse event (TRAE). TRAEs (≥10%) were diarrhea, nausea, fatigue, vomiting, abdominal pain, anemia, asthenia, and decreased platelet count. QD dosing showed sustained exposure with less peak-trough fluctuation compared to TIW dosing. Median time to maximum concentration was 1-2 hours and half-life was 15-19 hours. CDK7-target occupancy in skin and peripheral blood on day 15 was dose-dependent and reached near maximal occupancy of 75% at ≥15 mg QD. The maximum tolerated dose (MTD) was 20 mg QD. Twelve patients in part A1 (27.9%) and 5 patients in part A2 (45.5%) had a best overall response of stable disease. No complete response or partial response was observed. CONCLUSION: The MTD of LY3405105 monotherapy was 20 mg QD. The most common toxicities were gastrointestinal adverse events, myelosuppression, fatigue, and asthenia. Limited clinical activity was observed in this phase I trial, and there are no plans for further development. CLINICALTRIALS.GOV IDENTIFIER: NCT03770494.

Design, synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel potent CDK7 inhibitors.

The targeting of cyclin-dependent kinase 7 (CDK7) has become a highly desirable therapeutic approach in the field of oncology due to its dual role in regulating essential biological processes, encompassing cell cycle progression and transcriptional control. We have previously identified a highly selective thieno[3,2-d]pyrimidine-based CDK7 inhibitor with demonstrated efficacy and safety in animal model. In this study, we sought to optimize the thieno[3,2-d]pyrimidine core to discover a novel series of CDK7 inhibitors with improved potency and pharmacokinetic (PK) properties. Through extensive structure-activity relationship (SAR) studies, compound 20 has emerged as the lead candidate due to its potent inhibitory activity against CDK7 and remarkable efficacy on MDA-MB-453 cells, a representative triple negative breast cancer (TNBC) cell line. Furthermore, 20 has demonstrated favorable oral bioavailability and exhibited highly desirable pharmacokinetic (PK) properties, making it a promising lead candidate for further structural optimization.

TGF-ß/activin signaling promotes CDK7 inhibitor resistance in triple-negative breast cancer cells through upregulation of multidrug transporters.

Cyclin-dependent kinase 7 (CDK7) is a master regulatory kinase that drives cell cycle progression and stimulates expression of oncogenes in a myriad of cancers. Inhibitors of CDK7 (CDK7i) are currently in clinical trials; however, as with many cancer therapies, patients will most likely experience recurrent disease due to acquired resistance. Identifying targets underlying CDK7i resistance will facilitate prospective development of new therapies that can circumvent such resistance. Here we utilized triple-negative breast cancer as a model to discern mechanisms of resistance as it has been previously shown to be highly responsive to CDK7 inhibitors. After generating cell lines with acquired resistance, high-throughput RNA sequencing revealed significant upregulation of genes associated with efflux pumps and transforming growth factor-beta (TGF-ß) signaling pathways. Genetic silencing or pharmacological inhibition of ABCG2, an efflux pump associated with multidrug resistance, resensitized resistant cells to CDK7i, indicating a reliance on these transporters. Expression of activin A (INHBA), a member of the TGF-ß family of ligands, was also induced, whereas its intrinsic inhibitor, follistatin (FST), was repressed. In resistant cells, increased phosphorylation of SMAD3, a downstream mediator, confirmed an increase in activin signaling, and phosphorylated SMAD3 directly bound the ABCG2 promoter regulatory region. Finally, pharmacological inhibition of TGF-ß/activin receptors or genetic silencing of SMAD4, a transcriptional partner of SMAD3, reversed the upregulation of ABCG2 in resistant cells and phenocopied ABCG2 inhibition. This study reveals that inhibiting the TGF-ß/Activin-ABCG2 pathway is a potential avenue for preventing or overcoming resistance to CDK7 inhibitors.

Cyclin-dependent kinase 7 contributes to myelin maintenance in the adult central nervous system and promotes myelin gene expression.

Mechanisms regulating oligodendrocyte differentiation, developmental myelination and myelin maintenance in adulthood are complex and still not completely described. Their understanding is crucial for the development of new protective or therapeutic strategies in demyelinating pathologies such as multiple sclerosis. In this perspective, we have investigated the role of Cyclin-dependent kinase 7 (Cdk7), a kinase involved in cell-cycle progression and transcription regulation, in the oligodendroglial lineage. We generated a conditional knock-out mouse model in which Cdk7 is invalidated in post-mitotic oligodendrocytes. At the end of developmental myelination, the number and diameter of myelinated axons, as well as the myelin structure, thickness and protein composition, were normal. However, in young adult and in aged mice, there was a higher number of small caliber myelinated axons associated with a decreased mean axonal diameter, myelin sheaths of large caliber axons were thinner, and the level of some major myelin-associated proteins was reduced. These defects were accompanied by the appearance of an abnormal clasping phenotype. We also used an in vitro oligodendroglial model and showed that Cdk7 pharmacological inhibition led to an altered myelination-associated morphological modification combined with a decreased expression of myelin-specific genes. Altogether, we identified novel functions for Cdk7 in CNS myelination.

Pharmacological inhibition of CDK7 by THZ1 impairs tumor growth in p53-mutated HNSCC.

BACKGROUND: Cyclin-dependent kinase 7 (CDK7) has been critically linked to human cancer. However, the roles of CDK7 in head and neck squamous cell carcinoma (HNSCC) remain incompletely known. Here, we sought to dissect the functions of CDK7 underlying HNSCC tumorigenesis and explore whether pharmacological inhibition of CDK7 could induce anti-cancer effects. METHODS: CDK7 expression was measured in a panel of HNSCC cell lines with p53 mutation and 20 pairs of HNSCC samples and adjacent non-tumor tissues. Genetic targeting and pharmacological inhibition of CDK7 were conducted to dissect the biological roles of CDK7 in p53-mutated HNSCC cells. An HNSCC xenograft model was developed to determine the therapeutic effects of THZ1 in vivo. Potential genes and pathways responsible for therapeutic effects of THZ1 were identified by genome-wide RNA-sequencing and bioinformatics interrogations. RESULTS: CDK7 expression was significantly elevated in cancerous cells and samples as compared with their adjacent non-tumor counterparts. Impaired cell proliferation, migration, and invasion as well increased apoptosis were observed in cells upon CDK7 knockdown or THZ1 exposure. THZ1 administration potently inhibited tumor overgrowth in vivo. Mechanistically, hundreds of genes enriched in cell proliferation, apoptosis, and cancer-related categories were identified to be potentially mediated the therapeutic effects of THZ1 in HNSCC. CONCLUSION: Our findings reveal that CDK7 might serve as a novel putative pro-oncogenic gene underlying HNSCC tumorigenesis and therapeutic targeting of CDK7 might be a promising strategy for p53-mutated HNSCC.

Control of Expression of Key Cell Cycle Enzymes Drives Cell Line-Specific Functions of CDK7 in Human PDAC Cells.

Inhibition of the dual function cell cycle and transcription kinase CDK7 is known to affect the viability of cancer cells, but the mechanisms underlying cell line-specific growth control remain poorly understood. Here, we employed a previously developed, highly specific small molecule inhibitor that non-covalently blocks ATP binding to CDK7 (LDC4297) to study the mechanisms underlying cell line-specific growth using a panel of genetically heterogeneous human pancreatic tumor lines as model system. Although LDC4297 diminished both transcription rates and CDK T-loop phosphorylation in a comparable manner, some PDAC lines displayed significantly higher sensitivity than others. We focused our analyses on two well-responsive lines (Mia-Paca2 and Panc89) that, however, showed significant differences in their viability upon extended exposure to limiting LDC4297 concentrations. Biochemical and RNAseq analysis revealed striking differences in gene expression and cell cycle control. Especially the downregulation of a group of cell cycle control genes, among them CDK1/2 and CDC25A/C, correlated well to the observed viability differences in Panc89 versus Mia-Paca2 cells. A parallel downregulation of regulatory pathways supported the hypothesis of a feedforward programmatic effect of CDK7 inhibitors, eventually causing hypersensitivity of PDAC lines.

Targeting CDK7 suppresses super enhancer-linked inflammatory genes and alleviates CAR T cell-induced cytokine release syndrome.

BACKGROUND: Cytokine release syndrome (CRS) is a systemic inflammatory response characterized by the overexpression of inflammatory genes. Controlling CRS is essential for improving the therapeutic effects of chimeric antigen receptor (CAR) engineered T cells. However, current treatment options are limited given the complexity of cytokine interactions so it is important to seek a mild strategy with broad-spectrum inhibition to overcome this challenge. METHODS: Using THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), we demonstrated the transcriptional suppression of inflammatory genes in activated macrophages. RNA sequencing and ChIP sequencing were conducted to identify the key target genes of the inflammatory response. Pathogen- and CAR T cell-induced CRS models were also established to assess the efficacy and safety of targeting CDK7. RESULTS: CDK7 blockade attenuated cytokine release, mitigated hyperinflammatory states and rescued mice from lethal CRS. Targeting CDK7 preferentially suppressed a set of inflammatory genes, of which STAT1 and IL1 were the key targets associated with super enhancers. Furthermore, we confirmed the potent efficacy of THZ1 in alleviating the CRS induced by CAR T cell infusion without causing tissue injury or impairing antitumor effects. CONCLUSIONS: Our work indicates the CDK7-dependent transcription addiction of inflammatory genes. Targeting CDK7 is a promising strategy for treating CRS by inhibiting multiple cytokines.

Inhibition of cyclin-dependent kinase 7 down-regulates yes-associated protein expression in mesothelioma cells.

Cyclin-dependent kinase 7 (CDK7) is a protein kinase that plays a major role in transcription initiation. Yes-associated protein (YAP) is a main effector of the Hippo/YAP signalling pathway. Here, we investigated the role of CDK7 on YAP regulation in human malignant pleural mesothelioma (MPM). We found that in microarray samples of human MPM tissue, immunohistochemistry staining showed correlation between the expression level of CDK7 and YAP (n = 70, r = .513). In MPM cells, CDK7 expression level was significantly correlated with GTIIC reporter activity (r = .886, P = .019). Inhibition of CDK7 by siRNA decreased the YAP protein level and the GTIIC reporter activity in the MPM cell lines 211H, H290 and H2052. Degradation of the YAP protein was accelerated after CDK7 knockdown in 211H, H290 and H2052 cells. Inhibition of CDK7 reduced tumour cell migration and invasion, as well as tumorsphere formation ability. Restoration of the CDK7 gene rescued the YAP protein level and GTIIC reporter activity after siRNA knockdown in 211H and H2052 cells. Finally, we performed a co-immunoprecipitation analysis using an anti-YAP antibody and captured the CDK7 protein in 211H cells. Our results suggest that CDK7 inhibition reduces the YAP protein level by promoting its degradation and suppresses the migration and invasion of MPM cells. Cyclin-dependent kinase 7 may be a promising therapeutic target for MPM.

CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer.

OBJECTIVE: Cyclin-dependent kinase 7 (CDK7) engages tumor growth by acting as a direct link between the regulation of transcription and the cell cycle. Here, we investigated the clinical significance of CDK7 expression and its potential as a therapeutic target in epithelial ovarian cancer (EOC). METHODS: CDK7 expression was examined in 436 ovarian tissues including normal to metastatic ovarian tumors using immunohistochemistry, and its clinical implications were analyzed. Furthermore, we performed in vitro and in vivo experiments using CDK7 siRNA or a covalent CDK7 inhibitor (THZ1) to elucidate the effect of CDK7 inhibition on tumorigenesis in EOC cells. RESULTS: The patient incidence of high CDK7 expression (CDK7High) gradually increased from normal ovarian epithelium to EOC (P < 0.001). Moreover, CDK7High was associated with an advanced stage and high-grade histology (P = 0.035 and P = 0.011, respectively) in EOC patients and had an independent prognostic significance in EOC recurrence (P = 0.034). CDK7 inhibition with siRNA or THZ1 decreased cell proliferation and migration, and increased apoptosis in EOC cells, and this anti-cancer mechanism is caused by G0/G1 cell cycle arrest. In in vivo therapeutic experiments using cell-line xenograft and PDX models, CDK7 inhibition significantly decreased the tumor weight, which was mediated by cell proliferation and apoptosis. CONCLUSION: Mechanistic interrogation of CDK7 revealed that it is significantly associated with an aggressive phenotype of EOC, and it has independent prognostic power for EOC recurrence. Furthermore, CDK7 may be a potential therapeutic target for patients with EOC, whether platinum sensitive or resistant.

Characterization of cyclin dependent kinase-7 and its differential response to grafting challenge in the black shell colored selected line of pearl oyster Pinctada fucata martensii.

Cyclin dependent kinase-7 (Cdk-7) is a protein kinase associated with regulating the cell cycle, cell differentiation and proliferation, apoptosis and inflammatory response. This study characterized the full cDNA sequence of Cdk-7 in Pinctada fucata martensii (PmCdk-7). A full length sequence of 1473bp with an open reading frame (ORF) of 915bp and encodes a 304aa, 5'-UTR of 58bp and a 3'-UTR of 500bp was obtained. The construed amino acid sequence of PmCdk-7 comprised of a Serine/Threonine protein kinases, catalytic (S_TKc) domain with a protein kinases ATP-binding region signature (14-38aa) and the serine/Threonine protein kinases active-site signature (129-141aa) within the domain. Tissue distribution analysis revealed a high relative mRNA expression of PmCdk-7 within haemocytes. Following the insertion operation (grafting), the relative expression levels of PmCdk-7 in the haemocyte was expressed differentially among the studied groups; the black shell colored selected line (BS) and the control group (CG). High expression was recorded between 12 h and 5d with a peak at 3d suggesting a heightened level of DNA replication and inflammatory response during the pearl-sac formation and this expression was higher in BS than CS showcasing, the heightened immune capacity of BS to grafting operation. Immune stimulation experiment with bacterial endotoxin and a viral mimic revealed PmCdk-7 response to pathogenic stress. The results from our study showed that PmCdk-7 performs a vital function during the cell cycle by aiding DNA replication and also aid response to inflammations generated due to the incision from the grafting operation and long exposure to immune-stimulants (pathogens).

Blockade of CDK7 Reverses Endocrine Therapy Resistance in Breast Cancer.

Cyclin-dependent kinase (CDK)-7 inhibitors are emerging as promising drugs for the treatment of different types of cancer that show chemotherapy resistance. Evaluation of the effects of CDK7 inhibitor, THZ1, alone and combined with tamoxifen is of paramount importance. Thus, in the current work, we assessed the effects of THZ1 and/or tamoxifen in two estrogen receptor-positive (ER+) breast cancer cell lines (MCF7) and its tamoxifen resistant counterpart (LCC2) in vitro and in xenograft mouse models of breast cancer. Furthermore, we evaluated the expression of CDK7 in clinical samples from breast cancer patients. Cell viability, apoptosis, and genes involved in cell cycle regulation and tamoxifen resistance were determined. Tumor volume and weight, proliferation marker (Ki67), angiogenic marker (CD31), and apoptotic markers were assayed. Bioinformatic data indicated CDK7 expression was associated with negative prognosis, enhanced pro-oncogenic pathways, and decreased response to tamoxifen. Treatment with THZ1 enhanced tamoxifen-induced cytotoxicity, while it inhibited genes involved in tumor progression in MCF-7 and LCC2 cells. In vivo, THZ1 boosted the effect of tamoxifen on tumor weight and tumor volume, reduced Ki67 and CD31 expression, and increased apoptotic cell death. Our findings identify CDK7 as a possible therapeutic target for breast cancer whether it is sensitive or resistant to tamoxifen therapy.

Mechanistic insights into avian reovirus p17-modulated suppression of cell cycle CDK-cyclin complexes and enhancement of p53 and cyclin H interaction.

The avian reovirus p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remain unclear. This is the first report that avian reovirus p17 possesses broad inhibitory effects on cell cycle CDKs, cyclins, CDK-cyclin complexes, and CDK-activating kinase activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK-cyclin complexes; transcriptional down-regulation of CDKs; cytoplasmic retention of CDKs and cyclins; and inhibition of CDK-activating kinase activity by promoting p53-cyclin H interaction. p17 binds to CDK-cyclin except for CDK1-cyclin B1 and CDK7-cyclin H complexes. We have determined that the negatively charged 151LAVXDVDA(E/D)DGADPN165 motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1-cyclin B1, but it is required for other CDK-cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif, 125RXL127 Sequence and mutagenic analyses of p17 indicated that a 140WXFD143 motif and residues Asp-113 and Lys-122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication, whereas p17 mutants with low binding ability to cell cycle CDKs had no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle, benefiting virus replication. An in vivo tumorigenesis assay also showed a significant reduction in tumor size.

Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G1, S, and G2), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes.

Inhibition of cyclin-dependent kinase 7 suppresses human hepatocellular carcinoma by inducing apoptosis.

Increasing evidence has shown that THZ1, a covalent cyclin-dependent kinase 7 (CDK7) inhibitor, exhibits therapeutic effects in various tumors. However, the possible effect of THZ1 on hepatocellular carcinoma (HCC) remains unknown. Our study was to investigate the roles of THZ1 in HCC cells and in subcutaneous HCC model and illustrate the molecular mechanisms. The phosphorylation levels of Ser2, Ser5, and Ser7 within RNA polymerase II (RNAPII) C-terminal domain (CTD) and the expression levels of Ki67, Mcl-1, survivin, XIAP, and p53 in HCC cells under different conditions were examined by Western blot analysis. Cell growth and apoptosis were assessed via 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis, respectively. Tumor volume was assessed in HCC mice with THZ1 or vehicle treatment and immunohistochemical (IHC) analysis was conducted on excised tumors. THZ1 significantly inhibited the phosphorylation of Ser2, Ser5, and Ser7 within RNAPII-CTD in the dose-dependent and irreversible manner. MTT assay and flow cytometry analysis showed that THZ1 inhibited HCC cell proliferation and induced apoptosis, respectively. Western blot analysis indicated THZ1 significantly upregulated p53 expression and downregulated the expressions of Mcl-1, survivin, XIAP, and Ki67. THZ1 suppressed tumor growth in Hep3B xenografted mice in a time-dependent manner. IHC analysis indicated that tumors in THZ1 group had less Ki67+ cells and more cleaved caspase-3+ cells than those in vehicle group. THZ1 exhibited anti-HCC effects through irreversibly inhibiting CDK7 activity, decreasing RNAPII-CTD phosphorylation, inducing p53 expression and inhibiting antiapoptotic gene expressions, which subsequently induced apoptosis and inhibited proliferation of HCC cells.

Interaction with CCNH/CDK7 facilitates CtBP2 promoting esophageal squamous cell carcinoma (ESCC) metastasis via upregulating epithelial-mesenchymal transition (EMT) progression.

CtBP2, as a transcriptional corepressor of epithelial-specific genes, has been reported to promote tumor due to upregulating epithelial-mesenchymal transition (EMT) in cancer cells. CtBP2 was also demonstrated to contribute to the proliferation of esophageal squamous cell carcinoma (ESCC) cells through a negative transcriptional regulation of p16(INK4A). In this study, for the first time, we reported that CtBP2 expression, along with CCNH/CDK7, was higher in ESCC tissues with lymph node metastases than in those without lymph node metastases. Moreover, both CtBP2 and CCNH/CDK7 were positively correlated with E-cadherin, tumor grade, and tumor metastasis. However, the concrete mechanism of CtBP2's role in enhancing ESCC migration remains incompletely understood. We confirmed that CCNH/CDK7 could directly interact with CtBP2 in ESCC cells in vivo and in vitro. Furthermore, our data demonstrate for the first time that CtBP2 enhanced the migration of ESCC cells in a CCNH/CDK7-dependent manner. Our results indicated that CCNH/CDK7-CtBP2 axis may augment ESCC cell migration, and targeting the interaction of both may provide a novel therapeutic target of ESCC.

Inhibition of cyclin-dependent kinase 7 mitigates doxorubicin cardiotoxicity and enhances anticancer efficacy.

AIMS: The anthracycline family of anticancer agents such as doxorubicin (DOX) can induce apoptotic death of cardiomyocytes and cause cardiotoxicity. We previously reported that DOX-induced apoptosis is accompanied by cardiomyocyte cell cycle re-entry. Cell cycle progression requires cyclin-dependent kinase 7 (CDK7)-mediated activation of downstream cell cycle CDKs. This study aims to determine whether CDK7 can be targeted for cardioprotection during anthracycline chemotherapy. METHODS AND RESULTS: DOX exposure induced CDK7 activation in mouse heart and isolated cardiomyocytes. Cardiac-specific ablation of Cdk7 attenuated DOX-induced cardiac dysfunction and fibrosis. Treatment with the covalent CDK7 inhibitor THZ1 also protected against DOX-induced cardiomyopathy and apoptosis. DOX treatment induced activation of the proapoptotic CDK2-FOXO1-Bim axis in a CDK7-dependent manner. In response to DOX, endogenous CDK7 directly bound and phosphorylated CDK2 at Thr160 in cardiomyocytes, leading to full CDK2 kinase activation. Importantly, inhibition of CDK7 further suppressed tumour growth when used in combination with DOX in an immunocompetent mouse model of breast cancer. CONCLUSION: Activation of CDK7 is necessary for DOX-induced cardiomyocyte apoptosis and cardiomyopathy. Our findings uncover a novel proapoptotic role for CDK7 in cardiomyocytes. Moreover, this study suggests that inhibition of CDK7 attenuates DOX-induced cardiotoxicity but augments the anticancer efficacy of DOX. Therefore, combined administration of CDK7 inhibitor and DOX may exhibit diminished cardiotoxicity but superior anticancer activity.

CDK7/CDK9 mediates transcriptional activation to prime paraptosis in cancer cells.

BACKGROUND: Paraptosis is a programmed cell death characterized by cytoplasmic vacuolation, which has been explored as an alternative method for cancer treatment and is associated with cancer resistance. However, the mechanisms underlying the progression of paraptosis in cancer cells remain largely unknown. METHODS: Paraptosis-inducing agents, CPYPP, cyclosporin A, and curcumin, were utilized to investigate the underlying mechanism of paraptosis. Next-generation sequencing and liquid chromatography-mass spectrometry analysis revealed significant changes in gene and protein expressions. Pharmacological and genetic approaches were employed to elucidate the transcriptional events related to paraptosis. Xenograft mouse models were employed to evaluate the potential of paraptosis as an anti-cancer strategy. RESULTS: CPYPP, cyclosporin A, and curcumin induced cytoplasmic vacuolization and triggered paraptosis in cancer cells. The paraptotic program involved reactive oxygen species (ROS) provocation and the activation of proteostatic dynamics, leading to transcriptional activation associated with redox homeostasis and proteostasis. Both pharmacological and genetic approaches suggested that cyclin-dependent kinase (CDK) 7/9 drive paraptotic progression in a mutually-dependent manner with heat shock proteins (HSPs). Proteostatic stress, such as accumulated cysteine-thiols, HSPs, ubiquitin-proteasome system, endoplasmic reticulum stress, and unfolded protein response, as well as ROS provocation primarily within the nucleus, enforced CDK7/CDK9-Rpb1 (RNAPII subunit B1) activation by potentiating its interaction with HSPs and protein kinase R in a forward loop, amplifying transcriptional regulation and thereby exacerbating proteotoxicity leading to initiate paraptosis. The xenograft mouse models of MDA-MB-231 breast cancer and docetaxel-resistant OECM-1 head and neck cancer cells further confirmed the induction of paraptosis against tumor growth. CONCLUSIONS: We propose a novel regulatory paradigm in which the activation of CDK7/CDK9-Rpb1 by nuclear proteostatic stress mediates transcriptional regulation to prime cancer cell paraptosis.

An Antiherpesviral Host-Directed Strategy Based on CDK7 Covalently Binding Drugs: Target-Selective, Picomolar-Dose, Cross-Virus Reactivity.

The repertoire of currently available antiviral drugs spans therapeutic applications against a number of important human pathogens distributed worldwide. These include cases of the pandemic severe acute respiratory coronavirus type 2 (SARS-CoV-2 or COVID-19), human immunodeficiency virus type 1 (HIV-1 or AIDS), and the pregnancy- and posttransplant-relevant human cytomegalovirus (HCMV). In almost all cases, approved therapies are based on direct-acting antivirals (DAAs), but their benefit, particularly in long-term applications, is often limited by the induction of viral drug resistance or side effects. These issues might be addressed by the additional use of host-directed antivirals (HDAs). As a strong input from long-term experiences with cancer therapies, host protein kinases may serve as HDA targets of mechanistically new antiviral drugs. The study demonstrates such a novel antiviral strategy by targeting the major virus-supportive host kinase CDK7. Importantly, this strategy focuses on highly selective, 3D structure-derived CDK7 inhibitors carrying a warhead moiety that mediates covalent target binding. In summary, the main experimental findings of this study are as follows: (1) the in vitro verification of CDK7 inhibition and selectivity that confirms the warhead covalent-binding principle (by CDK-specific kinase assays), (2) the highly pronounced antiviral efficacies of the hit compounds (in cultured cell-based infection models) with half-maximal effective concentrations that reach down to picomolar levels, (3) a particularly strong potency of compounds against strains and reporter-expressing recombinants of HCMV (using infection assays in primary human fibroblasts), (4) additional activity against further herpesviruses such as animal CMVs and VZV, (5) unique mechanistic properties that include an immediate block of HCMV replication directed early (determined by Western blot detection of viral marker proteins), (6) a substantial drug synergism in combination with MBV (measured by a Loewe additivity fixed-dose assay), and (7) a strong sensitivity of clinically relevant HCMV mutants carrying MBV or ganciclovir resistance markers. Combined, the data highlight the huge developmental potential of this host-directed antiviral targeting concept utilizing covalently binding CDK7 inhibitors.

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.