Cell Cycle Cyclin-dependent Kinases Research Articles

Cryo-EM structure of SKP1-SKP2-CKS1 in complex with CDK2-cyclin A-p27KIP1

p27KIP1 (cyclin-dependent kinase inhibitor 1B, p27) is a member of the CIP/KIP family of CDK (cyclin dependent kinase) regulators that inhibit cell cycle CDKs. p27 phosphorylation by CDK1/2, signals its recruitment to the SCFSKP2 (S-phase kinase associated protein 1 (SKP1)-cullin-SKP2) E3 ubiquitin ligase complex for proteasomal degradation. The nature of p27 binding to SKP2 and CKS1 was revealed by the SKP1-SKP2-CKS1-p27 phosphopeptide crystal structure. Subsequently, a model for the hexameric CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex was proposed by overlaying an independently determined CDK2-cyclin A-p27 structure. Here we describe the experimentally determined structure of the isolated CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex at 3.4 Å global resolution using cryogenic electron microscopy. This structure supports previous analysis in which p27 was found to be structurally dynamic, transitioning from disordered to nascent secondary structure on target binding. We employed 3D variability analysis to further explore the conformational space of the hexameric complex and uncovered a previously unidentified hinge motion centred on CKS1. This flexibility gives rise to open and closed conformations of the hexameric complex that we propose may contribute to p27 regulation by facilitating recognition with SCFSKP2. This 3D variability analysis further informed particle subtraction and local refinement approaches to enhance the local resolution of the complex.

Abstract 1557: Defining modulators of response to samuraciclib, a CDK7 inhibitor for breast cancer

Abstract Breast cancer (BC) is the most prevalent cancer in women worldwide. Despite initial sensitivity to endocrine therapies in hormone receptor positive BC, high mortality is driven by treatment-resistant disease. While several highly effective targeted treatments have been developed, resistance is common in the metastatic setting, highlighting the need for new therapies. Cyclin-dependent kinases (CDKs) have emerged as important targets for cancer treatment due to their integral role in driving the cell cycle. CDK4/6 inhibitors have advanced strongly in ER+ BC and are now becoming standard-of-care. However, resistance is a major problem. To address this, we have targeted CDK7, a master regulator of the cell cycle CDKs and transcription processes, both commonly dysregulated in cancer. CDK7 inhibition selectively inhibits cancer cell growth due to their reliance on aberrant gene expression and/or unregulated cell cycle progression. ICEC0942 (Samuraciclib), discovered in our lab at Imperial College London, has recently progressed to Phase 2 clinical trials. Considering the current clinical development of this and other CDK7 inhibitors (CDK7i), it is important to determine which patients will best respond to Samuraciclib. Towards identifying modulators of response to Samuraciclib, we performed a genome-wide CRISPR/Cas9-knockout screen in MCF7 cells. The mTOR/PI3K pathway was identified as important for response to CDK7i. Knockout of mTOR activators reduced sensitivity to Samuraciclib, while knockout of mTOR repressors increased sensitivity. Next, we studied the role of mutations in the catalytic subunit of PI3K (PIK3CA), which occur in nearly 30% of BC cases, in response to Samuraciclib. We utilized an isogenic panel of MCF10A cell lines, which were either wild-type for PIK3CA or had a heterozygous knock-in of either of the two most common mutations (E545K or H1047R). Cell growth was monitored following long-term (42-day) treatment with Samuraciclib. The drug was washed out (day 15) to determine the role of PIK3CA mutations in drug-induced growth arrest. We find that although Samuraciclib effectively inhibits growth of all cell lines, regardless of PIK3CA mutation status, the longevity of growth arrest post-washout is promoted by activating PIK3CA mutations. Finally, we assessed the role of mTOR and its downstream effectors in Samuraciclib response. Cell proliferation of MCF7 cells treated with Samuraciclib or in combination with mTOR/PI3K/AKT inhibitors was measured via flow cytometry. Samuraciclib treatment arrested cell proliferation; however, proliferation was reverted in combination with an mTOR/PI3K/AKT inhibitor. In conclusion, our findings highlight the importance of an active mTOR/PI3K pathway in promoting response to Samuraciclib. This may help guide patient treatments and development of effective combination therapies. Citation Format: Kaste Jurgaityte, Georgina Sava, Chun Fui Lai, Hailing Fan, Van Nguyen, Laki Buluwela, Simak Ali. Defining modulators of response to samuraciclib, a CDK7 inhibitor for breast 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 1557.

Biolayer Interferometry Assay for Cyclin-Dependent Kinase-Cyclin Association Reveals Diverse Effects of Cdk2 Inhibitors on Cyclin Binding Kinetics.

Cyclin-dependent kinases (CDKs) are key mediators of cell proliferation and have been a subject of oncology drug discovery efforts for over two decades. Several CDK and activator cyclin family members have been implicated in regulating the cell division cycle. While it is thought that there are canonical CDK-cyclin pairing preferences, the extent of selectivity is unclear, and increasing evidence suggests that the cell-cycle CDKs can be activated by a pool of available cyclins. The molecular details of CDK-cyclin specificity are not completely understood despite their importance for understanding cancer cell cycles and for pharmacological inhibition of cancer proliferation. We report here a biolayer interferometry assay that allows for facile quantification of CDK binding interactions with their cyclin activators. We applied this assay to measure the impact of Cdk2 inhibitors on Cyclin A (CycA) association and dissociation kinetics. We found that Type I inhibitors increase the affinity between Cdk2 and CycA by virtue of a slowed cyclin dissociation rate. In contrast, Type II inhibitors and other small-molecule Cdk2 binders have distinct effects on the CycA association and dissociation processes to decrease affinity. We propose that the differential impact of small molecules on the cyclin binding kinetics arises from the plasticity of the Cdk2 active site as the kinase transitions between active, intermediate, and inactive states.

A comprehensive analysis of the role of molecular docking in the development of anticancer agents against the cell cycle CDK enzyme

Cancer is considered one of the most lethal diseases responsible for causing deaths worldwide. Although there have been many breakthroughs in anticancer development, cancer remains the major cause of death globally. In this regard, targeting cancer-causing enzymes is one of the efficient therapeutic strategies. Biological functions like cell cycle, transcription, metabolism, apoptosis, and other depend primarily on cyclin-dependent kinases (CDKs). These enzymes help in the replication of DNA in the normal cell cycle process, and deregulation in the functioning of any CDK can cause abnormal cell growth, which leads to cancer. This review is focused on anticancer drug discovery against cell cycle CDK enzyme using an in silico technique, i.e., molecular docking studies. Molecular docking helps in deciphering the key interactions formed within the inhibitor and the respective enzyme. This concise study provides an overview of the most current in silico research advancements made in the field of anticancer drug discovery. The findings presented in the current review article can help in understanding the nature of inhibitor-target interactions and provide information on the structural and molecular prerequisites for the inhibition of cell cycle CDKs.

Combination of androgen receptor inhibitor enzalutamide with the CDK4/6 inhibitor ribociclib in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) that currently lacks specific therapy options. Thus, chemotherapy continues to be the primary treatment, and developing novel targets is a top clinical focus. The androgen receptor (AR) has emerged as a therapeutic target in a subtype of TNBC, with substantial clinical benefits shown in various clinical studies. Numerous studies have shown that cancer is associated with changes in components of the cell cycle machinery. Although cell cycle cyclin-dependent kinase (CDK) 4/6 inhibitors are successful in the treatment of ER-positive BC, they are not helpful in the treatment of patients with TNBC. We investigated the possibility of combining CDK4/6 inhibitor(ribociclib) with AR inhibitor(enzalutamide) in the AR-positive TNBC cell line. Ribociclib showed an inhibitory effect in TNBC cells. Additionally, we found that enzalutamide reduced cell migration/invasion, clonogenic capacity, cell cycle progression, and cell growth in AR-positive cells. Enzalutamide therapy could increase the cytostatic impact of ribociclib in AR+ TNBC cells. Furthermore, dual inhibition of AR and CDK4/6 demonstrated synergy in an AR+ TNBC model compared to each treatment alone.

Promising Anticancer Activity of Multitarget Cyclin Dependent Kinase Inhibitors against Human Colorectal Carcinoma Cells.

Colorectal cancer (CRC) is the third leading cause of cancer death worldwide, and its incidence is steadily rising in developing nations. Cell cycle aberrations due to deregulation of cyclin dependent kinases (CDKs) and cyclins are common events during colorectal carcinogenesis. Yet, efficacy of multitarget CDK inhibitors as therapeutic agents has not been much explored against CRC. The anticancer potential of multitarget CDK inhibitor riviciclib (also known as P276-00), was investigated against CRC cell lines of varied genetic background. Cytotoxicity of riviciclib - potent CDK1, CDK4 and CDK9-specific inhibitor was evaluated in vitro. Further, its effect on clonogenic potential, cell cycle, apoptosis and transcription was tested using colony forming assay, flow cytometry and western blot analysis, respectively. Also, efficacy of riviciclib in combination with standard chemotherapeutic agents was assessed. Dependency of CRC cells on specific CDKs for their survival was confirmed using siRNA studies. Riviciclib exerted significant cytotoxicity against CRC cells and inhibited their colony forming potential. It induced apoptosis along with inhibition of cell cycle CDKs and cyclins as well as transcriptional CDKs and cyclins. Moreover, dual combination of riviciclib with standard chemotherapeutic drugs exhibited synergism in CRC cells. siRNA studies indicated that CRC cells are dependent on specific CDKs for their survival which are targets of riviciclib. This study provides evidence that multitarget CDK inhibitors can serve as promising therapeutic agents against CRC alone or in combination.

Abstract 1103: CDK4 copy number gain in BRAF V600E-mutated non-small cell lung cancers resistant to dabrafenib plus trametinib

Abstract Background: Since the discovery of the oncogenic mutation B-Raf proto-oncogene, serine threonine kinase (BRAF) V600E, dabrafenib and trametinib (DT) combination therapies have been shown to have great anti-tumor activities against V600E-mutated non-small cell lung cancers (NSCLC). However, the resistant mechanisms for DT therapies are not fully understood. Single nucleotide mutation in KRAS, NRAS, MEK1, which locate in the universal growth pathway RAF-RAS-MEK-ERK, or frameshift stop of PTEN had been reported as acquired resistances previously. We had been aimed to search a new therapeutic target for clinically DT resistant, and have been performed comprehensive cancer drug screening by establishment of the clinically resistant patient's derived cell lines. In the process, we focused on a positive regulator of cell cycle cyclin dependent kinase 4 (CDK4) as a potential role in resistant acquisition. Materials: Two patients' pleural effusion-derived NSCLC cell lines of BRAF V600E-mutant who were clinically resistant for DT therapies (LC-1, LC-6) were established and used for post-DT samples. The same patients' derived tissue samples collected at diagnosis (tLC-1, tLC-6) were used for pre-DT samples as comparison. Encompassing cancer drug screening were performed by MTS assay. Copy number variant (CNV) and single nucleotide variant (SNV) were analyzed by amplicon-captured next generation sequence (NGS). Each mRNA expression was quantified using TaqMan Array of human cancer drug panel. Protein expression were evaluated by western blotting and immunohistochemistry (IHC). As references for protein expression, we used patient's derived cell lines of BRAF K601E-mutant (LC-12), and BRAF V600E-mutant (OY) both of which were DT naive. Results: Patient 1 was treated with DT for 3 months and progressed with pleural effusions. Patient 6 was treated with DT for 11 months and progressed with pleural effusions. In both LC-1 and LC-6, dabrafenib and trametinib showed resistance in each 1microM screening in vitro. Analysis of CNV and SNV pre- and post-treatment in each patient, copy number of CDK4 was significantly gained in the patient 1 after DT therapies. qPCR and western blotting revealed high expression of CDK4 in LC-1 while not remarkable in LC-6, LC-12 and OY. Protein expression assessed by IHC revealed that in pre- and post-DT sample, increased protein expression of CDK4, cyclinD1 phosphorylated-Rb were observed in LC-1, but remarkable change were not observed in the patient 6. Conclusions: We highlight CDK4 copy number gain as a possible clinical resistant mechanism for DT therapies in BRAF V600E-mutated NSCLC. We are currently investigating how CDK4 contribute as the acquired resistance. Citation Format: Noriko Hirai, Takaaki Sasaki, Shinichi Chiba, Yuji Uno, Hidehiro Takei. CDK4 copy number gain in BRAF V600E-mutated non-small cell lung cancers resistant to dabrafenib plus trametinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1103.

CDK9: A Comprehensive Review of Its Biology, and Its Role as a Potential Target for Anti-Cancer Agents.

Cyclin-dependent kinases (CDKs) are proteins pivotal to a wide range of cellular functions, most importantly cell division and transcription, and their dysregulations have been implicated as prominent drivers of tumorigenesis. Besides the well-established role of cell cycle CDKs in cancer, the involvement of transcriptional CDKs has been confirmed more recently. Most cancers overtly employ CDKs that serve as key regulators of transcription (e.g., CDK9) for a continuous production of short-lived gene products that maintain their survival. As such, dysregulation of the CDK9 pathway has been observed in various hematological and solid malignancies, making it a valuable anticancer target. This therapeutic potential has been utilized for the discovery of CDK9 inhibitors, some of which have entered human clinical trials. This review provides a comprehensive discussion on the structure and biology of CDK9, its role in solid and hematological cancers, and an updated review of the available inhibitors currently being investigated in preclinical and clinical settings.

Activity of Combined Androgen Receptor Antagonism and Cell Cycle Inhibition in Androgen Receptor Positive Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype, with a peak recurrence rate within the first few years after diagnosis. Few targeted therapies are available to treat this breast cancer subtype, defined by the lack of estrogen receptor (ER) and progesterone receptor and without amplification of human epidermal growth factor receptor 2 (HER2). Although cell cycle cyclin-dependent kinase (CDK) 4/6 inhibitors are approved for treatment of ER-positive (ER+) breast cancer, they have not proven effective as monotherapy in patients with TNBC. The androgen receptor (AR) has emerged as a therapeutic target in a subset of TNBCs and with significant clinical benefit observed in multiple trials. The purpose of this study was to investigate the preclinical activity of the CDK4/6 inhibitor, abemaciclib, in combination with an agent that targets both androgen biosynthesis and AR activity, seviteronel, using TNBC cell lines expressing high AR, cell line xenografts, and an AR-positive (AR+), androgen-responsive TNBC patient-derived xenograft (PDX). Single-cell RNA sequencing demonstrated heterogeneity in AR levels, even in a highly AR+ cell line, and identified cell cycle pathway activation in ARHigh- versus ARLow-expressing cells. Combination treatment with the cell cycle CDK4/6 inhibitor, abemaciclib, and seviteronel showed synergy in an AR+ TNBC model compared with each drug alone. Although cell cycle inhibitors are FDA approved for use in ER+ breast cancer, our studies suggest that they may also be effective in AR+ TNBC, perhaps combined with AR-targeted agents.

MTBP phosphorylation controls DNA replication origin firing

Faithful genome duplication requires regulation of origin firing to determine loci, timing and efficiency of replisome generation. Established kinase targets for eukaryotic origin firing regulation are the Mcm2-7 helicase, Sld3/Treslin/TICRR and Sld2/RecQL4. We report that metazoan Sld7, MTBP (Mdm2 binding protein), is targeted by at least three kinase pathways. MTBP was phosphorylated at CDK consensus sites by cell cycle cyclin-dependent kinases (CDK) and Cdk8/19-cyclin C. Phospho-mimetic MTBP CDK site mutants, but not non-phosphorylatable mutants, promoted origin firing in human cells. MTBP was also phosphorylated at DNA damage checkpoint kinase consensus sites. Phospho-mimetic mutations at these sites inhibited MTBP’s origin firing capability. Whilst expressing a non-phospho MTBP mutant was insufficient to relieve the suppression of origin firing upon DNA damage, the mutant induced a genome-wide increase of origin firing in unperturbed cells. Our work establishes MTBP as a regulation platform of metazoan origin firing.

Targeting MM at the Nexus between Cell Cycle and Transcriptional Regulation Via CDK7 Inhibition

Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.

Abstract 1771: Targeting oncogenic transcription in prostate cancer with a novel, oral bioavailable, and ultra-selective CDK9 inhibitor

Abstract Castration resistant prostate cancers (CRPCs) lose sensitivity to hormone therapy, but remain dependent on oncogenic transcription programs driven by the androgen receptor (AR) and other oncogenic transcription factors such as MYC. Using small molecule microarrays (SMMs), we screened HEK293 cellular lysates for compounds binding to exogenously expressed ARv7, a mutant splice form of AR that drives castration resistance. Although transcription factors like ARv7 and MYC are considered classically undruggable, SMMs are able to identify small molecule interactors of druggable co-factors and other proteins in complex with the target protein – in this case ARv7. SMM hits were triaged for the ability to selectively inhibit an AR dependent transcriptional reporter, and also for their ability to reduce proliferation in AR dependent tumor cells. From this screen, we identified KI-ARv3, a potent and selective inhibitor of CDK9. CDK9 is a cyclin-dependent kinase (CDK) that functions primarily as a general co-factor in RNA Polymerase II (RNA Pol II) transcription elongation. CDK9 is a well-characterized and important cofactor for AR, MYC, and other oncogenic transcription factors. In prostate cancer, CDK9 has been shown to modulate and be required for AR-specific gene expression. More broadly, transcriptional CDK inhibitors including those selective for CDK9 have shown strong potential as therapeutic agents owing to their ability to selectively downregulate oncogenic transcription programs and target tumors addicted to transcription factors such as AR or MYC. However, as CDK9 also plays a global role in transcription, it is unclear whether there exists a sufficient therapeutic index for clinical benefit. Prior clinical investigation of transcriptional CDK inhibitors has also been confounded by off-target interactions with other kinases and especially other CDKs that also play important roles in transcription and the cell cycle. We found that KI-ARv3 demonstrated excellent selectivity for CDK9 versus other CDKs and kinases, and further optimization of KI-ARv3 resulted in KB-00130742, an oral bioavailable CDK9 inhibitor with a biochemical IC50 of 15nM against CDK9 and greater than 50-fold selectivity for all profiled CDKs and greater than 100-fold selectivity against cell cycle CDKs. Both KI-ARv3 and KB-00130742 exhibited potent anti-tumor activity in CRPC models, as well as other models known to be dependent on MYC-driven transcription. In 22Rv1 CRPC cells, KB-00130742 rapidly downregulated nascent transcription, and preferentially depleted short half-life transcripts and AR driven oncogenic programs. In vivo, oral administration of KB-00130742 was well-tolerated and significantly reduced tumor growth in models of CRPC and leukemia. Overall these data support CDK9 inhibition using KB-00130742 as a therapeutic strategy to target AR dependence in CRPC and oncogenic transcription in other tumor types. Citation Format: André Richters, David Freeman, Christina Lee, Florian Kabinger, Shelby Doyle, Becky Leifer, Peter Mikochik, Sajjeev Jagannathan, Jost Vrabic Koren, Kristen Karlin, Calla M. Olson, Christopher Wilfong, Charles Y. Lin, Doug Saffran, Joseph Vacca, Norbert Bischofberger, Marius Pop, Angela N. Koehler. Targeting oncogenic transcription in prostate cancer with a novel, oral bioavailable, and ultra-selective CDK9 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1771.

Abstract 3710: Combinatorial micro RNA therapy can be used as potential cell cycle inhibitors to treat lung cancer

Abstract Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Among the 18.1 million of total cancer patients last year, 2.1 million of the new cases were of LC worldwide. The poor prognosis, higher incidence, and mortality rate make this disease a devastating type of cancer. Cell cycle inhibition is one of the prominent choices among researchers in cancer treatment. Different cell cycle inhibitors are developed by scientists where the cyclin-dependent kinase (CDK) inhibitors are among the most frequently researched. Just recently, 3 CDK inhibitors received FDA-approval for treatment of specific breast cancer subtypes, but no CDK inhibitors currently exist for LC patient treatment. Thus, there is a need for molecules that regulate cell cycle progression and CDK expression. Micro RNAs (miRNAs) are gene-expression regulatory molecules. Among several miRs, we selected miR-143 and miR-506 to regulate CDK1 and CDK4/6, respectively. qPCR and RNA-sequencing indicated that this combinatorial miR therapy caused downregulation of the respective CDK1, 4, and 6 genes in H358 and H1975 lung cancer cell lines. This RNA inhibition corresponded to protein levels downregulation, as detected by western blot analysis. Further investigation found that this treatment caused significant halt of the cell cycle progression, which is markedly stronger than the commercially available CDK inhibitors, Ribociclib and Flavopiridol. We also identified the combined miR-143 and miR-506 caused significant increase of the apoptotic cell population at both 24 and 48 h post-treatment, as detected by an Annexin V/PI analysis. Interestingly, low or no apoptotic impact was observed in HFL1 normal lung fibroblast cells by the combinatorial miR treatment, indicating a cancer-cell specific activity. Upstream pathway analysis through RNA-sequencing using Ingenuity Pathway Analysis software (IPA; Qiagen) indicated a potential ERK-mediation on the activity of the combinatorial miR treatment. Our analysis of the ERK pathway using MAPK (ERK) inhibitors indicated an ERK-independent cell cycle inhibitory activity of the two miRs. Through further analysis of the RNA-Seq data, we observed a potential inhibition of the cellular migration and invasion activity of the cells, which was confirmed by a cell migration assay. In summary, combinatorial treatment of miR-143 and miR-506 has a significant impact on the cell cycle, overall survival, and migration of LC cells, which still merits further study. Citation Format: A K M Nawshad Hossian. Combinatorial micro RNA therapy can be used as potential cell cycle inhibitors to treat lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3710.

Transcription-Associated Cyclin-Dependent Kinases as Targets and Biomarkers for Cancer Therapy.

Drugs targeting the cell cycle-regulatory cyclin-dependent kinase (CDK) 4 and 6 have been approved for the treatment of hormone receptor-positive breast cancer, and inhibitors targeting other cell-cycle CDKs are currently in clinical trials. Another class of CDKs, the transcription-associated CDKs, including CDK7, CDK8, CDK9, CDK12 and CDK13, are critical regulators of gene expression. Recent evidence suggests several novel functions of these CDKs, including regulation of epigenetic modifications, intronic polyadenylation, DNA-damage responses, and genomic stability. Here, we summarize our current understanding of the transcriptional CDKs, their utility as biomarkers, and their potential as therapeutic targets. SIGNIFICANCE: CDK inhibitors targeting CDK4 and CDK6 have been approved in hormone receptor-positive breast cancer, and inhibitors targeting other cell-cycle CDKs are currently in clinical trials. Several studies now point to potential therapeutic opportunities by inhibiting the transcription-associated CDKs as well as therapeutic vulnerabilities with PARP inhibitors and immunotherapy in tumors deficient in these CDKs.

Novel CDKs inhibitors for the treatment of solid tumour by simultaneously regulating the cell cycle and transcription control

A novel series of cyclin-dependent kinases (CDKs) inhibitors, which play critical roles in the cell cycle control and regulation of cell transcription, were synthesised. A systematic study of enzymatic and cellular assays led to the identification of compound X22 with a nanomolar potency against CDK4 and CDK9 and potent antiproliferative activities against a panel of tumour cell lines. X22 could induce cell cycle arrest and cell apoptosis in cancer cell lines. X22 dose-dependently inhibits signalling pathways downstream of CDKs in cancer cells. In vivo antitumor activity assays, oral administration of X22 led to significant tumour regression in mouse model without obvious toxicity. Superior anti-cancer efficacy in vitro and in vivo of X22 demonstrated combined depletion of cell cycle and transcriptional CDK all contributed to antitumor activity. Taken together, concomitant inhibition of cell cycle and transcriptional CDK activities provided valuable guide for further structural optimisation.

Pan-CDK inhibition augments cisplatin lethality in nasopharyngeal carcinoma cell lines and xenograft models

In addition to their canonical roles in regulating cell cycle transition and transcription, cyclin-dependent kinases (CDKs) have been shown to coordinate DNA damage response pathways, suggesting a rational pairing of CDK inhibitors with genotoxic chemotherapeutic agents in the treatment of human malignancies. Here, we report that roniciclib (BAY1000394), a potent pan-CDK inhibitor, displays promising anti-neoplastic activity as a single agent and potentiates cisplatin lethality in preclinical nasopharyngeal carcinoma (NPC) models. Proliferation of the NPC cell lines HONE-1, CNE-2, C666-1, and HK-1 was effectively curbed by roniciclib treatment, with IC50 values between 11 and 38 nmol/L. These anticancer effects were mediated by pleiotropic mechanisms consistent with successful blockade of cell cycle CDKs 1, 2, 3, and 4 and transcriptional CDKs 7 and 9, ultimately resulting in arrest at G1/S and G2/M, downregulation of the transcriptional apparatus, and repression of anti-apoptotic proteins. Considerably enhanced tumor cell apoptosis was achieved following combined treatment with 10 nmol/L roniciclib and 2.0 μmol/L cisplatin; this combination therapy achieved a response over 250% greater than either drug alone. Although roniciclib chemosensitized NPC cells to cisplatin, it did not sensitize untransformed (NP69) cells. The administration of 0.5 mg/kg roniciclib to BALB/c xenograft mice was well tolerated and effectively restrained tumor growth comparable to treatment with 6 mg/kg cisplatin, whereas combining these two agents produced far greater tumor suppression than either of the monotherapies. In summary, these data demonstrate that roniciclib has strong anti-NPC activity and synergizes with cisplatin chemotherapy at clinically relevant doses, thus justifying further evaluation of this combinatorial approach in clinical settings.

Abstract 2352: Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells

Abstract Background. Cell cycle is regulated by cyclin-dependent kinases (CDKs) activity, whose deregulation can lead to uncontrolled proliferation and cancer. Other CDKs are engaged in the regulation of transcription and post-transcriptional mRNA processing through the phosphorylation of the C-terminal domain of RNA polymerase II, such as CDK9 and CDK12. Inhibitors of cell cycle CDKs have been developed as anticancer agents and some of them are under clinical validation (e.g. palbociclib). While recent data would suggest that inhibition of CDK9 is feasible and has antitumor effect, the data on the therapeutic role of CDK12 inhibition are very scanty. Methods. Ovarian cancer cell lines were maintained in RPMI medium supplemented with 5% glutamine and 10% FBS. Cells were treated with different drug concentrations and after 72 hours cell survival was evaluated by MTS assay (Promega). IC50 values were calculated by interpolation method. Cell cycle analysis and apoptosis were performed with standard flow cytometric methods. A2780 and SKOV3 ovarian cancer cell lines knocked out for CDK12 were generated with CRISPR/CAS9 genome editing tool. Results. The cytotoxicity of palbociclib (a CDK4/6 inhibitor) and LDC000067 (a CDK9 inhibitor) was tested in a panel of ovarian cancer cell lines (A2780, SKOV3, OVCAR3, OVCAR5, OVCAR8, OVCA432, OVCA433, IGROV1, EFO27). Sensitivity of cells was similar for palbociclib and LDC000067, ranging from 10 to 33 and from 8 to 60 μM, respectively. A preferential G1 block was observed with palbociclib, while LDC000067 caused a S-G2 block. A higher induction of apoptosis was observed after LDC000067 than after palbociclib treatment in both A2780 and SKOV3. The palbociclib-induced G1 block was associated with decreased Rb phosphorylation, while no modulation of the Ser2 in the carboxyterminal domain of RNA polymerase II was observed after LDC000067 treatment. We generated CDK12 knocked out cells transfecting CRISPR/CAS9 engineered plasmid in both A2780 and SKOV3 ovarian cancer lines. The biological and pharmacological characterization of these clones is under study. Conclusions. Palbociclib and LDC000067 showed a dose dependent cytotoxic effect in the panel of ovarian cancer cell lines tested and were active in the μM range. Preliminary data of treatment induced cell cycle perturbation and apoptosis suggest that the two drugs behave in a different manner and have distinct molecular effects on cells. Citation Format: Rosaria Chilà, Nicolò Panini, Eugenio Erba, Giovanna Damia, Massimo Broggini. Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2352. doi:10.1158/1538-7445.AM2017-2352

JNKs function as CDK4-activating kinases by phosphorylating CDK4 and p21

Cyclin D-CDK4/6 are the first cyclin-dependent kinase (CDK) complexes to be activated by mitogenic/oncogenic pathways. They have a central role in the cell multiplication decision and in its deregulation in cancer cells. We identified T172 phosphorylation of CDK4 rather than cyclin D accumulation as the distinctly regulated step determining CDK4 activation. This finding challenges the view that the only identified metazoan CDK-activating kinase, cyclin H-CDK7-Mat1 (CAK), which is constitutively active, is responsible for the activating phosphorylation of all cell cycle CDKs. We previously showed that T172 phosphorylation of CDK4 is conditioned by an adjacent proline (P173), which is not present in CDK6 and CDK1/2. Although CDK7 activity was recently shown to be required for CDK4 activation, we proposed that proline-directed kinases might specifically initiate the activation of CDK4. Here, we report that JNKs, but not ERK1/2 or CAK, can be direct CDK4-activating kinases for cyclin D-CDK4 complexes that are inactivated by p21-mediated stabilization. JNKs and ERK1/2 also phosphorylated p21 at S130 and T57, which might facilitate CDK7-dependent activation of p21-bound CDK4, however, mutation of these sites did not impair the phosphorylation of CDK4 by JNKs. In two selected tumor cells, two different JNK inhibitors inhibited the phosphorylation and activation of cyclin D1-CDK4-p21 but not the activation of cyclin D3-CDK4 that is mainly associated to p27. Specific inhibition by chemical genetics in MEFs confirmed the involvement of JNK2 in cyclin D1-CDK4 activation. Therefore, JNKs could be activating kinases for cyclin D1-CDK4 bound to p21, by independently phosphorylating both CDK4 and p21.

Inhibitor Selectivity for Cyclin-Dependent Kinase 7: A Structural, Thermodynamic, and Modelling Study.

Deregulation of the cell cycle by mechanisms that lead to elevated activities of cyclin-dependent kinases (CDK) is a feature of many human diseases, cancer in particular. We identified small-molecule inhibitors that selectively inhibit CDK7, the kinase that phosphorylates cell-cycle CDKs to promote their activities. To investigate the selectivity of these inhibitors we used a combination of structural, biophysical, and modelling approaches. We determined the crystal structures of the CDK7-selective compounds ICEC0942 and ICEC0943 bound to CDK2, and used these to build models of inhibitor binding to CDK7. Molecular dynamics (MD) simulations of inhibitors bound to CDK2 and CDK7 generated possible models of inhibitor binding. To experimentally validate these models, we gathered isothermal titration calorimetry (ITC) binding data for recombinant wild-type and binding site mutants of CDK7 and CDK2. We identified specific residues of CDK7, notably Asp155, that are involved in determining inhibitor selectivity. Our MD simulations also show that the flexibility of the G-rich and activation loops of CDK7 is likely an important determinant of inhibitor specificity similar to CDK2.

Abstract 2821: Characterization of CDK inhibitors in a biochemical assay using a comprehensive panel of human CDK-cyclin complexes

Abstract Members of the family of cyclin dependent kinases (CDKs) have been recognized as pivotal regulators of cell cycle progression for more than 20 years. Concordant to their central role in the control of cell division they have been in the focus of research of proliferation associated diseases ever since, most prominently amongst these cancer. Although initial results obtained from first and second generation, low specificity CDK inhibitors (e.g. Flavopyridol, Roscovitine, Dinaciclib, AT7519, R547) have been sobering the recent approval of the first CDK-inhibitor Palbociclib for the treatment of certain forms of breast cancer clearly demonstrates the suitability of cell cycle CDKs as targets in oncology. Furthermore, in addition to cell cycle CDKs a second group of CDKs have been shown to have important roles in the regulation of gene transcription, and several of the “transcriptional” CDKs have become interesting targets in oncology. Recent results underline the notion that for being effective in the treatment of cancer, CDK inhibition requires very high specificity towards the respective target CDK(s). For example CDK1 knockdown or CDK9 inhibition have been shown to be synthetically lethal in combination with MYC overexpression. Selectivity of compounds within the family of CDKs could so far only be tested using a quite limited number of CDK-Cyclin complexes expressed in human cells. To date there are 20 CDK genes and at least 17 different Cyclin genes described, many of which give rise to different variants, e.g. there are 3 D-type cyclins, two A- and E-type cyclins etc.. Experimental data indicates that at least 50-60 different, biologically relevant CDK-Cyclin complexes may exist, but only a limited number of these are available for biochemical testing of drug candidates so far. We have recombinantly expressed and purified 28 different CDK-Cyclin complexes, covering a significant part of the CDK family, and established in-vitro kinase-activity assays for these recombinant enzymes. The resulting CDK panel represents the most comprehensive array for biochemical testing of this enzyme group currently available. We characterized the specificity of several CDK inhibitors that have been or are currently in preclinical or clinical development with this CDK collection. Results will be presented showing the specificity of these inhibitors not only for CDKs but also for CDKs complexed to different Cyclins. In several cases we could detect signifcant differences in the inhibition of the same CDK complexed to different Cyclins, e.g. a 10fold difference was seen for CDK6 complexes with Cyclin D1-3. A >100 fold difference was detected for CDK3 complexed to either Cyclin E1 or Cyclin C. This screening panel allows generating data on compound selectivity early in development, diminishing the risk of designing a compound with suboptimal target specificity. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Christian Beisenherz-Huss, Diane Kraemer, Carolin Heidemann-Dinger, Constance Ketterer, Chris Eckert, Michael H.G. Kubbutat. Characterization of CDK inhibitors in a biochemical assay using a comprehensive panel of human CDK-cyclin complexes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2821.