Cyclin-dependent Kinase 9 Inhibitor Research Articles

Preclinical metabolism and disposition of [14C]GFH009, a novel selective CDK9 inhibitor

1. GFH009 is a potent and highly selective cyclin-dependent kinase 9 (CDK9) inhibitor currently under phase II clinical trials. In this study, we investigated the metabolism and disposition of GFH009 in Sprague-Dawley (SD) rats, as well as in vitro metabolism of CD-1 mouse, SD rat, beagle dog, cynomolgus monkey and human. 2. A radiolabelled study indicated that [14C]GFH009 was quickly and widely distributed throughout the body, but presented low levels in brain, testis and epididymis after a single intravenous dose of 6 mg (100 µCi)/kg to SD rats. 3. GFH009 undergoes systemic metabolic changes, primarily throughO-demethylation, oxidation to carboxylic acid and N-dealkylation, cleavage off the methoxyisopropyl moiety being a minor pathway. These metabolic pathways were found to be mainly consistent both in vitro and in vivo. 4. In SD rats, GFH009 was rapidly and completely eliminated, with feces serving as the major excretion pathway and urine serving as the minor one. Besides, the major clearance pathway for GFH009 was excretion and the minor one was metabolism. 5. GFH009 exhibits favourable drug metabolism and pharmacokinetics (DMPK) properties, which provides valuable insights into the disposition of GFH009 and can be used to guide future clinical studies.

Discovery of a Novel Macrocyclic Noncovalent CDK7 Inhibitor for Cancer Therapy.

Cyclin-dependent kinase 7 (CDK7) is a key regulator of the cell cycle and transcription, making it a promising target for cancer therapy. Although current CDK7 inhibitors have improved in their selectivity and druglike properties, CDK7 inhibitors have failed to progress through clinical development due to severe gastrointestinal and hematotoxic side effects. To mitigate these limitations, we have developed novel, macrocyclic, noncovalent CDK7 hit compounds 2 and 3 using a macrocyclization platform that has optimized these compounds from SY-5609, a leading clinical asset. We conducted extensive structure-activity relationship (SAR) studies to improve their potency, enhance oral bioavailability, and reduce intestinal distribution, which resulted in compound 13. Compound 13 exhibits potent in vitro activity, good ADME properties, and robust in vivo antitumor activity in xenograft models as a monotherapy. Notably, compound 13 with lower basicity demonstrated improved Caco-2 permeability, reduced blood/plasma ratio, and reduced intestinal distribution in rats, thus mitigating gastrointestinal and hematotoxic side effects.

Development of novel CDK9 and CYP3A4 inhibitors for cancer therapy through field and computational approaches.

Cyclin-dependent kinase 9 (CDK9) and cytochrome P450 3A4 (CYP3A4) have emerged as promising targets in the development of anticancer drugs, presenting a consistent challenge in the quest for potent inhibitors. CDK9 inhibitors can selectively target fast-growing cancer cells by disrupting transcription elongation, which in turn hinders the production of proteins essential for cell cycle progression and survivaŚ. Understanding how CYP3A4 metabolizes specific chemotherapy drugs allows for personalized treatment plans, optimizing drug dosages according to a patient's metabolic profile. Since many cancer patients undergo combination therapies, and CYP3A4 is vital in drug metabolism, its inhibition or induction by one drug can alter the plasma levels of others, potentially leading to treatment failure or increased toxicity. Therefore, managing CYP3A4 activity is critical for effective cancer treatment. Employing a range of computational methodologies, this study systematically investigated the binding mechanisms of pyrimidine derivatives against CDK9 and CYP3A4. The field-based model demonstrated high R 2 values (0.99), with Q2 (0.66), demonstrating its ability to predict in silico inhibitory activity against the target of this study. The screening process followed in this work led to the discovery of powerful new inhibitor compounds. Of the 15 new compounds designed, three have a high affinity with the target (ranging from -8 to -9kcal/molkcal/mol) and were singled out through docking filtration for more detailed investigation. As well as, a reference compound with a substantial pIC50 value of 8.4, serving as the foundation for the development of the new compounds, was included for comparative analysis. To elucidate the essential features of CDK9 and CYP3A4 inhibitor design, a comparative analysis was conducted between 3D-QSAR-generated contours and molecular docking conformations of ligands. Molecular dynamics simulations were carried out for a duration of 100 ns on selected docked complexes, specifically those involving novel compounds with CDK9 and CYP3A4 enzymes. Additionally, the binding free energy for these complexes was assessed using the MM/PBSA method, which evaluates the free energy landscape of protein-ligand interactions. The results of MM/PBSA highlighted the strength of the new compounds in enhancing interactions with the target protein, which favors the results of molecular docking and MD simulation. These insights contribute to a deeper understanding of the mechanisms underlying CDK9 and CYP3A4 inhibition, offering potential avenues for the development of innovative and effective CDK9 inhibitors.

Compromised CDK12 activity causes dependency on the high activity of O-GlcNAc transferase.

O-GlcNAc transferase (OGT) coordinates with regulators of transcription, including cyclin-dependent kinase 12 (CDK12), the major transcription elongation kinase. Here, we use inhibitor- and knockdown-based strategies to show that co-targeting of OGT and CDK12 is toxic to prostate cancer cells. OGT catalyzes all nucleocytoplasmic O-GlcNAcylation and due to its essentiality in higher eukaryotes, it is not an ideal drug target. Our glycoproteomics-data revealed that short-term CDK12 inhibition induces hyper-O-GlcNAcylation of the spliceosome-machinery in different models of prostate cancer. By integrating our glycoproteomics-, gene essentiality- and clinical-data from CDK12 mutant prostate cancer patients, we identify the non-essential serine-arginine protein kinase 1 (SRPK1) as a synthetic lethal partner with CDK12-inactivation. Both normal and cancer cells become highly sensitive against inhibitors of OGT and SRPK1 if they have lowered activity of CDK12. Inactivating mutations in CDK12 are enriched in aggressive prostate cancer, and we propose that these patients would benefit from therapy targeting the spliceosome.

Predictors of response to venetoclax and therapeutic potential of CDK7 inhibition in multiple myeloma

AbstractVenetoclax, a selective BCL2 inhibitor, has emerged as a promising therapeutic agent for multiple myeloma (MM), particularly in patients harboring the t(11;14) translocation. In this study, we set out to identify markers of sensitivity and resistance to venetoclax in a real-world patient population, aiming to facilitate the development of personalized therapeutic strategies. Through the analysis of RNA-seq data from relapsed/refractory patients treated with venetoclax, either as a single agent or in combination with other drugs, we unveiled a novel 6-gene signature that significantly stratified patients into risk groups for relapse and further validated its clinical relevance in 2 independent clinical and ex vivo data sets. Our analysis also highlighted the negative impact of chromosome 1q gain, which harbors the MCL1 gene, on progression-free survival, even in t(11;14)-positive patients. Encouraged by the well-documented role of MCL1 in resistance to venetoclax in various malignancies and the prognostic importance of the BCL2/MCL1 ratio in our cohort, we explored Cyclin-Dependent Kinase 7 (CDK7) inhibition as a potential strategy to overcome venetoclax resistance. In vitro experiments demonstrated that CRISPR-Cas9–mediated CDK7 depletion led to decreased MCL1 levels, enhancing the sensitivity of MM cells to venetoclax. Moreover, the combination of the CDK7 inhibitor THZ1 with venetoclax markedly induced cell death in venetoclax-resistant MM cells harboring 1q gain, thus offering a rational therapeutic approach, particularly for patients with this aberration. Overall, these findings provide important insights for optimizing venetoclax-based therapeutic strategies in MM.

Abstract A084: CDK12 inhibition drives mitotic catastrophe, triggering genome instability and innate immune response in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited therapies. In our screening using patient-derived organoids, we identified cyclin-dependent kinase 12 (CDK12) as a promising target in a subset of PDAC. Both pharmacological and genetic inhibition of CDK12 exerted potent antitumor activity in vitro and in vivo, inducing double-strand DNA breaks and apoptosis. CDK12 is a member of cyclin-dependent kinases that is involved in gene expression regulations at multiple levels. Recently, CDK12 has gained attention as a therapeutic target in several types of cancers including triple-negative breast cancer and Ewing sarcoma. However, little is known about the impact and possibility of CDK12 inhibition in pancreatic cancer treatment. As the lack of specific inhibitors that selectively target CDK12, studying CDK12 has long been challenging. To overcome this, we used CRISPR-Cas9 mediated genome editing to generate FKBP12F36V (dTAG) knock-in PDAC cell lines, enabling rapid elimination of endogenous CDK12 using a degrader against dTAG (dTAG-13). Quantitative-image-based cytometry and time lapse imaging showed that CDK12 ablation induces mitotic arrest, DNA damage and apoptosis (mitotic catastrophe). Specifically, loss of CDK12 kinase activity in the S phase was sufficient to slow DNA replication speed, leading to spindle assembly checkpoint activation and mitotic catastrophe. Importantly, cells that escaped mitotic death accumulate micronuclei, triggering immune response signaling that is mediated by the cGAS-STING-type I interferon pathway. Our results suggest that CDK12 is a promising target in a subset of PDAC patients, where CDK12 inhibition exerts antitumor activity both in cell autonomous and non-cell autonomous ways through inducing mitotic catastrophe as well as triggering immune response. Citation Format: Dosuke Iwadate, Keisuke Yamamoto, Hiroyuki Kato, Keisuke Tateishi, Mitsuhiro Fujishiro. CDK12 inhibition drives mitotic catastrophe, triggering genome instability and innate immune response in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A084.

Suppression of neuronal CDK9/p53/VDAC signaling provides bioenergetic support and improves post-stroke neuropsychiatric outcomes

Bioenergy decline occurs with reperfusion following acute ischemic stroke. However, the molecular mechanisms that limit energy metabolism and their impact on post-stroke cognitive and emotional complications are still unclear. In the present study, we demonstrate that the p53 transcriptional response is responsible for neuronal adenosine triphosphate (ATP) deficiency and progressively neuropsychiatric disturbances, involving the downregulation of mitochondrial voltage-dependent anion channels (VDACs). Neuronal p53 transactivated the promoter of microRNA-183 (miR-183) cluster, thereby upregulating biogenesis of miR-183-5p (miR-183), miR-96-5p (miR-96), and miR-182-5p. Both miR-183 and miR-96 directly targeted and post-transcriptionally suppressed VDACs. Neuronal ablation of p53 protected against ATP deficiency and neurological deficits, whereas post-stroke rescue of miR-183/VDAC signaling reversed these benefits. Interestingly, cyclin-dependent kinase 9 (CDK9) was found to be enriched in cortical neurons and upregulated the p53-induced transcription of the miR-183 cluster in neurons after ischemia. Post-treatment with the CDK9 inhibitor oroxylin A promoted neuronal ATP production mainly through suppressing the miR-183 cluster/VDAC axis, further improved long-term sensorimotor abilities and spatial memory, and alleviated depressive-like behaviors in mice following stroke. Our findings reveal an intrinsic CDK9/p53/VDAC pathway that drives neuronal bioenergy decline and underlies post-stroke cognitive impairment and depression, thus highlighting the therapeutic potential of oroxylin A for better outcomes.

Therapeutic Path to Triple Knockout: Investigating the Pan-inhibitory Mechanisms of AKT, CDK9, and TNKS2 by a Novel 2-phenylquinazolinone Derivative in Cancer Therapy- An In-silico Investigation Therapy.

Blocking the oncogenic Wnt//β-catenin pathway has of late been investigated as a viable therapeutic approach in the treatment of cancer. This involves the multi-targeting of certain members of the tankyrase-kinase family; Tankyrase 2 (TNKS2), Protein Kinase B (AKT), and Cyclin- Dependent Kinase 9 (CDK9), which propagate the oncogenic Wnt/β-catenin signalling pathway. During a recent investigation, the pharmacological activity of 2-(4-aminophenyl)-7-chloro- 3H-quinazolin-4-one was repurposed to serve as a 'triple-target' inhibitor of TNKS2, AKT and CDK9. Yet, the molecular mechanism that surrounds its multi-targeting activity remains unanswered. As such, this study aims to explore the pan-inhibitory mechanism of 2-(4-aminophenyl)-7-chloro-3H-quinazolin- 4-one towards AKT, CDK9, and TNKS2, using in silico techniques. Results revealed favourable binding affinities of -34.17 kcal/mol, -28.74 kcal/mol, and -27.30 kcal/mol for 2-(4-aminophenyl)-7-chloro-3H-quinazolin-4-one towards TNKS2, CDK9, and AKT, respectively. Pan-inhibitory binding of 2-(4-aminophenyl)-7-chloro-3H-quinazolin-4-one is illustrated by close interaction with specific residues on tankyrase-kinase. Structurally, 2-(4-aminophenyl)-7-chloro- 3H-quinazolin-4-one had an impact on the flexibility, solvent-accessible surface area, and stability of all three proteins, which was illustrated by numerous modifications observed in the unbound as well as the bound states of the structures, which evidenced the disruption of their biological function. Prediction of the pharmacokinetics and physicochemical properties of 2-(4-aminophenyl)-7-chloro-3H-quinazolin-4- one further established its inhibitory potential, evidenced by the favourable absorption, metabolism, excretion, and minimal toxicity properties. The following structural insights provide a starting point for understanding the paninhibitory activity of 2-(4-aminophenyl)-7-chloro-3H-quinazolin-4-one. Determining the criticality of the interactions that exist between the pyrimidine ring and catalytic residues could offer insight into the structure-based design of innovative tankyrase-kinase inhibitors with enhanced therapeutic effects.

Uncovering potential CDK9 inhibitors from natural compound databases through docking-based virtual screening and MD simulations.

Cyclin-dependent kinase 9 (CDK9) plays a significant role in gene regulation and RNA polymerase II transcription under basal and stimulated conditions. The upregulation of transcriptional homeostasis by CDK9 leads to various malignant tumors and therefore acts as a valuable drug target in addressing cancer incidences. Ongoing drug development endeavors targeting CDK9 have yielded numerous clinical candidate molecules currently undergoing investigation as potential CDK9 modulators, though none have yet received Food and Drug Administration (FDA) approval. In this study, we employ in silico approaches including the molecular docking and molecular dynamics simulations for the virtual screening over the natural compounds library to identify novel promising selective CDK9 inhibitors. The compounds derived from the initial virtual screening were subsequently employed for molecular dynamics simulations and binding free energy calculations to study the compound's stability under virtual physiological conditions. The first-generation CDK inhibitor Flavopiridol was used as a reference to compare with our novel hit compound as a CDK9 antagonist. The 500-ns molecular dynamics simulation and binding free energy calculation showed that two natural compounds showed better binding affinity and interaction mode with CDK9 receptors over the reference Flavopiridol. They also showed reasonable figures in the predicted absorption, distribution, metabolism, excretion, and toxicity (ADMET) calculations as well as in computational cytotoxicity predictions. Therefore, we anticipate that the proposed scaffolds could contribute to developing potential and selective CDK9 inhibitors subjected to further validations.

Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9.

RNA polymerase II (RNAPII) transcription initiates bidirectionally at many human protein-coding genes. Sense transcription usually dominates and leads to messenger RNA production, whereas antisense transcription rapidly terminates. The basis for this directionality is not fully understood. Here, we show that sense transcriptional initiation is more efficient than in the antisense direction, which establishes initial promoter directionality. After transcription begins, the opposing functions of the endonucleolytic subunit of Integrator, INTS11, and cyclin-dependent kinase 9 (CDK9) maintain directionality. Specifically, INTS11 terminates antisense transcription, whereas sense transcription is protected from INTS11-dependent attenuation by CDK9 activity. Strikingly, INTS11 attenuates transcription in both directions upon CDK9 inhibition, and the engineered recruitment of CDK9 desensitises transcription to INTS11. Therefore, the preferential initiation of sense transcription and the opposing activities of CDK9 and INTS11 explain mammalian promoter directionality.

Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9

RNA polymerase II (RNAPII) transcription initiates bidirectionally at many human protein-coding genes. Sense transcription usually dominates and leads to messenger RNA production, whereas antisense transcription rapidly terminates. The basis for this directionality is not fully understood. Here, we show that sense transcriptional initiation is more efficient than in the antisense direction, which establishes initial promoter directionality. After transcription begins, the opposing functions of the endonucleolytic subunit of Integrator, INTS11, and cyclin-dependent kinase 9 (CDK9) maintain directionality. Specifically, INTS11 terminates antisense transcription, whereas sense transcription is protected from INTS11-dependent attenuation by CDK9 activity. Strikingly, INTS11 attenuates transcription in both directions upon CDK9 inhibition, and the engineered recruitment of CDK9 desensitises transcription to INTS11. Therefore, the preferential initiation of sense transcription and the opposing activities of CDK9 and INTS11 explain mammalian promoter directionality.

Cyclin dependent kinase 9 inhibition reduced programmed death-ligand 1 expression and improved treatment efficacy in hepatocellular carcinoma

The anti-programmed death-ligand 1 (PD-L1) antibody is a standard therapy for advanced hepatocellular carcinoma (HCC). Tumor expression of PD-L1 can be induced upon stimulus. Because cyclin-dependent kinase 9 (CDK9) inhibition reduces the expression of inducible proteins, we explored the influence of CDK9 inhibition on PD-L1 expression in HCC cells. We found that PD-L1 expression was low in HCC cells; however, IFN-γ treatment increased this expression. CDK9 inhibitors AZD4573 and atuveciclib reduced the IFN-γ induced PD-L1 expression in a dose-dependent manner. CDK9 knockdown yielded similar results, but CDK9 overexpression reversed the influence of the CDK9 inhibitors. In the orthotopic mouse model, mice treated with a CDK9 inhibitor and an anti-PD-L1 antibody had significantly smaller tumors and exhibited longer survival than mice treated with either agent. In conclusion, CDK9 inhibition could reduce the expression of PD-L1 in HCC cells. Using both CDK9 inhibitors and anti-PD-L1 antibodies is more effective than using either agent alone.

DIPG-74. TARGETING TRANSCRIPTIONAL DYSREGULATION IN DMG THROUGH CDK9 AND HDAC INHIBITOR COMBINATION THERAPY

Abstract BACKGROUND With no successful treatment options available, diffuse midline glioma (DMG) are fatal brain tumors with a median survival of ∼12 months. DMG are characterized by transcriptional dysregulation induced by the H3K27M driver mutations. Since these histone mutations cannot yet be directly targeted pharmacologically, our goal is to identify druggable targets of aberrant transcription in DMG. Previous studies identified RNA polymerase 2 (Pol2)-targeting cyclin-dependent kinase 9 inhibitors (CDK9i) and histone deacetylase inhibitors (HDACi) as potential DMG therapeutics, and CDK9i and HDACi are under investigation in ongoing glioma clinical trials. This study aims to develop CDK9i+HDACi combination therapies for DMG. METHODS We determined the consequences of CDK9i+HDACi combination treatment in DMG using in vitro dose curve analyses, orthotopic xenograft models, and molecular profiling through immunofluorescence imaging, Western blotting, flow cytometry and RNA-seq analysis. RESULTS We find that CDK9i+HDACi combination therapy synergistically reduces DMG growth in vitro and alters DMG protein post-translational modifications associated with gene regulation by reducing phos-Pol2-Ser2 and increasing histone acetylation. Further RNA-seq analysis identified unique transcriptional changes induced by CDK9i+HDACi, including the downregulation of genes associated with mitochondrial function, cell division, and DNA repair. In line with these gene expression changes, additional studies suggest that CDK9i+HDACi treatment induces DNA damage and cell cycle defects. Finally, preliminary pre-clinical studies using an orthotopic DMG xenograft model reveal that CDK9i and HDACi treatment reduces DMG tumor growth as measured by bioluminescent imaging. CONCLUSIONS Together, these studies suggest that CDK9i and HDACi co-treatment alters DMG protein modifications and gene expression leading to cell cycle defects, DNA damage, and reduced tumor growth. In view of the proposed synergistic role of CDK9i and HDACi in DMG, additional ongoing studies are examining if this combination may represent a novel approach for the treatment of DMG.

MODL-12. REPURPOSING SSRI/SNRI TARGETING NEUROTRANSMITTER SIGNALING TO SENSITIZE DMG TO CDK9 INHIBITORS

Abstract BACKGROUND Diffuse Midline Glioma (DMG) comprise a subset of aggressive pediatric brain tumors with few effective therapies. Cyclin-dependent kinase 9 inhibitors (CDK9i) like zotiraciclib (ZTR) are in clinical trials for adult and pediatric glioma, but resistance frequently develops. In this study, we find that co-treatment with CDK9i and brain penetrant, FDA-approved drugs targeting neurotransmitter signaling alters DMG transcription and reduces DMG growth and tumorigenesis. METHODS We conducted drug screens to assess DMG response to a sub-threshold dose of ZTR combined with a library of ~220 bioactive and FDA-approved compounds. Combinatorial effects of CDK9i+SSRI/SNRI treatment were confirmed through in vitro dose curve studies and in orthotopic DMG xenograft models. We identified downstream targets of CDK9i+SSRI/SNRI treatment through phospho-proteomic profiling, RNAseq analysis, immunofluorescence staining, and Western blotting. RESULTS Our targeted drug screens identified SSRIs (selective serotonin reuptake inhibitors) and SNRIs (serotonin and norepinephrine reuptake inhibitors) that increase DMG sensitivity to a low dose of zotiraciclib (ZTR). Follow-up studies confirm that combinations of structurally distinct CDK9i (ZTR, AZD4573) and SSRI/SNRI (sertraline, duloxetine) synergistically reduce DMG growth in neurospheres, induce the apoptotic marker cleaved caspase 3, and reduce the expression of proliferation markers. Additional proteogenomic analyses define downstream targets of CDK9i+SSRI co-treatment, including differential phosphorylation of chromatin regulators and RNA Polymerase 2 (Pol2)-associated elongation factors and splicing machinery. Further RNAseq studies reveal robust transcriptional changes induced by CDK9i+SSRI treatment characterized by altered expression of developmental and synaptic signaling genes. Finally, pilot pre-clinical studies find that CDK9i+SSRI treatment reduces tumor growth and increases progression free survival in a DMG xenograft model. CONCLUSIONS We have identified synergistic CDK9i+SSRI drug combinations that can be applied to alter malignant gene regulation and reduce DMG growth in vitro and in xenografts. These studies provide a basis for a drug-repurposing strategy to combine SSRI/SNRI and CDK9 inhibitors for DMG treatment.

Discovery of Orally Bioavailable and Potent CDK9 Inhibitors for Targeting Transcription Regulation in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents a highly aggressive and heterogeneous malignancy. Currently, effective therapies for TNBC are very limited and remain a significant unmet clinical need. Targeting the transcription-regulating cyclin-dependent kinase 9 (CDK9) has emerged as a promising avenue for therapeutic treatment of TNBC. Herein, we report the design, synthesis, optimization, and evaluation of a new series of aminopyrazolotriazine compounds as orally bioavailable, potent, and CDK9/2 selectivity-improved inhibitors, enabling efficacious inhibition of TNBC cell growth, as well as notable antitumor effect in TNBC models. The compound C35 demonstrated low-nanomolar potency with substantially improved CDK9/2 selectivity, downregulated the CDK9-downstream targets (e.g., MCL-1), and induced apoptosis in TNBC cell lines. Moreover, with the desired oral bioavailability, oral administration of C35 could significantly suppress the tumor progression in two TNBC mouse models. This study demonstrates that target transcriptional regulation is an effective strategy and holds promising potential as a targeted therapy for the treatment of TNBC.

Discovery of novel CDK9 inhibitor with tridentate ligand: Design, synthesis and biological evaluation

Cyclin-dependent kinase 9 (CDK9) plays a role in transcriptional regulation, which had become an attractive target for discovery of antitumor agent. In this work, beyond traditional CDK9 inhibitor with bidentate ligands in ATP binding domain, a series of novel CDK9 inhibitor with tridentate ligand were designed and synthesized. Surprisingly, this unique tridentate ligand structure endows better CDK9 inhibition selectivity compared to other CDK subtypes, and the lead candidate compound Z4-7a showed effective proliferation inhibition in HCT116 cells with acceptable pharmacokinetic properties. Research on the mechanism indicated that Z4-7a could induce apoptosis in the HCT116 cell line by inhibiting phosphorylation of RNA polymerase II at Ser2, which resulted in the inhibition of apoptosis-related genes and proteins expression. In brief, introduction of tridentate ligand might work as a promising strategy for the development of novel selective CDK9 inhibitor.

Fulvestrant with or without the cyclin-dependent kinase 7 (CDK7) inhibitor samuraciclib in advanced hormone receptor positive (HR+) breast cancer after CDK4/6 inhibition: Phase II SUMIT-BC study.

TPS1126 Background: CDK7 enhances oncogene transcription and upregulates anti-apoptotic genes, accelerates the cell cycle via CDK phosphorylation, and activates estrogen receptors (ERs) that can drive resistance to hormonal therapy in cancer. CDK7 inhibition therefore represents a potential anticancer strategy in cancer. Samuraciclib (CT7001) is a small molecule, ATP competitive, selective oral inhibitor of CDK7 that inhibits these key effects of CDK7. Samuraciclib had a favorable safety profile and showed clinical activity when combined with fulvestrant in a phase I study in patients with HR+/HER2− advanced breast cancer (BC) who had previously been treated with a CDK4/6 inhibitor. Patients with no detectable TP53 mutation in ctDNA at baseline and those without baseline liver metastases appeared to have better outcomes. The instant release capsule formulation of samuraciclib used in this study released large amounts of samuraciclib high in the gastrointestinal (GI) tract, which may have contributed to the observed low grade GI adverse events, which were managed with prophylaxis and counselling (1). Methods: A phase 2 open-label randomized study has been designed to evaluate the efficacy, safety, pharmacokinetics (PK), and quality of Life (QoL) of samuraciclib combined with fulvestrant compared to fulvestrant monotherapy (SUMIT-BC; NCT05963984). Eligible patients (n=60) are ≥18 years, have histologically or cytologically confirmed ER+/HER2− advanced or metastatic BC not amenable to resection or radiotherapy of curative intent, have received an aromatase inhibitor combined with a CDK4/6 inhibitor in the adjuvant or advanced setting, are receiving a luteinizing hormone-releasing hormone agonist if pre/perimenopausal, and have RECIST v1.1 evaluable disease. Prior SERD, mTOR inhibition, or chemotherapy for advanced BC are not permitted. All patients undergo baseline Guardant360 ctDNA analysis to establish TP53 and other mutation status. Patients are randomized 1:1:1 to fulvestrant alone (500 mg IM administered on days 1, 15 and 29 and then monthly), fulvestrant and samuraciclib 240 mg QD, or fulvestrant and samuraciclib 360 mg QD. In preparation for phase 3 development, a novel single tablet formulation of samuraciclib is used, which may enhance GI tolerability. Patients undergo RECIST v1.1 evaluation at baseline, every 8 weeks until week 48, and every 12 weeks thereafter. The primary endpoint is clinical benefit response at 24 weeks. Secondary endpoints are tolerability, progression free survival, overall response rate, duration of response, and PK of fulvestrant and samuraciclib. QoL (Functional Assessment of Cancer Therapy - Breast questionnaire [FACT-B]) and correlations between TP53 mutation and efficacy/safety will be assessed. 1. Coombes et al. Nature Comms 2023. Clinical trial information: NCT05963984 .

The role of cyclin-dependent kinase inhibitor 3 in the proliferation and migration of renal cell carcinoma

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, is over expressed in renal cell carcinoma (RCC). However, the cell biology functions of RCC are not well understood. The present study aimed to verify the ability of CDKN3 to promote the proliferation and migration of RCC through in vitro experiments. Subsequently, the clinical prognostic effects were analyzed using The Cancer Genome Atlas (TCGA; https://www.cancer.gov/) and Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/). The chelators, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), an analogue of the anti-tumor agent, were screened through bioinformatics analysis. The expression of CDKN3 is positively correlated with the IC50 of Dp44mT. In two RCC cell lines, 786-0 and Caki-1, we conducted small interfering RNA (siRNA) knockdown of CDKN3 and overexpression of CDKN3 by transfection plasmid. Subsequently, we administered Dp44mT to examine the resulting alterations in cell proliferation, migration, and apoptosis, thereby elucidating the role of CDKN3 and Dp44mT in these processes. The results of the experiment revealed a positive association between CDKN3 expression and the proliferation of RCC cell lines. Down-regulating CDKN3 significantly increased the apoptosis rate and inhibited cell migration in 786-0 and Caki-1 cells. Furthermore, bioinformatics analysis revealed a high expression of CDKN3 in RCC and a negative association between CDKN3 expression and survival. Gene set enrichment analysis (GSEA) revealed a significant association between high CDKN3 expression and the cell cycle pathway. Furthermore, we identified Dp44mT as a drug highly correlated with CDKN3 through the database. Subsequent addition of Dp44mT resulted in similar findings to those observed upon CDKN3 knockdown. Our findings have important implications for the diagnosis and treatment of CDKN3 in RCC. Additionally, Dp44mT is likely to be a promising candidate for future clinical applications.

CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release

Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.

N6-Methyladenosine modified circ-NAB1 modulates cell cycle and epithelial-mesenchymal transition via CDKN3 in endometrial cancer.

Endometrial cancer (EC) is a common malignant tumor in the female reproductive system. Circular RNAs (circRNAs) and N6-methyladenosine (m6A) modification are widely involved in cancer progression. Nevertheless, the cross-talk between circ-NAB1 and m6A as well as the biological functions of circ-NAB1 in EC remain unclear. Circ-NAB1 was observed to be upregulated in EC tissues and cells by RT-qPCR. MeRIP and RNA pull-down assays were utilized for detecting the m6A modification of circ-NAB1. The interaction between circ-NAB1 and RNAs was also detected. Colony formation, transwell, flow cytometry, and western blot were utilized for measuring EC cell behaviors. Mechanically, we proved the m6A demethylase alkylation repair homolog protein 5 (ALKBH5) can mediate circ-NAB1 expression through an m6A-YTHDF2-dependent manner. Circ-NAB1 overexpression can promote cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) process, and cell cycle through functional assays. Circ-NAB1 knockdown exerts the opposite function on EC cells. Furthermore, we proved that circ-NAB1 can sponge miR-876-3p to upregulate the target gene cyclin-dependent kinase inhibitor 3 (CDKN3) in EC cells. CDKN3 overexpression can reverse the impacts of circ-NAB1 depletion on EC cell behaviors. Collectively, we proved that ALKBH5-mediated m6A modification of circ-NAB1 promoted EMT process and cell cycle in EC via targeting the miR-876-3p/CDKN3 axis.