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Gastric cancer remains one of the most dangerous cancers, bringing suffering and economic burden to people worldwide. Long noncoding RNAs (lncRNAs) exhibit great potentials for targeted therapy of various cancers. In this investigation, we tested mechanisms by which LINC01021 may regulate gastric cancer progression. We collected gastric cancer tissues and procured cell lines to explore the potential factors by which LINC01021 had effects on angiogenesis, invasion, and migration, by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), Transwell assay, and western blot analysis. Relationships among LINC01021, Caudal-type homeobox 2 (CDX2), and KISS1 were validated by dual-luciferase gene reporter, RNA pull-down, and RNA immunoprecipitation assays. Additionally, a murine model was developed to further explore the impact of LINC01021 on tumors in vivo. LINC01021 was upregulated in gastric cancer tissues and cells. LINC01021 regulated KISS1 through CDK2, which promoted phosphorylation and nuclear export in CDX2. Inhibition of LINC01021 suppressed the tumorigenesis of gastric cancer. Further, silencing LINC01021 exerted an inhibitory effect on cancer cell migration, invasion, and angiogenesis by promoting the binding between CDX2 and KISS1, while inhibiting that between CDK2 and CDX2. Taken altogether, high LINC01021 expression in gastric cancer promotes malignant cell migration and angiogenesis by downregulation of KISS1 through CDK2-mediated CDX2 phosphorylation., Graphical Abstract, In this investigation, the mechanisms by which LINC01021 regulates gastric cancer progression were investigated by in vivo and in vitro experiments. It was found that high LINC01021 expression in gastric cancer promoted malignant cell migration and angiogenesis by downregulation of KISS1 through CDK2-mediated CDX2 phosphorylation.

Extracellular vesicles (EVs) are nanosized vesicles that act as snapshots of cellular components and mediate cellular communications, but they may contain cargo contents with undesired effects. We developed a model to improve the effects of endometrium-derived EVs (Endo-EVs) on the porcine embryo attachment in feeder-free culture conditions. Endo-EVs cargo contents were analyzed using conventional and real-time PCR for micro-RNAs, messenger RNAs, and proteomics. Porcine embryos were generated by parthenogenetic electric activation in feeder-free culture conditions supplemented with or without Endo-EVs. The cellular uptake of Endo-EVs was confirmed using the lipophilic dye PKH26. Endo-EVs cargo contained miR-100, miR-132, and miR-155, together with the mRNAs of porcine endogenous retrovirus (PERV) and β-catenin. Targeting PERV with CRISPR/Cas9 resulted in reduced expression of PERV mRNA transcripts and increased miR-155 in the Endo-EVs, and supplementing these in embryos reduced embryo attachment. Supplementing the medium containing Endo-EVs with miR-155 inhibitor significantly improved the embryo attachment with a few outgrowths, while supplementing with Rho-kinase inhibitor (RI, Y-27632) dramatically improved both embryo attachment and outgrowths. Moreover, the expression of miR-100, miR-132, and the mRNA transcripts of BCL2, zinc finger E-box-binding homeobox 1, β-catenin, interferon-γ, protein tyrosine phosphatase non-receptor type 1, PERV, and cyclin-dependent kinase 2 were all increased in embryos supplemented with Endo-EVs + RI compared to those in the control group. Endo-EVs + RI reduced apoptosis and increased the expression of OCT4 and CDX2 and the cell number of embryonic outgrowths. We examined the individual and combined effects of RI compared to those of the miR-155 mimic and found that RI can alleviate the negative effects of the miR-155 mimic on embryo attachment and outgrowths. EVs can improve embryo attachment and the unwanted effects of the de trop cargo contents (miR-155) can be alleviated through anti-apoptotic molecules such as the ROCK inhibitor.

CDK2 forms a complex with cyclin A and cyclin E to promote the progress of cell cycle, but when cyclin A and cyclin E are dissociated from the complex and degraded by the ubiquitin proteasome pathway, the fate of the inactive CDK2 is unclear. In this study, we found that the inactive CDK2 protein was degraded by autophagy-lysosome pathway. In the classic model of G0/G1 phase arrest induced by serum starvation, we found that the mRNA level in CDK2 did not change but the protein level decreased. Subsequently, using PI3K and AKT inhibitors and gene knockout methods, it was found that CDK2 degradation was mediated by the inhibition of PI3Kα/AKT

Engulfment and cell motility 1 (ELMO1) plays a crucial role in the process of migration, chemotaxis, and metastasis of tumor cells. ELMO1 has been implicated in the pathogenesis of various cancers. However, the distinct function of ELMO1 in colorectal cancer (CRC) is unclear. We determined whether ELMO1 affects the oncogenic behavior of CRC cells and investigated its prognostic value in CRC patients.We investigated the impact of ELMO1 on tumor cell behavior using small interference RNA (siRNA) in CRC cell lines, including SW480 and DLD1. The expression of ELMO1 was investigated by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) in cancer tissues and sera obtained from CRC patients.ELMO1 knockdown suppressed tumor cell proliferation in SW480 and DLD1 cells. ELMO1 knockdown-induced apoptosis through up-regulation of caspase-3, -7, and PARP activities and down-regulation of the anti-apoptotic Mcl-1 protein. ELMO1 knockdown-induced cell-cycle arrest by decreasing cyclin D1, cyclin-dependent kinase 2, 4 and 6, and the 25C cell division cycle (CDC25C). ELMO1 knockdown suppressed tumor cell invasion and migration. The expression of E-cadherin was increased, while that of Vimentin and Claudin 1 decreased following ELMO1 knockdown. The phosphorylation levels of PDK1, Akt, and GSK-3β and were down-regulated after ELMO1 knockdown. The expression of ELMO1 was found up-regulated in cancer tissues and sera taken from CRC patients. ELMO1 expression was significantly associated with tumor stage, lymph node metastasis, distant metastases, and poor survival.ELMO1 mediates tumor progression by increasing tumor cell motility and inhibiting apoptosis in human CRC.

Tumors are still one of the main causes of death; therefore, the search for new therapeutic agents that will enable the implementation of effective treatment is a significant challenge for modern pharmacy. One of the important factors contributing to the development of neoplastic diseases is the overexpression of enzymes responsible for the regulation of cell division processes such as cyclin-dependent kinases. Numerous studies and examples of already-developed drugs confirm that isatin is a convenient basis for the development of new groups of inhibitors for this class of enzyme. Therefore, in this work, a new group of potential inhibitors of the CDK2 enzyme, utilizing isatin derivatives and substituted benzoylhydrazines, has been designed based on the application of computational chemistry methods, such as docking and molecular dynamics, and their inhibiting ability was assessed. In the cases of the selected compounds, a synthesis method was developed, and the selected physicochemical properties of the newly synthesized derivatives were estimated. As part of the completed project, new compounds are developed which are potential inhibitors of the CDK2 enzyme.

A new series of 1

3,5,7-Trisubstituted pyrazolo[4,3

Objective To investigate the effect of over-expression of nudix hydrolase 21 (NUDT21) on the proliferation of colon cancer HCT-116 cells and its mechanism. Methods The NUDT21 over-expression plasmid was constructed by Gibson assembly. The colony formation assay and CCK-8 assay were used to detect cell proliferation. The cell cycle of HCT-116 cells was detected by flow cytometry. Western blot was performed to detect the expressions of P53, cyclin-dependent kinase 2 (CDK2), phosphorylated retinoblastoma protein at serine 780 (p-Rb-Ser780), and p-Rb-Ser608. Results The sequencing results showed that the NUDT21 over-expression plasmid was successfully constructed. After the NUDT21 over-expression plasmid was transfected into HCT-116 cells, the expressions of NUDT21 mRNA and protein in the cells were significantly increased. The over-expression of NUDT21 inhibited the proliferation of HCT-116 cells and arrested the cell cycle in G0/G1 phase. The expressions of CDK2, p-Rb-Ser608, and p-Rb-Ser780 proteins decreased while the expression of P53 protein increased. Conclusion Over-expression of NUDT21 inhibits the proliferation of HCT-116 cells by blocking P53/CDK2/Rb signal pathway.

Cyclins and cyclin-dependent kinases (CDKs) regulate the cell cycle, which is important for cell proliferation and development. Cyclins bind to and activate CDKs, which then drive the cell cycle. The expression of cyclins periodically changes throughout the cell cycle, while that of CDKs remains constant. To elucidate the mechanisms underlying the constant expression of CDKs, we search for compounds that alter their expression and discover that the natural product fucoxanthinol downregulates CDK2, 4, and 6 expression. We then develop a method to immobilize a compound with a hydroxyl group onto FG beads® and identify human ribosomal protein uS7 (also known as ribosomal protein S5) as the major fucoxanthinol-binding protein using the beads and mass spectrometry. The knockdown of uS7 induces G1 cell cycle arrest with the downregulation of CDK6 in colon cancer cells. CDK6, but not CDK2 or CDK4, is degraded by the depletion of uS7, and we furthermore find that uS7 directly binds to CDK6. Fucoxanthinol decreases uS7 at the protein level in colon cancer cells. By identifying the binding proteins of a natural product, the present study reveals that ribosomal protein uS7 may contribute to the constant expression of CDK6 via a direct interaction.

Background: Azolopyrimidines are imposed on the arena of drugs treated for cancer. The urgent need to discover new selective anticancer agents, paved the way to explore the antitumor significance of such fused systems. From the synthetic point of view, Microwave facilitated technique for synthesis is very strongly associated with green method in chemistry field. Aim: Our aim is to synthesize bioactive compounds using docking simulation run by MOE program to explore the binding mode of the most active enzyme inhibitor among the target compounds. Method: In addition to the use of conventional heating, the MARS system of CEM utilized for Microwave irradiation that is equipped with a multi-mode platform with a magnetic stirring plate and a rotor that allows the parallel processing of many vessels per batch. All the synthesized compounds were tested for their anticancer activity against hepatic cancer (HepG-2), breast cancer (MCF-7) and colon cancer (HCT-116). Screening against the cancer cell lines was performed, using doxorubicin as a reference drug. Docking studies were conducted using MOE software. Result: A novel series of fluorinated fused-pyrimidine namely, pyrazolopyrimidine, triazolopyrimidine and pyrimidobenzimidazole were designed and synthesized conventionally and under microwave irradiations. The mechanistic pathways as well as the structure of all products were debated and demonstrated based on all possible spectral data. In-vitro examination of the novel prepared derivatives versus the three different human cancer cell lines [hepatic cancer (HepG-2), breast cancer (MCF-7) and colon cancer (HCT-116)] was evaluated to estimate their actual activity. Conclusion: We have developed a simple, facile, and efficient procedure for the formation of new series of azolopyrimidines. All spectra of all products were investigated deliberately to confirm their structures. The anti-cancer activity has been examined against three cancer cell lines e.g. HepG-2, MCF-7 and HCT116. Molecular modeling study was carried out in order to rationalize the in vitro anti-tumor results.

Salivary gland cancers (SGCs) are rare yet aggressive malignancies with significant histological heterogeneity, which has made prediction of prognosis and development of targeted therapies challenging. In majority of patients, local recurrence and/or distant metastasis are common and systemic treatments have minimal impact on survival. Therefore, identification of novel targets for treatment that can also be used as predictors of recurrence for multiple histological subtypes of SGCs is an area of unmet need. In this study, we developed a novel transgenic mouse model of SGC, efficiently recapitulating the major histological subtype (adenocarcinomas of the parotid gland) of human SGC. CDK2 knock out (KO) mice crossed with MMTV-low molecular weight forms of cyclin E (LMW-E) mice generated the transgenic mouse models of SGC, which arise in the parotid region of the salivary gland, similar to the common site of origin seen in human SGCs. To identify the CDK2 independent catalytic partner(s) of LMW-E, we used LMW-E expressing cell lines in mass spectrometric analysis and subsequent biochemical validation in pull down assays. These studies revealed that in the absence of CDK2, LMW-E preferentially binds to CDK5. Molecular targeting of CDK5, using siRNA, resulted in inhibition of cell proliferation of human SGCs overexpressing LMW-E. We also provide clinical evidence of significant association of LMW-E/CDK5 co-expression and decreased recurrence free survival in human SGC. Immunohistochemical analysis of LMW-E and CDK5 in 424 patients representing each of the four major histological subtypes of human salivary cancers (Aci, AdCC, MEC, and SDC) revealed that LMW-E and CDK5 are concordantly (positive/positive or negative/negative) expressed in 70% of these patients. The co-expression of LMW-E/CDK5 (both positive) robustly predicts the likelihood of recurrence, regardless of the histological classification of these tumors. Collectively, our results suggest that CDK5 is a novel and targetable biomarker for the treatment of patients with SGC presenting with LMW-E overexpressing tumors.

Capturing the dynamic processes of biomolecular systems in atomistic detail remains difficult despite recent experimental advances. Although molecular dynamics (MD) techniques enable atomic-level observations, simulations of “slow” biomolecular processes (with timescales longer than submilliseconds) are challenging because of current computer speed limitations. Therefore, we developed a method to accelerate MD simulations by high-frequency ultrasound perturbation. The binding events between the protein CDK2 and its small-molecule inhibitors were nearly undetectable in 100-ns conventional MD, but the method successfully accelerated their slow binding rates by up to 10–20 times. Hypersound-accelerated MD simulations revealed a variety of microscopic kinetic features of the inhibitors on the protein surface, such as the existence of different binding pathways to the active site. Moreover, the simulations allowed the estimation of the corresponding kinetic parameters and exploring other druggable pockets. This method can thus provide deeper insight into the microscopic interactions controlling biomolecular processes., Molecular dynamics (MD) techniques enable atomic-level observations, but simulations of “slow” biomolecular processes are challenging because of current computer speed limitations. Here, the authors develop a method to accelerate MD simulations by high-frequency ultrasound perturbation and reveal binding events between the protein CDK2 and its small-molecule inhibitors.

The hepatitis B virus X protein (HBx) is involved in the process of hepatocellular carcinoma via the activation of various oncogenes. Our previous study indicated that ARBB1 (arrestin beta 1) promotes hepatocellular carcinogenesis (HCC). However, the role of ARRB1 in HBx-related HCC remains unclear. Herein, we identified that ARRB1 was upregulated by HBx in vivo and in vitro. Arrb1 deficiency suppressed HBx-induced hepatocellular carcinogenesis in several mouse models. Furthermore, knockdown of ARRB1 blocked HBx-induced macroautophagic/autophagic flux and disrupted the formation of autophagosomes. ARRB1 interacted with HBx, and the autophagic core protein MAP1LC3/LC3, a scaffolding protein, was essential for complete autophagy. Inhibition of autophagy by 3-methyladenine or interference of ATG5 or ATG7 attenuated HBx-induced cell cycle acceleration and the subsequent proliferative response via the induction of G1/S arrest. The absence of autophagy abolished the phosphorylation of CDK2 and the activity of the CDK2-CCNE1 complex. Our results demonstrate that ARRB1 plays a critical role in HBV-related HCC via modulating autophagy and the CDKN1B-CDK2-CCNE1-E2F1 axis and indicate that ARRB1 may be a potential therapeutic target for HCC.

The major cause of Hepatocellular carcinoma (HCC) is acute or chronic infection caused by hepatotropic viruses and HBV infection is the main cause. UHRF2, a ubiquitin-protein ligase E3, is associated with cancer development. This study aimed to investigate the connection and mechanism between UHRF2 and HBV-associated HCC. The expression of UHRF2 in human HBV-positive HCC tissues and paracancerous tissues was detected by western blot and tissue microarray. The effects of UHRF2 on invasion, migration and proliferation were detected in HBV-positive hepatoma cell lines. Furthermore, western blot, immunofluorescence, Co-immunoprecipitation and ubiquitination assays were used to explore the relationship and mechanism between UHRF2 and HBV-associated HCC. HBV-positive HCC tissues had higher UHRF2 expression levels than adjacent non-tumor tissues. The HBV-positive HCC patients with a low UHRF2 level in cancer tissues had longer overall and recurrence-free survival compared with those with a high UHRF2 level. UHRF2 induced invasion, migration and proliferation in human HBV-positive HCC cell lines HepG2.2.15 and Hep AD38(−). HBx, an encoding protein of HBV, maintained the stability of UHRF2 by blocking the ubiquitination of UHRF2. HBx up-regulated CDK2 expression through ETS1. UHRF2 bound to CDK2 directly and enhanced UHRF2 phosphorylation at serine 643. These results suggest that HBx-ETS1-CDK2-UHRF2 pathway plays an important role in the pathogenesis of HBV-associated HCC and represents new therapeutic targets for human HCC. ChiCTR2000041416.

Targeted protein degradation using heterobifunctional proteolysis-targeting chimera (PROTAC) compounds, which recruit E3 ligase machinery to a target protein, is increasingly becoming an attractive pharmacologic strategy. PROTAC compounds are often developed from existing inhibitors, and assessing selectivity is critical for understanding on-target and off-target degradation. We present here an in-depth kinetic degradation study of the pan-kinase PROTAC, TL12-186, applied to 16 members of the cyclin-dependent kinase (CDK) family. Each CDK family member was endogenously tagged with the 11-amino-acid HiBiT peptide, allowing for live cell luminescent monitoring of degradation. Using this approach, we found striking differences and patterns in kinetic degradation rates, potencies, and Dmax values across the CDK family members. Analysis of the responses revealed that most of the CDKs showed rapid and near complete degradation, yet all cell cycle-associated CDKs (1, 2, 4, and 6) showed multimodal and partial degradation. Further mechanistic investigation of the key cell cycle protein CDK2 was performed and revealed CDK2 PROTAC-dependent degradation in unsynchronized or G1-arrested cells but minimal loss in S or G2/M arrest. The ability of CDK2 to form the PROTAC-mediated ternary complex with CRBN in only G1-arrested cells matched these trends, despite binding of CDK2 to TL12-186 in all phases. These data indicate that target subpopulation degradation can occur, dictated by the formation of the ternary complex. These studies additionally underscore the importance of profiling degradation compounds in cellular systems where complete pathways are intact and target proteins can be characterized in their relevant complexes.

Preeclampsia (PE) is a severe pregnancy-specific complication responsible for a majority of maternal and fetal mortality. The dysfunction of trophoblast cells is known to be associated with the etiology of PE. Moreover, elevated expression of hsa_circ_0001326 was found in patients with PE without elucidating specific mechanisms. Thus, this study aimed to investigate the role of hsa_circ_0001326 in the dysfunction of trophoblast cells in vitro. Human trophoblast SWAN71 cells were used in this study. Cell proliferation, apoptosis and cell cycle were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining, cell counting kit-8 assay, Annexin V/propidium iodide (PI) staining and flow cytometry, respectively. Dual luciferase assay was performed to validate the predicted targets. Additionally, Western blot was conducted for protein detection. The results indicated overexpression (OE) of hsa_circ_0001326 remarkably decreased the viability and proliferation of SWAN71 cells. MiR-186-5p was identified as the target of hsa_circ_0001326. Meanwhile, p27 Kip1 was validated as the target of hsa_miR-186-5p. Moreover, the increased apoptosis and decreased migration induced by hsa_circ_0001326 OE were inhibited by p27 Kip1 knockdown. Hsa_circ_0001326 OE upregulated p27 Kip1 and cleaved caspase3 and downregulated CDK2 and cyclin E1 in cells, while these phenomena were reversed by p27 Kip1 knockdown. In addition, hsa_circ_0001326 OE induced G0/G1 cell cycle arrest was also attenuated in the presence of p27 Kip1 knockdown. Taken together, hsa_circ_0001326 OE contributed to the decreased viability of SWAN71 cells by targeting miR-186-5p via upregulation of p27 Kip1. Our findings were helpful to uncover the pathophysiological process of PE, as well as inspire the development of novel targeted therapy against PE.

Lung cancer remains the leading cause of cancer death worldwide. Late diagnosis, chemoresistance, and metastasis are the main reasons for the high mortality rate of lung cancer. Therefore, the development of other treatments is urgent. Cediranib (CED), a vascular endothelial growth factor receptor (VEGFR) kinase inhibitor, shows promising antitumour activities in various cancers including lung cancer. Here, we explored the effects and the underlying molecular mechanism of CED on non-small-cell lung cancer (NSCLC) cell line A549 cells in vitro. Our results show that CED could inhibit A549 cell proliferation and cloning formation. Meanwhile, G1 phase cell cycle arrest was also found, as featured by the increased proportion of G1 phase cells as well as the reduction of G1 phase relative proteins CDK4/cyclin D1 and CDK2/cyclin E. Moreover, the ratio of LC3-II/LC3-I was elevated significantly in CED-treated groups compared with the controls. Furthermore, the expression of p-Akt, p-P38, p-Erk1/2, and p-mTOR proteins was decreased obviously in the treatment groups. These results suggest that CED could induce apoptosis and G1 phase cell cycle arrest in A549 cells. Meanwhile, CED may induce autophagy through MAPK/Erk1/2 and Akt/mTOR signal pathway in A549 cells.

Pancreatic cancer (PC) is highly malignant and has a high mortality with a 5-year survival rate of less than 8%. As a member of the roundabout immunoglobulin superfamily of proteins, ROBO1 plays an important role in embryogenesis and organogenesis and also inhibits metastasis in PC. Our study was designed to explore whether ROBO1 has effects on the proliferation of PC and its specific mechanism. The expression of ROBO1 was higher in cancer tissues than in matched adjacent tissues by immunohistochemistry (IHC) and qRT-PCR. Low ROBO1 expression is associated with PC progression and poor prognosis. Overexpression of ROBO1 can inhibit the proliferation of PC cells in vitro, and the S phase fraction can also be induced. Further subcutaneous tumor formation in nude mice showed that ROBO1 overexpression can significantly inhibit tumor growth. YY1 was found to directly bind to the promoter region of ROBO1 to promote transcription by a luciferase reporter gene assay, a chromatin immunoprecipitation (ChIP) and an electrophoretic mobility shift assay (EMSA). Mechanistic studies showed that YY1 can inhibit the development of PC by directly regulating ROBO1 via the CCNA2/CDK2 axis. Taken together, our results suggest that ROBO1 may be involved in the development and progression of PC by regulating cell proliferation and shows that ROBO1 may be a novel and promising therapeutic target for PC.

Cyclin-dependent kinase 2 (CDK2) antagonism inhibits clustering of excessive centrosomes at mitosis, causing multipolar cell division and apoptotic death. This is called anaphase catastrophe. To establish induced anaphase catastrophe as a clinically tractable antineoplastic mechanism, induced anaphase catastrophe was explored in different aneuploid cancers after treatment with CYC065 (Cyclacel), a CDK2/9 inhibitor. Antineoplastic activity was studied in preclinical models. CYC065 treatment augmented anaphase catastrophe in diverse cancers including lymphoma, lung, colon, and pancreatic cancers, despite KRAS oncoprotein expression. Anaphase catastrophe was a broadly active antineoplastic mechanism. Reverse phase protein arrays (RPPAs) revealed that along with known CDK2/9 targets, focal adhesion kinase and Src phosphorylation that regulate metastasis were each repressed by CYC065 treatment. Intriguingly, CYC065 treatment decreased lung cancer metastases in in vivo murine models. CYC065 treatment also significantly reduced the rate of lung cancer growth in syngeneic murine and patient-derived xenograft (PDX) models independent of KRAS oncoprotein expression. Immunohistochemistry analysis of CYC065-treated lung cancer PDX models confirmed repression of proteins highlighted by RPPAs, implicating them as indicators of CYC065 antitumor response. Phospho-histone H3 staining detected anaphase catastrophe in CYC065-treated PDXs. Thus, induced anaphase catastrophe after CYC065 treatment can combat aneuploid cancers despite KRAS oncoprotein expression. These findings should guide future trials of this novel CDK2/9 inhibitor in the cancer clinic.

Hepatocellular carcinoma is one of the most fatal cancers, and the majority of patients die within three years. However, a small proportion of patients overcome this fatal disease and survive for more than five years. To determine the molecular characteristics of long-term survivors (survival ≥ 5 years), we analyzed the genomic and clinical data of hepatocellular carcinoma patients from The Cancer Genome Atlas and the International Cancer Genome Consortium databases, and identified molecular features that were strongly associated with the patients' prognosis. Genes involved in the cell cycle were expressed at lower levels in tumor tissues from long-term survivors than those from short-term survivors (survival ≤ 1 years). High levels of positive regulators of the G1/S cell cycle transition (cyclin-dependent kinase 2 [CDK2], CDK4, Cyclin E2 [CCNE2], E2F1, E2F2) were potential markers of poor prognosis. Hepatocellular carcinoma patients with TP53 mutations were mainly belonged to the short-term survivor group. Abemaciclib, an FDA-approved selective inhibitor of CDK4/6, inhibited the cell proliferation and tumor growth of hepatocellular carcinoma cells in vitro and in vivo. Thus, high G1/S transition-related gene levels and TP53 mutations are promising diagnostic biomarkers for short-term survivals, and abemaciclib may be a potential targeted drug for hepatocellular carcinoma.

Despite the increasing number of effective anti-cancer therapies, successful treatment is limited by the development of drug resistance. While the contribution of genetic factors to drug resistance is undeniable, little is known about how drug-sensitive cells first evade drug action to proliferate in drug. Here we track the responses of thousands of single melanoma cells to BRAF inhibitors and show that a subset of cells escapes drug via non-genetic mechanisms within the first three days of treatment. Cells that escape drug rely on ATF4 stress signalling to cycle periodically in drug, experience DNA replication defects leading to DNA damage, and yet out-proliferate other cells over extended treatment. Together, our work reveals just how rapidly melanoma cells can adapt to drug treatment, generating a mutagenesis-prone subpopulation that expands over time., BRAF inhibitors are used to treat late-stage melanoma patients harbouring BRAF mutations. Here the authors track the responses of single melanoma cells to BRAF inhibitors and show that a subset of cells rapidly escapes drug via non-genetic mechanisms and incurs DNA damage.

DNA damage activates cell cycle checkpoint proteins ATR and CHK1 to arrest cell cycle progression, providing time for repair and recovery. Consequently, inhibitors of ATR (ATRi) and CHK1 (CHK1i) enhance damage-induced cell death. Intriguingly, both CHK1i and ATRi alone elicit cytotoxicity in some cell lines. Sensitivity has been attributed to endogenous replications stress, but many more cell lines are sensitive to ATRi than CHK1i. Endogenous activation of the DNA damage response also did not correlate with drug sensitivity. Sensitivity correlated with the appearance of γH2AX, a marker of DNA damage, but without phosphorylation of mitotic markers, contradicting suggestions that the damage is due to premature mitosis. Sensitivity to ATRi has been associated with ATM mutations, but dysfunction in ATM signaling did not correlate with sensitivity. CHK1i and ATRi circumvent replication stress by reactivating stalled replicons, a process requiring a low threshold activity of CDK2. In contrast, γH2AX induced by single agent ATRi and CHK1i requires a high threshold activity CDK2. Hence, phosphorylation of different CDK2 substrates is required for cytotoxicity induced by replication stress plus ATRi/CHK1i as compared to their single agent activity. In summary, sensitivity to ATRi and CHK1i as single agents is elicited by premature hyper-activation of CDK2.

Cyclin-dependent kinases (CDKs) 4/6 inhibitors are a powerful class of therapeutic drugs for the treatment of advanced metastatic breast cancer. However, the currently approved CDK4/6 inhibitors palbociclib, ribociclib and abemaciclib have dose-limiting toxicities that require treatment holidays or reductions to sub-optimal doses, thus limiting full target inhibition. The residual CDK4/6 activity, together with persistent signaling through CDK2/cyclin E are among key resistant mechanisms that can compromise full clinical benefit. RGT-419B is a 3rd generation CDK inhibitor with an optimized kinase activity spectrum that has been discovered employing a Computer Accelerated Rational Design technology platform. RGT-419B has potent sub-nM CDK4 activity with desired degrees of selectivity against kinases such as CDK6, CDK9 and GSK3β to enable full target engagement with an improved safety profile. Furthermore, single digit nM CDK2 kinase activity has been incorporated into the design of RGT-419B to combat Cyclin E/CDK2-driven resistance. In vitro, RGT-419B showed more robust activity against palbociclib-resistant ER+ breast cancer cells than abemaciclib. In ER+ T47D breast cancer cells with overexpression of Cyclin E1, RGT-419B exhibited better antiproliferation activity than either abemaciclib or palbociclib. RGT-419B also demonstrated more durable in vivo tumor growth inhibition when compared with abemaciclib in an ER+ breast cancer xenograft model. The optimized kinase activity spectrum of RGT-419B provides an opportunity to treat ER+ breast cancer patients refractory to the existing CDK4/6 inhibitors, as either a single agent or in combination with other therapies. Citation Format: Zhi Xie, Jing Han, Zhilong Hu, Hu He, Xianqiang Sun, Fei Zhang, Jing Lin, Lili Yao, Xinjuan Wang, Liufeng Mei, Yangyang Liu, Guoyun Zhu, Xi Chen, Xiaotian Zhu, Wenge Zhong. Targeting resistance to current CDK4/6 therapies by RGT-419B, an inhibitor with optimized kinase activity spectrum [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-22.

Background/aim Benzimidazoles are considered potential anticancer candidates. We herein studied the anticancer activity of CCL299, 4-(1H-1,3-benzodiazol-1-yl) benzonitrile. Materials and methods In this in vitro study, we used ATP assays, flow cytometry, western blotting, and caspase-3/7 assays to evaluate the effects of CCL299 on cell proliferation, cell-cycle progression and apoptosis. Results ATP assays showed that CCL299 inhibited cell growth in the hepatoblastoma cell line HepG2 and the cervical cancer cell line HEp-2, without exhibiting cytotoxic effects on non-cancer cells and TIG-1-20 fibroblasts. Flow cytometry, western blotting, and caspase-3/7 assays revealed that CCL299 induced G1-phase cell-cycle arrest followed by apoptosis that was associated with up-regulation of p-p53 (Ser15) and p21 expression and the down-regulation of p-CDK2 (Thr160) expression. Conclusion CCL299 exhibits cytotoxic activity via apoptosis in a subset of cancer cells, and should be considered as a promising anticancer candidate agent.

Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL–positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2–AP1–TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand–induced killing that can potentially be exploited for acute leukemia treatment. Significance: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.

Background N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) RNA editing are two of the most abundant RNA modification events affecting adenosines in mammals. Both these RNA modifications determine mRNA fate and play a pivotal role in tumor development and progression. Results Here, we show that METTL3, upregulated in glioblastoma, methylates ADAR1 mRNA and increases its protein level leading to a pro-tumorigenic mechanism connecting METTL3, YTHDF1, and ADAR1. We show that ADAR1 plays a cancer-promoting role independently of its deaminase activity by binding CDK2 mRNA, underlining the importance of ADARs as essential RNA-binding proteins for cell homeostasis as well as cancer progression. Additionally, we show that ADAR1 knockdown is sufficient to strongly inhibit glioblastoma growth in vivo. Conclusions Hence, our findings underscore METTL3/ADAR1 axis as a novel crucial pathway in cancer progression that connects m6A and A-to-I editing post-transcriptional events.

The serine/threonine protein kinases CDK2 and GSK-3β are key oncotargets in breast cancer cell lines, therefore, in the present study three series of oxindole-benzofuran hybrids were designed and synthesised as dual CDK2/GSK-3β inhibitors targeting breast cancer (5a–g, 7a–h, and 13a–b). The N1-unsubstituted oxindole derivatives, series 5, showed moderate to potent activity on both MCF-7 and T-47D breast cancer cell lines. Compounds 5d–f showed the most potent cytotoxic activity with IC50 of 3.41, 3.45 and 2.27 μM, respectively, on MCF-7 and of 3.82, 4.53 and 7.80 μM, respectively, on T-47D cell lines, in comparison to the used reference standard (staurosporine) IC50 of 4.81 and 4.34 μM, respectively. On the other hand, the N1-substituted oxindole derivatives, series 7 and 13, showed moderate to weak cytotoxic activity on both breast cancer cell lines. CDK2 and GSK-3β enzyme inhibition assay of series 5 revealed that compounds 5d and 5f are showing potent dual CDK2/GSK-3β inhibitory activity with IC50 of 37.77 and 52.75 nM, respectively, on CDK2 and 32.09 and 40.13 nM, respectively, on GSK-3β. The most potent compounds 5d–f caused cell cycle arrest in the G2/M phase in MCF-7 cells inducing cell apoptosis because of the CDK2/GSK-3β inhibition. Molecular docking studies showed that the newly synthesised N1-unsubstituted oxindole hybrids have comparable binding patterns in both CDK2 and GSK-3β. The oxindole ring is accommodated in the hinge region interacting through hydrogen bonding with the backbone CO and NH of the key amino acids Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135, respectively, in GSK-3β. Whereas, in series 7 and 13, the N1-substitutions on the oxindole nucleus hinder the compounds from achieving these key interactions with hinge region amino acids what rationalises their moderate to low anti-proliferative activity.

Cyclin dependent kinase 2 (CDK2) inhibition is a well-established strategy for treating cancer. Different series of novel thiazolone (1, 7-9) together with fused thiazolthione (2-6, and 10) derivatives were designed, then synthesized and evaluated for their biological inhibitory activity against CDK2. Additionally, the cytotoxicity of the new compounds was explored against breast and colon cancer cell lines. The novel thiazolone and the fused thiazolthione derivatives exhibited potent CDK2/cyclin A2 inhibitory effect of an IC50 values ranging 105.39-742.78 nM. Amongst them compounds 4 and 6 revealed highest IC50 of 105.39 and 139.27 nM, respectively. Most compounds showed significant inhibition on both breast cancer and colon cancer cell lines with IC50 range 0.54-5.26 and 0.83-278 µM, respectively. Further investigations involved flow cytometry analysis on MCF-7 cancer cell line for compounds 5 and 7 which resulted in arrest cell-cycle at two phases Pre G1/G2-M and re-enforced apoptosis via activation of caspase-7. Molecular modeling simulation of the designed compounds revealed that they were well fitted into CDK2 active site and their complexes were stabilized through the essential hydrogen bonding. Three dimensional quantitative structure activity relationship (3D QSAR) pharmacophore, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies were also carried out showing proper pharmacokinetic and drug-likeness which aided in the prediction of the structure requirements responsible for the observed antitumor activity.

Xanthorrhizol (XNT), is a bioactive compound found in Curcuma xanthorrhiza Roxb. This study aimed to determine the potential targets of the XNT via computational target fishing method. This compound obeyed Lipinski’s and Veber’s rules where it has a molecular weight (MW) of 218.37 gmol-1, TPSA of 20.23, rotatable bonds (RBN) of 4, hydrogen acceptor and donor ability is 1 respectively. Besides, it also has half-life (HL) values 3.5 h, drug-likeness (DL) value of 0.07, oral bioavailability (OB) of 32.10, and blood–brain barrier permeability (BBB) value of 1.64 indicating its potential as therapeutic drug. Further, 20 potential targets were screened out through PharmMapper and DRAR-CPI servers. Co-expression results derived from GeneMANIA revealed that these targets made connection with a total of 40 genes and have 744 different links. Four genes which were RXRA, RBP4, HSD11B1 and AKR1C1 showed remarkable co-expression and predominantly involved in steroid metabolic process. Furthermore, among these 20 genes, 13 highly expressed genes associated with xenobiotics by cytochrome P450, chemical carcinogenesis and steroid metabolic pathways were identified through gene ontology (GO) and KEGG pathway analysis. In conclusion, XNT is targeting multiple proteins and pathways which may be exploited to shape a network that exerts systematic pharmacological effects.

Necroptosis has emerged as an important mode of programmed cell death (PCD), which can be enhanced by cell cycle arrest. As an alternative destruction mode in cancer therapy, necroptosis induced by chemotherapeutic agents can be a powerful weapon in combating drug-resistant cancers. Herein, two iridium(III) complexes, NecroIr1 and NecroIr2, were synthesized as necroptosis inducers in cisplatin-resistant lung cancer cells (A549R). NecroIr1 and NecroIr2 selectively accumulated in mitochondria, leading to oxidative stress and loss of mitochondrial membrane potential (MMP). Necroptosis induced by the Ir(III) complexes was characterized by the annexin V/propidium iodide costaining assay and western blot, the results of which showed the activation of receptor-interacting serine–threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL). Other necroptotic events, such as extracellular calcium influx and leakage of lactate dehydrogenase (LDH), were observed, thus confirming NecroIr1 and NecroIr2 as necroptosis inducers in A549R cells. Most importantly, NecroIr1 and NecroIr2 interfered with the progression of the cell cycle and caused cell cycle arrest in the G0/G1 phase by the downregulation of cyclin-dependent kinases (CDK1, CDK2, CDK4 and CDK6) and cyclin A2 and D2. The combined effect of necroptosis and cell cycle arrest makes both NecroIr1 and NecroIr2 potential antitumor agents that circumvent drug-resistant cancer cells.

Seliciclib is a cyclin-dependent kinase (CDK) inhibitor that has been assayed in phase II clinical trials as an anticancer agent. This paper describes the synthesis of novel derivatives of seliciclib with improved potency, metabolic stability, aqueous solubility, and anti-proliferative activity. The new derivatives showed a novel CDKs selectivity profile. Replacement of ethyl alcohol at position 2 of purine with dimethylaminopropyl and fluorination of benzyl at position 6 of purine of seliciclib resulted in the formation of a derivative that potently and selectively inhibited CDK9 (26 nM vs. CDK9 and > 60-fold selectivity vs. CDK2/5/7). In comparison to seliciclib, this derivative shows lower metabolic clearance (25% lower in Clint), higher aqueous solubility and is more cytotoxic in androgen-independent prostate cancer cells.

Lung cancer is the leading cause of cancer death in the world and classified into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). As tyrosine kinase inhibitors (TKIs), several triterpenoid saponins can target to epidermal growth factor receptor (EGFR), a widely used molecular therapeutic target, to exhibit remarkable anti-proliferative activities in cancer cells. As one of triterpenoid saponins, 20([Formula: see text])-ginsenoside Rg3 [20([Formula: see text])-Rg3] was confirmed to be an EGFR-TKI in this work. According to the quantitative real-time reverse transcription-PCR (qRT-PCR) and immunoblotting analysis, 20([Formula: see text])-Rg3 was certified to play a key role on EGFR/Ras/Raf/MEK/ERK signal pathway regulation. Our data demonstrated that 20([Formula: see text])-Rg3 might block the cell cycle at the G0/G1 phase by downregulating CDK2, Cyclin A2, and Cyclin E1. Molecular docking suggested that the combination of both hydrophobic and hydrogen-bonding interactions may help stabilizing the 20([Formula: see text])-Rg3-EGFR binding. Furthermore, their binding stability was assessed by molecular dynamics simulation. Taken together, these data provide the evidence that 20([Formula: see text])-Rg3 could prohibit A549 cell proliferation, probably by arresting the cell cycle at the G0/G1 phase via the EGFR/Ras/Raf/MEK/ERK pathway.

Cudrania tricuspidata Bureau (CTB), a species of the Moraceae plant, has been used as a bruise recovery treatment. This study aimed to determine whether the 75 kDa phytoglycoprotein extracted from CTB has a regulatory effect on the proliferation of human colon epithelial cells and the pathological process of inflammatory bowel disease (IBD). We found that CTB glycoprotein significantly induces the proliferation of human colon epithelial HT-29 cells by activating protein kinase C. CTB glycoprotein stimulated the phosphorylation of c-Jun N-terminal kinase and transcription factor nuclear factor-κB, which are responsible for the expression of cell-cycle-related proteins (CDK2, CDK4, cyclin D1 and cyclin E) during its promotion of cell proliferation. Experimental colitis was induced in mice by adding dextran sulfate sodium to their drinking water at a concentration of 4% (W/V) for seven days. We found that CTB glycoprotein ameliorates the pathological process of IBD and lowers the disease activity index score, which was composed of body weight change, diarrhea, and hematochezia in ICR mice treated with dextran sulfate sodium. Hence, we suggest that CTB glycoprotein has the ability to prevent IBD by promoting cell proliferation signaling events via the activation of PKC, JNK and NF-κB in colon epithelial cells.

CDK2 is one of the most important members of Cyclin-dependent kinases. It is a critical modulator of various oncogenic signaling pathways, and its activity is vital for loss of proliferative control during oncogenesis. This work has focused on developing a pharmacophore model for CDK2 inhibitors by using a dataset of known inhibitors as a pre-filter throughout the virtual screening and docking process. Consequently, the best pharmacophore model was made of one hydrogen bond acceptor, and two aromatic ring features with a high correlation value of 0.906. The validation findings proved out that the selected model can be used as a filter to screen new molecules like Enamine kinase hinge region directed library against CDK2. As a result, 69 hits were subjected to molecular docking studies. Eventually, three compounds (5909, 701 and 8397) scored good interaction energy values and strong molecular interactions. Hence, they were identified as leads for novel CDK2 inhibitors as anticancer drugs.

Objective: Natural extracts, including nobiletin, have been reported to enhance the efficacy and sensitivity of chemotherapeutic drugs. However, whether and how nobiletin affects tumor growth and progression in renal cell carcinoma (RCC) are still unclear. Methods: Cell proliferation, cell cycle and apoptosis analyses, colony-formation assays, immunoblotting analysis, and qRT-PCR analysis were performed to investigate how nobiletin affected RCC cell proliferation in vitro. The nude mouse model was used to test the efficacy of nobiletin alone or in combination with palbociclib. Results: Nobiletin inhibited cell proliferation by inducing G1 cell cycle arrest and cell apoptosis in RCC cells. Mechanistically, nobiletin decreased SKP2 protein expression by reducing its transcriptional level. The downregulated SKP2 caused accumulation of its substrates, p27 and p21, which further inhibited the activity of the G1 phase-related protein, CDK2, leading to inhibition of cell proliferation and tumor formation. A higher SKP2 protein level indicated less sensitivity to the CDK4/6 inhibitor, palbociclib. A combination of nobiletin and palbociclib showed a synergistic tumor inhibition in vitro and in an in vivo model. Conclusions: Nobiletin downregulated the SKP2-p21/p27-CDK2 axis to inhibit tumor progression and showed synergistic tumor inhibition effects with the CDK4/6 inhibitor, palbociclib, on RCC, which indicates a potential new therapeutic strategy.

Mechanisms of breast cancer progression and invasion, often involve alteration of hormonal signaling, and upregulation and/or activation of signal transduction pathways that input to cell cycle regulation. Herein, we describe a rationally designed first-in-class novel small molecule inhibitor for targeting oncogenic and hormonal signaling in ER-positive breast cancer. BC-N102 treatment exhibits dose-dependent cytotoxic effects against ER+ breast cancer cell lines. BC-N102 exhibited time course- and dose-dependent cell cycle arrest via downregulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), phosphatidylinositol 3-kinase (PI3K), phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-Akt, CDK2, and CDK4 while increasing p38 mitogen-activated protein kinase (MAPK), and mineralocorticoid receptor (MR) signaling in breast cancer cell line. In addition, we found that BC-N102 suppressed breast cancer tumorigenesis in vivo and prolonged the survival of animals. Our results suggest that the proper application of BC-N102 may be a beneficial chemotherapeutic strategy for ER+ breast cancer patients.

Cyclin Dependent Kinases-2 (CDK2) are members of serine/threonine protein kinases family. They play an important role in the regulation events of the eukaryotic cell division cycle, especially during the G1 to S phase transition. Experimental evidence indicate that excessive expression of CDK2s should cause abnormal cell cycle regulation. Therefore, since a long time, CDK2s have been considered potential therapeutic targets for cancer therapy. In this work, onehundred and forty-nine complexes of inhibitors bound in the CDK2-ATP pocket were submitted to short MD simulations (10ns) and free energy calculation. Comparison with experimental data (Ki, Kd and pIC50) revealed that short simulations are exhaustive to examine the crucial ligand-protein interactions within the complexes. Information collected on MD simulations of protein-ligand complexes has been used to perform a molecular modelling approach that incorporates flexibility into structure-based pharmacophore modelling (Common Hits Approach, CHA). The high number of pharmacophore models resulting from the MD simulation was thus reduced to a few representative groups of pharmacophore models. The performance of the models has been assessed by using the ROC curves analysis. This definitive set of validated pharmacophore models could be used to screen in-house and/or commercial datasets for detection of new CDK-2 inhibitors. We provide the models to all the researchers involved in this field.

Polyploidy is an integral part of development and is associated with cellular stress, aging, and pathological conditions. The endocycle, comprised of successive rounds of G and S phases without mitosis, is widely employed to produce polyploid cells in plants and animals. In Drosophila, maintenance of the endocycle is dependent on E2F-governed oscillations of Cyclin E (CycE)–Cdk2 activity, which is known to be largely regulated at the level of transcription. In this study, we report an additional level of E2F-dependent control of CycE–Cdk2 activity during the endocycle. Genetic experiments revealed that an alternative isoform of Drosophila de2f1, dE2F1b, regulates the expression of the p27CIP/KIP-like Cdk inhibitor Dacapo (Dap). We provide evidence showing that dE2F1b-dependent Dap expression in endocycling tissues is necessary for setting proper CycE–Cdk2 activity. Furthermore, we demonstrate that dE2F1b is required for proliferating cell nuclear antigen expression that establishes a negative feedback loop in S phase. Overall, our study reveals previously unappreciated E2F-dependent regulatory networks that are critical for the periodic transition between G and S phases during the endocycle.

Mg2+ ions are essential for the proper functioning of protein kinases, and their roles in kinase activity have been studied for years. However, recent investigations have shed light on how these me...

Background Induction therapy for acute myeloid leukemia (AML) is an anthracycline-based chemotherapy regimen. However, many patients experience a relapse or exhibit refractory disease (R/R). There is an urgent need for more effective regimens to reverse anthracycline resistance in these patients. Methods In this paper, Twenty-seven R/R AML patients with anthracycline resistance consecutively received chidamide in combination with anthracycline-based regimen as salvage therapy at the Chinese PLA General Hospital. Results Of the 27 patients who had received one course of salvage therapy, 13 achieved a complete response and 1 achieved a partial response. We found that the HDAC3-AKT-P21-CDK2 signaling pathway was significantly upregulated in anthracycline-resistant AML cells compared to non-resistant cells. AML patients with higher levels of HDAC3 had lower event-free survival (EFS) and overall survival (OS) rates. Moreover, anthracycline-resistant AML cells are susceptible to chidamide, a histone deacetylase inhibitor which can inhibit cell proliferation, increase cell apoptosis and induce cell-cycle arrest in a time- and dose-dependent manner. Chidamide increases the sensitivity of anthracycline-resistant cells to anthracycline drugs, and these effects are associated with the inhibition of the HDAC3-AKT-P21-CDK2 signaling pathway. Conclusion Chidamide can increase anthracycline drug sensitivity by inhibiting HDAC3-AKT-P21-CDK2 signaling pathway, thus demonstrating the potential for application.

Deregulated expression of the MYC oncogene is a frequent event during tumorigenesis and generally correlates with aggressive disease and poor prognosis. While MYC is a potent inducer of apoptosis, it often suppresses cellular senescence, which together with apoptosis is an important barrier against tumor development. For this latter function, MYC is dependent on cyclin-dependent kinase 2 (CDK2). Here, we utilized a MYC/BCL-XL-driven mouse model of acute myeloblastic leukemia (AML) to investigate whether pharmacological inhibition of CDK2 can inhibit MYC-driven tumorigenesis through induction of senescence. Purified mouse hematopoietic stem cells transduced with MYC and BCL-XL were transplanted into lethally irradiated mice, leading to the development of massive leukemia and subsequent death 15–17 days after transplantation. Upon disease onset, mice were treated with the selective CDK2 inhibitor CVT2584 or vehicle either by daily intraperitoneal injections or continuous delivery via mini-pumps. CVT2584 treatment delayed disease onset and moderately but significantly improved survival of mice. Flow cytometry revealed a significant decrease in tumor load in the spleen, liver and bone marrow of CVT2584-treated compared to vehicle-treated mice. This was correlated with induced senescence evidenced by reduced cell proliferation, increased senescence-associated β-galactosidase activity and heterochromatin foci, expression of p19ARF and p21CIP1, and reduced phosphorylation (activation) of pRb, while very few apoptotic cells were observed. In addition, phosphorylation of MYC at Ser-62 was decreased. In summary, inhibition of CDK2 delayed MYC/BCL-XL-driven AML linked to senescence induction. Our results suggest that CDK2 is a promising target for pro-senescence cancer therapy, in particular for MYC-driven tumors, including leukemia.

Background/aim Forkhead box O4 (FOXO4) has been demonstrated to be a tumor suppressor and proposed as target for treatment of a variety of cancer types. However, the role of FOXO4 in cholangiocarcinoma (CCA), a dangerous cancer of bile-duct epithelium, has rarely been explored. Materials and methods The proliferative rate of CCA cell lines KKU-213B, KKU-055 and KKK-D068 was investigated using the sulforhodamine B (SRB) assay. Levels of FOXO4, cyclin E1 (CCNE1), CCNE2, cyclin-dependent kinase 2 (CDK2) and cell division cycle 25A (CDC25A) expression were measured using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The cell-cycle profile was explored using flow cytometry. Results The SRB assay demonstrated that KKU-213B expressed low levels of FOXO4 but its proliferative rate was highest of all cell lines tested. Interestingly, ectopic expression of FOXO4 significantly suppressed proliferation of KKU-213B cells. Cell-cycle analysis revealed that the cell population in the G0/G1 phase was significantly higher in FOXO4-transfected KKU-213B cells than in controls. RT-qPCR analysis demonstrated that the levels of expression of genes that play a role in the G1/S transition, namely CCNE1, CCNE2, CDK2 and CDC25A, were significantly lower in FOXO4-transfected KKU-213B cells compared to controls. Conclusion FOXO4 suppressed CCA cell proliferation partly via down-regulating the expression of genes involved in the G1/S transition, leading to G0/G1 arrest. Our findings suggest that induction of FOXO4 expression might be an alternative approach for the treatment of CCA.

Endometrial cancer (EC) is the most common gynecological cancer, and one of the most important causes of cancer‑related deaths in women worldwide. The long‑term survival rate is lower in advanced‑stage and recurrent EC, therefore it is important to identify new anticancer drugs. Garcinol, a polyisoprenylated benzophenone, is a promising anticancer drug for various cancer types but its effects on EC remain unclear. To investigate the anticancer effects of garcinol on EC, cell proliferation and cell cycle were assessed by real‑time cell proliferation, cell counting, and colony formation assays, flow cytometric analysis, and 5‑ethynyl‑2'‑deoxyuridine (EdU) incorporation assay, in EC Ishikawa (ISH) and HEC‑1B cell lines. Western blotting was used to evaluate the expression of cell cycle‑related protein cyclins, cyclin‑dependent kinase and tumor suppression proteins. Garcinol inhibited ISH and HEC‑1B cell proliferation in a dose‑dependent manner, and induced ISH and HEC‑1B cell cycle arrest at the G1 phase and G2/M phase, respectively, and decreased the S phase and DNA synthesis in these two cell lines. Following garcinol treatment the expression levels of p53 and p21 were increased, while the expression levels of CDK2, CDK4, cyclin D1 and cyclin B1 were gradually decreased in a dose‑dependent manner in both ISH and HEC‑1B cells. In addition, the expression levels of phosphorylated c‑JUN N‑terminal kinase (JNK) and p‑c‑JUN were significantly increased in both types of cells. Collectively, garcinol can induce EC cell cycle arrest and may be a promising candidate for EC chemotherapy.

Here, we demonstrated that the multikinase framework including ERK/AKT and CDK2 promotes the proliferation and invasion of lung adenocarcinoma cell lines through activating DRP1. Our results further uncovered the prognostic significance of DRP1 in early‐stage lung adenocarcinoma, showing that the expression and activation of DRP1 are both significantly associated with an increased risk of postoperative recurrence., Recent studies revealed the role of dynamin‐related protein 1 (DRP1), encoded by the DNM1L gene, in regulating the growth of cancer cells of various origins. However, the regulation, function, and clinical significance of DRP1 remain undetermined in lung adenocarcinoma. Our study shows that the expression and activation of DRP1 are significantly correlated with proliferation and disease extent, as well as an increased risk of postoperative recurrence in stage I to stage IIIA lung adenocarcinoma. Loss of DRP1 in lung adenocarcinoma cell lines leads to an altered mitochondrial morphology, fewer copies of mitochondrial DNA, decreased respiratory complexes, and impaired oxidative phosphorylation. Additionally, the proliferation and invasion are both suppressed in DRP1‐depleted lung adenocarcinoma cell lines. Our data further revealed that DRP1 activation through serine 616 phosphorylation is regulated by ERK/AKT and CDK2 in lung adenocarcinoma cell lines. Collectively, we propose the multikinase framework in activating DRP1 in lung adenocarcinoma to promote the malignant properties. Biomarkers related to mitochondrial reprogramming, such as DRP1, can be used to evaluate the risk of postoperative recurrence in early‐stage lung adenocarcinoma.

Cyclin-dependent kinase 2 (CDK2) complex is significantly over-activated in many cancers. While it makes CDK2 an attractive target for cancer therapy, most inhibitors against CDK2 are ATP competitors that are either nonspecific or highly toxic, and typically fail clinical trials. One alternative approach is to develop non-ATP competitive inhibitors; they disrupt interactions between CDK2 and either its partners or substrates, resulting in specific inhibition of CDK2 activities. In this report, we identify two potential druggable pockets located in the protein-protein interaction interface (PPI) between CDK2 and Cyclin A. To target the potential druggable pockets, we perform a LIVS in silico screening of a library containing 1925 FDA approved drugs. Using this approach, homoharringtonine (HHT) shows high affinity to the PPI and strongly disrupts the interaction between CDK2 and cyclins. Further, we demonstrate that HHT induces autophagic degradation of the CDK2 protein via tripartite motif 21 (Trim21) in cancer cells, which is confirmed in a leukemia mouse model and in human primary leukemia cells. These results thus identify an autophagic degradation mechanism of CDK2 protein and provide a potential avenue towards treating CDK2-dependent cancers.

The success of the CDK4/6 inhibitor Ibrance™ (Palbociclib) as an anticancer agent inspired and directed more efforts toward the discovery of selective cyclin-dependent kinase (CDKs) inhibitors. CDK2 is a member of the CDKs family that plays an important role in regulating the progression of cells into both S- and M-phases of the cell cycle. Studies suggest that overexpression of CDK2 may be implicated in tumor growth in cancer.This review covers the patent literature of CDK2 inhibitors published between 2017 and 2021. We searched the online databases of the European Patent Office, American Chemical Society, and Google patents.Developing selective CDK2 inhibitors is challenging due to the absence of a previously approved selective CDK2 inhibitor. However, ongoing efforts by Incyte Corporation and Pfizer Inc., which are reported herein, may stand out as a new starting point and bring novel information critical for the medicinal chemistry and drug design scientists in the field of CDK2 inhibitors' development.

Crossover formation is essential for proper segregation of homologous chromosomes during meiosis. Here, we show that Caenorhabditis elegans cyclin-dependent kinase 2 (CDK-2) partners with cyclin-like protein COSA-1 to promote crossover formation by promoting conversion of meiotic double-strand breaks into crossover–specific recombination intermediates. Further, we identify MutSγ component MSH-5 as a CDK-2 phosphorylation target. MSH-5 has a disordered C-terminal tail that contains 13 potential CDK phosphosites and is required to concentrate crossover–promoting proteins at recombination sites. Phosphorylation of the MSH-5 tail appears dispensable in a wild-type background, but when MutSγ activity is partially compromised, crossover formation and retention of COSA-1 at recombination sites are exquisitely sensitive to phosphosite loss. Our data support a model in which robustness of crossover designation reflects a positive feedback mechanism involving CDK-2–mediated phosphorylation and scaffold-like properties of the MSH5 C-terminal tail, features that combine to promote full recruitment and activity of crossover–promoting complexes.

The coronavirus disease 2019 (COVID-19) pandemic has devastated global health. Identifying key host factors essential for SARS-CoV-2 RNA replication is expected to unravel cellular targets for the development of broad-spectrum antiviral drugs which have been quested for the preparedness of future viral outbreaks. Here, we have identified host proteins that associate with nonstructural protein 12 (nsp12), the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 using a mass spectrometry (MS)-based proteomic approach. Among the candidate factors, CDK2 (Cyclin-dependent kinase 2), a member of cyclin-dependent kinases, interacts with nsp12 and causes its phosphorylation at T20, thus facilitating the assembly of the RdRp complex consisting of nsp12, nsp7 and nsp8 and promoting efficient synthesis of viral RNA. The crucial role of CDK2 in viral RdRp function is further supported by our observation that CDK2 inhibitors potently impair viral RNA synthesis and SARS-CoV-2 infection. Taken together, we have discovered CDK2 as a key host factor of SARS-CoV-2 RdRp complex, thus serving a promising target for the development of SARS-CoV-2 RdRp inhibitors.