Cyclin-dependent Kinase Family Research Articles

Advances in Structural Types and Pharmacochemistry of CDK12 Inhibitors.

Cyclin-Dependent Kinase (CDK) 12 is a member of the 20-membered CDK family (CDK1-20) and plays a vital role in regulating gene transcription, mRNA splicing, translation, cell cycle, and repair of DNA damage. CDK12 is an emerging therapeutic target due to its role in regulating the transcription of DNA Damage Response (DDR) genes in Cyclin-Dependent Kinase (CDK). However, the development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. So far, no CDK12 inhibitors approved by the US FDA have been found, and more novel CDK12 inhibitors have been reported for the treatment of prostate cancer, breast cancer, ovarian cancer, lung adenocarcinoma, stomach cancer, cervical cancer, etc. This review has attempted to summarize the structural characteristics and biological activities of various novel CDK12 inhibitors reported since 2020. Meanwhile, we collated and analyzed the reported CDK12 inhibitors from the perspective of structure, summarized the current clinical application potential of CDK12 inhibitors, and further analyzed their current challenges and future development trends.

CDK5 interacts with MST2 and modulates the Hippo signalling pathway.

MST2 (STK3) is a major upstream kinase in the Hippo signalling pathway, an evolutionary conserved pathway in regulation of organ size, self-renewal and tissue homeostasis. Its downstream effectors are the transcriptional regulators YAP and TAZ. This pathway is regulated by a variety of factors, such as substrate stiffness or cell-cell contacts. Using a yeast two-hybrid screen, we detected a novel interaction between the kinases MST2 and CDK5, which we further confirmed by co-immunoprecipitation experiments. Cyclin-dependent kinase 5 (CDK5) is an unusual member of the family of cyclin-dependent kinases, involved in tumour growth and angiogenesis. Although a link between CDK5 and Hippo has been previously postulated, the mode of action is still elusive. Here, we show that knockdown of CDK5 causes reduced transcriptional activity of YAP and that CDK5 influences the phosphorylation levels of the Hippo upstream kinase LATS1. Moreover, a phosphoproteomics approach revealed that CDK5 interferes with the phosphorylation of DLG5, another upstream kinase, which regulates the Hippo pathway. Hence, CDK5 seems to act as a signalling hub for integrating the Hippo pathway and other signalling cascades. These interactions might have important implications for the use of CDK5 inhibitors, which are already in clinical use for tumour diseases.

Cyclin-dependent kinases (CDKs) are key genes regulating early development of Neptunea arthritica cumingii: evidence from comparative transcriptome and proteome analyses

In this study, we applied comparative transcriptomics and proteomics techniques to systematically investigate the dynamic expression patterns of genes and proteins at various stages of early embryonic development of the gastropod Neptunea arthritica cumingii. Twelve cyclin-dependent kinase (CDKs) genes and five downstream proteins associated with these CDKs were identified. Through techniques such as qRT-PCR, our data elucidate for the first time the regulatory functions of CDK family genes and establish CDKs as a pivotal gene cluster in the early embryonic development of N. cumingii. These findings not only enhance the understanding of molecular developmental biology in N. cumingii and marine gastropods in general but also provide significant insights into the mechanisms involved in early embryonic development in N. cumingii. Furthermore, our results provide theoretical guidance for advancing artificial breeding technology for N. cumingii.

Targeting the cyclin-dependent kinase family in anticancer drug discovery: From computational to experimental studies

Uncontrolled cell proliferation, primarily regulated by cyclin-dependent kinases (CDKs), is a critical driver of cancer progression, with dysregulation of CDKs contributing to various cancer types. CDKs have emerged as well-established targets for cancer therapy; however, traditional drug development methods have often proven to be time-consuming, challenging, and expensive. Recent advancements in CDK inhibitors (CDKIs) have shown immense clinical potential but many first-generation CDKIs face issues of non-selectivity and significant toxicity, limiting their clinical approval. To address these challenges, innovative computational approaches, particularly pharmacophore modeling, have the potential to streamline drug discovery. These methods can guide the selection of small molecules through target-specific structure-activity relationship (SAR) models and chemotypes screening across databases, thereby accelerating the identification of effective CDKIs. This review paper summarizes the latest developments on CDK inhibitors, highlights their structural features, and the methodologies (key databases & software tools) that can provide further suggestions for future drug development.

Comprehensive analysis of the expression and prognosis for cyclin-dependent protein kinase family in osteosarcoma

Background and Objective Cyclin-dependent protein kinases (CDKs) have been suggested as prospective therapeutic targets because they control processes vital to the survival and growth of cancer cells. However, research on the varied CDK expression profiles and prognostic factors in osteosarcoma is still lacking. Methods The osteosarcoma microRNA (GSE65071) and gene expression profiles were retrieved from the Gene Expression Omnibus (GEO) database (GSE42352). A substantial variation in prognosis was discovered in CDKs using the TARGET database. Cytoscape was used to construct the miRNAs-CDKs network, and functional and pathway enrichment analyses were completed. It was looked at how immune checkpoint genes, m6A-related genes, and CDKs interact. Results In patients with osteosarcoma compared to normal samples, CDK1-5, CDK18, CDK16, and CDK17 gene expression levels were considerably greater, whereas CDK7-9, CDK11B, CDK16, and CDK20 gene expression levels were significantly lower. Patients with osteosarcoma who had low CDK3 and 18 gene levels or high CDK6, 9 gene levels were predicted to have a favorable prognosis and a long-life expectancy. Immune checkpoint genes, m6A-related gene expression, and CDKs expression all showed some connection. Finally, a network of crucial CDKs and miRNAs was constructed. Conclusion According to our research, CDK3, 6, 9, and 18 have been identified as possible therapeutic targets for osteosarcoma, and CDKs may have a role in controlling m6A mutations in tumor cells as well as immune checkpoint regulation.

A mathematical treatment of the impact of p53 signalling on the expression of the transcription factor E2F in the CycE/Cdk2 subsystem in cancerous cells

Complex networks of proteins and genes control cell growth until mitosis. These components play crucial roles in determining the characteristics and timing of each reaction in the cell. A family of cyclins and cyclin-dependent kinases regulates the progression of the cell from one stage to the next, while certain inhibitors regulate cell proliferation, significantly impacting this process. We examine the impact of p53 protein production in the cyclin E/cyclin-dependent kinase 2 (CycE/CDK2) subsystem. Although the precise functioning of the p53 protein is not fully understood, it is known to exhibit pulsatile behaviour, triggered in response to DNA damage. Consequently, the expression of the p53 protein within the cell system increases with the severity of DNA damage. Similarly, levels of CycE/CDK2 are substantially elevated in instances of DNA damage.To investigate this subsystem, we have developed a mathematical model of the CycE/CDK2 subsystem, presented as non-linear ordinary differential equations. Our primary aim is to determine the conditions that could theoretically result in tumorigenesis. We conduct a dynamical systems analysis to investigate the presence of stable limit cycles, which indicate the standard operational states of cells. Our investigation reveals the levels of p53 protein at which the cell can correct defects, undergo apoptosis, or enter quiescence, and where the system exhibits a Hopf-bifurcation (limit cycles). This is the novelty of our study, as previous research has primarily focused on numerical simulations.Using MATLAB simulations, we produced various results in response to different expressions and concentrations of the p53 protein, allowing us to assess its influence on malignant cells. By altering the concentration of protein p53, we ascertain the specific cellular circumstances that facilitate its natural growth, self-correction in the presence of defective activity, and entry into a state of quiescence.This investigation is motivated by the significant role of oscillations in driving, controlling, and managing the cellular oversight machinery. These aspects are incorporated into developing a mathematical model that emulates cellular operations. From this research, we establish that E2F is a critical target to mitigate oncogenic activity, achievable by managing the pulsatile behaviour of the inhibitor p53 or inhibiting its production process within the cell machinery.

Investigating the inhibitory potential of natural bioactive compounds against cyclin-dependent kinase 13: virtual high throughput screening and MD simulation studies to target CDK signaling

Cyclin-dependent kinase 13 (CDK13) belongs to the cyclin-dependent kinase (CDK) family that is actively involved in transcription regulation and RNA splicing. CDK13 binds with its partner, cyclin K, to regulate several biological processes. CDK13 and cyclin K complex phosphorylates RNA pol II carboxyl-terminal domain (CTD) at several serine residues, creating transcription elongation. The upregulation of the kinase contributes to tumor growth and cell proliferation, and is highly associated with various cancers, including skin, stomach, and ovarian. Thus, it can be considered an efficient therapeutic target for the development of drugs against cancer. In this work, a virtual high throughput screening (vHTS) of the ZINC library was carried out to elucidate the initial potent compounds. Further, filters were applied to identify the hit compounds among the ∼90,000 compound library. Based on the docking scores and binding affinity, the top 100 hits were elucidated, and they were further narrowed down to 50 compounds based on ADMET and Lipinski’s RO5 filter. Finally, 10 compounds were chosen that showed appreciable biological activity. Among them, ZINC02136558 was selected as a potent lead compound that showed strong interaction with the amino acid residues of active and binding sites of CDK13. Furthermore, the all-atom molecular dynamic simulation was performed at 200 ns to explore the dynamic evolution of the system. Finally, the results showed that the ZINC02136558 may be considered as a potential lead molecule to inhibit CDK13 and implicated in therapeutic management of cancer and associated diseases.

P21/Zbtb18 repress the expression of cKit to regulate the self-renewal of hematopoietic stem cells.

The maintenance of hematopoietic stem cells (HSCs) is a complex process involving numerous cell-extrinsic and -intrinsic regulators. The first member of the cyclin-dependent kinase family of inhibitors to be identified, p21, has been reported to perform a wide range of critical biological functions, including cell cycle regulation, transcription, differentiation, and so on. Given the previous inconsistent results regarding the functions of p21 in HSCs in a p21-knockout mouse model, we employed p21-tdTomato (tdT) mice to further elucidate its role in HSCs during homeostasis. The results showed that p21-tdT+ HSCs exhibited increased self-renewal capacity compared to p21-tdT- HSCs. Zbtb18, a transcriptional repressor, was upregulated in p21-tdT+ HSCs, and its knockdown significantly impaired the reconstitution capability of HSCs. Furthermore, p21 interacted with ZBTB18 to co-repress the expression of cKit in HSCs and thus regulated the self-renewal of HSCs. Our data provide novel insights into the physiological role and mechanisms of p21 in HSCs during homeostasis independent of its conventional role as a cell cycle inhibitor.

Unveiling the impact of CDK8 on tumor progression: mechanisms and therapeutic strategies.

CDK8 is an important member of the cyclin-dependent kinase family associated with transcription and acts as a key "molecular switch" in the Mediator complex. CDK8 regulates gene expression by phosphorylating transcription factors and can control the transcription process through Mediator complex. Previous studies confirmed that CDK8 is an important oncogenic factor, making it a potential tumor biomarker and a promising target for tumor therapy. However, CDK8 has also been confirmed to be a tumor suppressor, indicating that it not only promotes the development of tumors but may also be involved in tumor suppression. Therefore, the dual role of CDK8 in the process of tumor development is worth further exploration and summary. This comprehensive review delves into the intricate involvement of CDK8 in transcription-related processes, as well as its role in signaling pathways related to tumorigenesis, with a focus on its critical part in driving cancer progression.

Abstract 602: Targeting CDK9 with a novel potent and selective inhibitor, YX0798, in CAR-T-relapsed mantle cell lymphoma models

Abstract Introduction: Cyclin-dependent kinase (CDK) family members play critical roles in multiple cellular processes. CDK9 is a central regulator at several stages of PolII transcription, including initiation, elongation, and termination. CDK9 dysfunction resulted in transcriptomic reprogramming and promotion of the development of cancers, including lymphoma and leukemia. We and others reported that transcriptomic reprogramming is associated with disease progression and therapeutic resistance in mantle cell lymphoma (MCL). Targeting CDK9 with its inhibitors AZD4573 and enitociclib is safe and effective for treatment in preclinical MCL models, and they are currently under clinical investigation to assess their efficacy and safety. The data demonstrated that CDK9 is a promising therapeutic target. In this study, we developed a novel CDK9 inhibitor, YX0798, and assessed its potency and safety in preclinical MCL models. Method: Cell viability assays were conducted in a time- and dose-dependent manner to assess the in vitro efficacy of YX0798 as well as AZD4573. Apoptosis assays and western blots were used to determine the mechanism of action in MCL cells. Patient-derived xenograft models from primary MCL patient samples were established in immunodeficient mice and used to determine the therapeutic potential YX0798. Results: Next generation sequencing showed that CDK9 was upregulated and cancer hallmarks MYC-TARGETS-v1 and -v2 were highly enriched in CAR-T-relapsed MCL patients compared to CAR-T-naïve patients. We hypothesized that CDK9 overexpression and activity may greatly contribute to the enrichment of MYC-TARGETS-v1 and -v2 in patients with CAR-T resistance, so targeting CDK9 may overcome that resistance. YX0798 was developed to be a potent and selective CDK9 inhibitor. It markedly inhibited cell viability and induced robust apoptosis in MCL cell lines. As expected, it inhibited CDK9 kinase activity and suppressed the expression of short-lived proteins like c-MYC and MCL-1 that are critical for MCL cell survival and growth. YX0798 at 2 mg/kg for continuous 7 days via pump or at 5 mg/kg daily by oral administration potently inhibited the tumor growth of xenografts derived from a patient with therapeutic relapse to the CAR-T therapy in vivo (P<0.0001). This treatment efficacy was found to be similar at that of AZD4573 but at a higher dosage (15+15 mg/kg, 2 hours split, QW, IP). No adverse effects in mice were observed for any of treatments. Conclusion: These data show that our novel CDK9 inhibitor YX0798 is potent and efficacious in treating aggressive MCL models and overcame CAR-T resistance at a lower dosage than AZD4573. Citation Format: Vivian Jiang, Yu Xue, Hong Kim, Joseph McIntosh, Haiying Chen, Tianci Zhang, Yang Liu, Jia Zhou, Michael Wang. Targeting CDK9 with a novel potent and selective inhibitor, YX0798, in CAR-T-relapsed mantle cell lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 602.

Abstract 3301: Translational research of CDK12/13 inhibitor, CTX-439, informing clinical trial strategy

Abstract Gene dysregulation is a hallmark of cancer, arising from genetic alterations that invariably lead to dysregulated transcriptional programs. This dysregulation renders cancer cells highly dependent on certain gene expression regulators, leading to a potential for novel therapeutic strategies. Consequently, ongoing research aims to develop small molecules targeting RNA transcriptional processes. Cyclin-dependent kinase 12/13 (CDK12/13) is a member of the cyclin-dependent kinase family of serine/threonine protein kinases. CDK12/13 regulates the RNA transcription elongation by phosphorylating RNA polymerase II and promotes the elongation of transcripts, specifically those involved in DNA damage responses, such as BRCA1/2. The inhibition of CDK12/13 is expected to exert a synergistic effect when combined with PARP inhibitors and chemotherapeutic reagents. Therefore, we developed a novel small molecule inhibitor, CTX-439 (former name: CRD-1835439). Additionally, we demonstrated that CTX-439 exhibits a preclinical efficacy in vitro and in vivo [Cancer Res. (2022) 82 (12_Supplement): 5485]. To identify potential strategies for a clinical trial of CTX-439, in the current study, we conducted translational research to elucidate (1) pharmacodynamics (PD) markers to monitor the suppression of CDK12/13 in humans, (2) patient selection biomarkers to determine sensitivity to CTX-439, and (3) combination strategies with other key medications. Firstly, to identify PD markers of CTX-439, we performed a comprehensive analysis employing RNA-seq, Pol II-ChIP-seq, and Poly(A)-seq. We confirmed that CTX-439 induced the usage of intronic polyadenylation sites by inhibiting CDK12, resulting in the production of shortened mRNA. Of these transcripts, PCF11 emerged as a potential PD marker for clinical trials, which was confirmed using qPCR analysis of human blood. Secondly, to clarify the indication of CTX-439, we performed an in vitro survey involving 400 cell lines and 100 PDX-derived organoids (PDXOs) to identify biomarkers displaying sensitivity and insensitivity to CTX-439. Furthermore, a significant anti-tumor effect of CTX-439 was revealed in PDX models that showed sensitivity in the survey. CTX-439 exhibited a synergistic effect in combination with PARP inhibitors both in vitro and in vivo, highlighting its potential for use in combination strategies. Additionally, CTX-439 showed an anti-tumor effect when combined with other chemotherapy and molecular-targeted drugs. Collectively, the results from these translational research studies provide a foundation for developing a clinical trial by clearly defining PD monitoring, patient stratification biomarkers, and combination strategies. These findings highlight the therapeutic potential of CTX-439 as a single-agent and in combination with inhibitors, such as PARP, for the treatment of cancers characterized by transcriptional addiction. Citation Format: Hiroko Yamakawa, Shunsuke Ebara, Akio Mizutani, Misaki Yoshida, Midori Sugiyama, Koji Yamamoto, Daisuke Komura, Miwako Kakiuchi, Shumpei Ishikawa, Kosuke Yusa, Daisuke Morishita. Translational research of CDK12/13 inhibitor, CTX-439, informing clinical trial strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3301.

CDK4/6 inhibitor-induced bone marrow micronuclei might be caused by cell cycle arrest during erythropoiesis

BackgroundA micronucleus test is generally used to evaluate the genotoxic potential of chemicals. Exaggerated erythropoiesis, as occurs following bleeding, may induce an unexpected increase in micronucleus frequency. This false positive result would be typical in a genotoxicity study due to the enhanced progression of the cell cycle that restores decreased blood cells. The cyclin-dependent kinase (CDK) family is known to play an essential role in preventing genomic instability. Conversely, a selective CDK4/6 inhibitor PD0332991, clinically named Palbociclib, is reported to have genotoxic potential, shown by positive results in both in vitro and in vivo micronucleus studies. To clarify the mechanism by which cell cycle arrest induced by a CDK4/6 inhibitor increases micronucleus frequency, we investigated the positive results of the bone marrow micronucleus test conducted with PD0332991.ResultsRats treated with PD0332991 exhibited increased micronucleus frequency in an in vivo bone marrow micronucleus test whereas it was not increased by treatment in human lymphoblastoid TK6 cells. In addition, all other genotoxicity tests including the Ames test and the comet assay showed negative results with PD0332991. Interestingly, PD0332991 treatment led to an increase in erythrocyte size in rats and affected the size distribution of erythrocytes, including the micronucleus. The mean corpuscular volume of reticulocytes (MCVr) in the PD0332991 treatment group was significantly increased compared to that of the vehicle control (83.8 fL in the PD0332991, and 71.6 fL in the vehicle control.). Further, the average micronucleated erythrocytes (MNE) size of the PD0332991 group and vehicle control was 8.2 and 7.3 µm, respectively. In the histogram, the vehicle control showed a monomodal distribution with a peak near 7.3 µm. In contrast, the PD0332991 group showed a bimodal distribution with peaks around 7.5 and 8.5 µm. Micronucleated erythrocytes in the PD0332991 group were significantly larger than those in the vehicle control.These results suggest that the increase in micronucleus frequency induced by the CDK4/6 inhibitor is not due to genotoxicity, but is attributable to disturbance of the cell cycle, differentiation, and enucleation of erythroblasts.ConclusionsIt was suggested that the positive outcome of the in vivo bone marrow micronucleus test resulting from treatment with PD0332991 could not be attributed to its genotoxicity. Further studies to clarify the mechanism of action can contribute to the development of drug candidate compounds lacking intrinsic genotoxic effects.

Ribociclib-induced phototoxicity - the era of new drugs and new toxicities

Introduction. Ribociclib belongs to the family of cyclin-dependent kinases 4/6 inhibitors and it has been approved in 2017 for the treatment of patients with hormone receptor-positive (ER+) and human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer. As a drug that has been used for treatment of breast cancer for only few years its rare side effects are described through different case reports. Skin photosensitivity represents a range of dermatological conditions that are caused or exacerbated by sunlight exposure. Drug-induced photosensitivity can be seen in patients treated with all kinds of oncology treatments, including chemotherapeutic agents, targeted anticancer therapies and immune checkpoint inhibitors. Case Outline. We present a case of a patient with metastatic hormone receptor positive and HER2 negative metastatic breast cancer who developed phototoxic skin changes during the treatment with ribociclib/letrozole. During the treatment she developed mild redness of the skin in a strict photosensitive distribution and rash changed to erythematous confluent plaques on the neck and upper part of the chest, face and forearms. After treatment cessation the skin changes did not reappear. Conclusion. As the observed cutaneous adverse effect subsided after the treatment cessation and did not reappear after the initiation of palbociclib/letrozole treatment, it can be concluded that the adverse effects were the consequence of ribociclib action. Management of cutaneous adverse effects is different depending mostly of grade of cutaneous adverse effect and its severity.

Cancer therapy by cyclin-dependent kinase inhibitors (CDKIs): bench to bedside.

A major characteristic of cancer is dysregulated cell division, which results in aberrant growth of cells. Consequently, medicinal targets that prevent cell division would be useful in the fight against cancer. The primary regulator of proliferation is a complex consisting of cyclin and cyclin-dependent kinases (CDKs). The FDA has granted approval for CDK inhibitors (CDKIs) to treat metastatic hormone receptor-positive breast cancer. Specifically, CDK4/6 CDKIs block the enzyme activity of CDK4 and CDK6. Unfortunately, the majority of first-generation CDK inhibitors, also known as pan-CDK inhibitors because they target multiple CDKs, have not been authorized for clinical use owing to their serious side effects and lack of selection. In contrast to this, significant advancements have been created to permit the use of pan-CDK inhibitors in therapeutic settings. Notably, the toxicity and negative consequences of pan-CDK inhibitors have been lessened in recent years thanks to the emergence of combination therapy tactics. Therefore, pan-CDK inhibitors have renewed promise for clinical use when used in a combination regimen. The members of the CDK family have been reviewed and their primary roles in cell cycle regulation were covered in this review. Next, we provided an overview of the state of studies on CDK inhibitors.

Resistance gene-guided genome mining reveals the roseopurpurins as inhibitors of cyclin-dependent kinases.

With the significant increase in the availability of microbial genome sequences in recent years, resistance gene-guided genome mining has emerged as a powerful approach for identifying natural products with specific bioactivities. Here, we present the use of this approach to reveal the roseopurpurins as potent inhibitors of cyclin-dependent kinases (CDKs), a class of cell cycle regulators implicated in multiple cancers. We identified a biosynthetic gene cluster (BGC) with a putative resistance gene with homology to human CDK2. Using targeted gene disruption and transcription factor overexpression in Aspergillus uvarum, and heterologous expression of the BGC in Aspergillus nidulans, we demonstrated that roseopurpurin C (1) is produced by this cluster and characterized its biosynthesis. We determined the potency, specificity, and mechanism of action of 1 as well as multiple intermediates and shunt products produced from the BGC. We show that 1 inhibits human CDK2 with a Kiapp of 44 nM, demonstrates selectivity for clinically relevant members of the CDK family, and induces G1 cell cycle arrest in HCT116 cells. Structural analysis of 1 complexed with CDK2 revealed the molecular basis of ATP-competitive inhibition.

PFTK1 kinase regulates axogenesis during development via RhoA activation

BackgroundPFTK1/Eip63E is a member of the cyclin-dependent kinases (CDKs) family and plays an important role in normal cell cycle progression. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. We sought to understand the function of Eip63E in the CNS by studying the fly ventral nerve cord during development.ResultsOur results demonstrate that Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. Functional interaction studies performed using the same system identify an interaction between Eip63E and the small GTPase Rho1. Furthermore, deficiency of Eip63E homolog in mice, PFTK1, in a newly generated PFTK1 knockout mice results in increased axonal outgrowth confirming that the developmental defects observed in the fly model are due to defects in axogenesis. Importantly, RhoA phosphorylation and activity are affected by PFTK1 in primary neuronal cultures. We report that GDP-bound inactive RhoA is a substrate of PFTK1 and PFTK1 phosphorylation is required for RhoA activity.ConclusionsIn conclusion, our work establishes an unreported neuronal role of PFTK1 in axon development mediated by phosphorylation and activation of GDP-bound RhoA. The results presented add to our understanding of the role of Cdks in the maintenance of RhoA-mediated axon growth and its impact on CNS development and axonal regeneration.

PCTAIRE Protein Kinase 1 (PCTK1) Suppresses Proliferation, Stemness, and Chemoresistance in Colorectal Cancer through the BMPR1B-Smad1/5/8 Signaling Pathway.

Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality worldwide. Even with advances in therapy, CRC mortality remains high. Therefore, there is an urgent need to develop effective therapeutics for CRC. PCTAIRE protein kinase 1 (PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family, and the function of PCTK1 in CRC is poorly understood. In this study, we found that patients with elevated PCTK1 levels had a better overall survival rate in CRC based on the TCGA dataset. Functional analysis also showed that PCTK1 suppressed cancer stemness and cell proliferation by using PCTK1 knockdown (PCTK1-KD) or knockout (PCTK1-KO) and PCTK1 overexpression (PCTK1-over) CRC cell lines. Furthermore, overexpression of PCTK1 decreased xenograft tumor growth and knockout of PCTK1 significantly increased in vivo tumor growth. Moreover, knockout of PCTK1 was observed to increase the resistance of CRC cells to both irinotecan (CPT-11) alone and in combination with 5-fluorouracil (5-FU). Additionally, the fold change of the anti-apoptotic molecules (Bcl-2 and Bcl-xL) and the proapoptotic molecules (Bax, c-PARP, p53, and c-caspase3) was reflected in the chemoresistance of PCTK1-KO CRC cells. PCTK1 signaling in the regulation of cancer progression and chemoresponse was analyzed using RNA sequencing and gene set enrichment analysis (GSEA). Furthermore, PCTK1 and Bone Morphogenetic Protein Receptor Type 1B (BMPR1B) in CRC tumors were negatively correlated in CRC patients from the Timer2.0 and cBioPortal database. We also found that BMPR1B was negatively correlated with PCTK1 in CRC cells, and BMPR1B expression was upregulated in PCTK1-KO cells and xenograft tumor tissues. Finally, BMPR1B-KD partially reversed cell proliferation, cancer stemness, and chemoresistance in PCTK1-KO cells. Moreover, the nuclear translocation of Smad1/5/8, a downstream molecule of BMPR1B, was increased in PCTK1-KO cells. Pharmacological inhibition of Smad1/5/8 also suppressed the malignant progression of CRC. Taken together, our results indicated that PCTK1 suppresses proliferation and cancer stemness and increases the chemoresponse of CRC through the BMPR1B-Smad1/5/8 signaling pathway.

A multi-omics analysis reveals CLSPN is associated with prognosis, immune microenvironment and drug resistance in cancers

BackgroundImmunotherapy is effective only in limited patients. It is urgent to discover a novel biomarker to predict immune cells infiltration status and immunotherapy response of different cancers. CLSPN has been reported to play a pivotal role in various biological processes. However, a comprehensive analysis of CLSPN in cancers has not been conducted.MethodsTo show the whole picture of CLSPN in cancers, a pan-cancer analysis was conducted in 9125 tumor samples across 33 cancer types by integrating transcriptomic, epigenomic and pharmacogenomics data. Moreover, the role of CLSPN in cancer was validated by CCK-8, EDU, colony formation and flow cytometry in vitro and tumor cell derived xenograft model in vivo.ResultsCLSPN expression was generally upregulated in most cancer types and was significantly associated with prognosis in different tumor samples. Moreover, elevated CLSPN expression was closely correlated with immune cells infiltration, TMB (tumor mutational burden), MSI (microsatellite instability), MMR (mismatch repair), DNA methylation and stemness score across 33 cancer types. Enrichment analysis of functional genes revealed that CLSPN participated in the regulation of numerous signaling pathways involved in cell cycle and inflammatory response. The expression of CLSPN in LUAD patients were further analyzed at the single-cell level. Knockdown CLSPN significantly inhibited cancer cell proliferation and cell cycle related cyclin-dependent kinase (CDK) family and Cyclin family expression in LUAD (lung adenocarcinoma) both in vitro and in vivo experiments. Finally, we conducted structure-based virtual screening by modelling the structure of CHK1 kinase domain and Claspin phosphopeptide complex. The top five hit compounds were screened and validated by molecular docking and Connectivity Map (CMap) analysis.ConclusionOur multi-omics analysis offers a systematic understanding of the roles of CLSPN in pan-cancer and provides a potential target for future cancer treatment.

Large epigenome-wide association study identifies multiple novel differentially methylated CpG sites associated with suicidal thoughts and behaviors in veterans.

The U.S. suicide mortality rate has steadily increased during the past two decades, particularly among military veterans; however, the epigenetic basis of suicidal thoughts and behaviors (STB) remains largely unknown. To address this issue, we conducted an epigenome-wide association study of DNA methylation (DNAm) of peripheral blood samples obtained from 2,712 U.S. military veterans. Three DNAm probes were significantly associated with suicide attempts, surpassing the multiple testing threshold (FDR q-value <0.05), including cg13301722 on chromosome 7, which lies between the genes SLC4A2 and CDK5; cg04724646 in PDE3A; and cg04999352 in RARRES3. cg13301722 was also found to be differentially methylated in the cerebral cortex of suicide decedents in a publicly-available dataset (p = 0.03). Trait enrichment analysis revealed that the CpG sites most strongly associated with STB in the present sample were also associated with smoking, alcohol consumption, maternal smoking, and maternal alcohol consumption, whereas pathway enrichment analysis revealed significant associations with circadian rhythm, adherens junction, insulin secretion, and RAP-1 signaling, each of which was recently associated with suicide attempts in a large, independent genome-wide association study of suicide attempts of veterans. Taken together, the present findings suggest that SLC4A2, CDK5, PDE3A, and RARRES3 may play a role in STB. CDK5, a member of the cyclin-dependent kinase family that is highly expressed in the brain and essential for learning and memory, appears to be a particularly promising candidate worthy of future study; however, additional work is still needed to replicate these finding in independent samples.

Abstract 4989: Discovery and evaluation of ISM6466A, a novel covalent CDK12 inhibitor for the treatment of cancer

Abstract Cyclin-dependent kinase 12 (CDK12) belongs to the cyclin-dependent kinase (CDK) family of serine/threonine protein kinases. The CDK12/cyclin K complex regulates the elongation step of RNA transcription by phosphorylating Ser2 on the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II, and selectively affects the expression of genes, such as BRCA1 and BRCA2, involved in DNA Damage Response (DDR). CDK12 mutations as well as overexpression have been reported in different types of malignancies, making it an attractive cancer target. In this study, a series of covalent CDK12 inhibitors were designed and profiled in biochemical and cellular assays. ISM6466A was identified as a lead compound, demonstrating good in vitro efficacy, ADME properties, safety pharmacology profile (e.g., CYP, hERG), and a reasonable in vivo PK profile. Biochemically, ISM6466A exhibited reasonable selectivity over other CDKs, and inhibited cell proliferation in a panel of tumor cell lines with IC50 values in the nanomolar range (e.g., IC50 of ~100nM in the TNBC and AML cell lines, SUM149PT and MV4-11, respectively). Cell-based assays showed that ISM6466A has a selective inhibitory action for the phosphorylation of Ser2 on the CTD, but not for that of Ser5 or Ser7. Notably, ISM6466A demonstrated excellent in vivo anti-tumor efficacy in the SUM149PT breast cancer CDX model (Tumor Growth Inhibition, TGI, of 114% at 60 mg/kg QD) and in the MV4-11 AML CDX model (TGI of 95% at 30 mg/kg QD), without body weight loss. Data from an endpoint PKPD study in the MV4-11 CDX model revealed that ISM6466A inhibited phosphorylation of Ser2 on the CTD, and also inhibited DDR genes such as BRCA1 and ATR. Results from a 28-day rat DRF study revealed that the compound did not show evident signs of toxicity at the tested doses, suggesting a reasonable safety window for ISM6466A.In summary, the novel, covalent, small-molecule CDK12 inhibitor, ISM6466A, demonstrated excellent preclinical efficacy along with favorable drug-like properties. Our results underscore the therapeutic potential of ISM6466A for the treatment of cancer. Citation Format: Lihua Min, Hongfu Lu, Yihong Zhang, Jianping Wu, Xin Cai, Min Zheng, Hui Cui, Junwen Qiao, Xiao Ding, Sujata Rao, Feng Ren, Alex Zhavoronkov. Discovery and evaluation of ISM6466A, a novel covalent CDK12 inhibitor for the treatment of cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4989.