2-Anilino-4-(1-methyl-1H-pyrazol-4-yl)pyrimidine-derived CDK2 inhibitors as anticancer agents: Design, synthesis & evaluation.

Deregulation of cyclin-dependent kinase 2 (CDK2) and its activating partners, cyclins A and E, is associated with the pathogenesis of a myriad of human cancers and with resistance to anticancer drugs including CDK4/6 inhibitors. Thus, CDK2 has become an attractive target for the development of new anticancer therapies and for the amelioration of the resistance to CDK4/6 inhibitors. Bioisosteric replacement of the thiazole moiety of CDKI-73, a clinically trialled CDK inhibitor, by a pyrazole group afforded 9 and 19 that displayed potent CDK2-cyclin E inhibition (Ki = 0.023 and 0.001 µM, respectively) with submicromolar antiproliferative activity against a panel of cancer cell lines (GI50 = 0.025-0.780 µM). Mechanistic studies on 19 with HCT-116 colorectal cancer cells revealed that the compound reduced the phosphorylation of retinoblastoma at Ser807/811, arrested the cells at the G2/M phase, and induced apoptosis. These results highlight the potential of the 2-anilino-4-(1-methyl-1H-pyrazol-4-yl)pyrimidine series in developing potent and selective CDK2 inhibitors to combat cancer.

From cyclins to CDKIs: Cell cycle regulation of skeletal muscle stem cell quiescence and activation.

After extensive proliferation during development, the adult skeletal muscle cells remain outside the cell cycle, either as post-mitotic myofibers or as quiescent muscle stem cells (MuSCs, also known as satellite cells). Despite its terminally differentiated state, adult skeletal muscle has a remarkable regeneration potential, driven by MuSCs. Upon injury, MuSC quiescence is reversed to support tissue growth and repair and it is re-established after the completion of muscle regeneration. The distinct cell cycle states and transitions observed in the different myogenic populations are orchestrated by elements of the cell cycle machinery. This consists of i) complexes of cyclins and Cyclin-Dependent Kinases (CDKs) that ensure cell cycle progression and ii) their negative regulators, the Cyclin-Dependent Kinase Inhibitors (CDKIs). In this review we discuss the roles of these factors in developmental and adult myogenesis, with a focus on CDKIs that have emerging roles in stem cell functions.

Stabilization of p27Kip1/CDKN1B by UBCH7/UBE2L3 catalyzed ubiquitinylation: a new paradigm in cell-cycle control.

Ubiquitinylation drives many cellular processes by targeting proteins for proteasomal degradation. Ubiquitin conjugation enzymes promote ubiquitinylation and, thus, degradation of protein substrates. Ubiquitinylation is a well-known posttranslational modification controlling cell-cycle transitions and levels or/and activation levels of ubiquitin-conjugating enzymes change during development and cell cycle. Progression through the cell cycle is tightly controlled by CDK inhibitors such as p27Kip1. Here we show that, in contrast to promoting its degradation, the ubiquitin-conjugating enzyme UBCH7/UBE2L3 specifically protects p27Kip1 from degradation. Overexpression of UBCH7/UBE2L3 stabilizes p27Kip1 and delays the G1-to-S transition, while depletion of UBCH7/UBE2L3 increases turnover of p27Kip1. Levels of p21Cip1/Waf1, p57Kip2, cyclin A and cyclin E, all of which are also involved in regulating the G1/S transition are not affected by UBCH7/UBE2L3 depletion. The effect of UBCH7/UBE2L3 on p27Kip1 is not due to alteration of the levels of any of the ubiquitin ligases known to ubiquitinylate p27Kip1. Rather, UBCH7/UBE2L3 catalyzes the conjugation of heterotypic ubiquitin chains on p27Kip1 that are proteolytically incompetent. These data reveal new controls and concepts about the ubiquitin proteasome system in which a ubiquitin-conjugating enzyme selectively inhibits and may even protect, rather than promote degradation of a crucial cell-cycle regulatory molecule.-Whitcomb, E. A., Tsai, Y. C., Basappa, J., Liu, K., Le Feuvre, A. K., Weissman, A. M., Taylor, A. Stabilization of p27Kip1/CDKN1B by UBCH7/UBE2L3 catalyzed ubiquitinylation: a new paradigm in cell-cycle control.

CDKI-73: an orally bioavailable and highly efficacious CDK9 inhibitor against acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common form of acute leukemia with dismal long-term prognosis with age. The most aggressive subtype of AML is MLL-AML that is characterized by translocations of the mixed-lineage leukemia gene (MLL) and resistance to conventional chemotherapy. Cyclin dependent kinase 9 (CDK9) plays a crucial role in the MLL-driven oncogenic transcription, and hence, inhibiting activity of CDK9 has been proposed as a promising strategy for treatment of AML. We investigated the therapeutic potential of CDKI-73, one of the most potent CDK9 inhibitors, against a panel of AML cell lines and samples derived from 97 patients. CDKI-73 induced cancer cells undergoing apoptosis through transcriptional downregulation of anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP by majorly targeting CDK9. Contrastively, it was relatively low toxic to the bone marrow cells of healthy donors. In MV4-11 xenograft mouse models, oral administration of CDKI-73 resulted in a marked inhibition of tumor growth (p < 0.0001) and prolongation of animal life span (P < 0.001) without causing body weight loss and other overt toxicities. The study suggests that CDKI-73 can be developed as a highly efficacious and orally deliverable therapeutic agent for treatment of AML.

Disruption of a conserved CAP-D3 threonine alters condensin loading on mitotic chromosomes leading to chromosome hypercondensation.

The condensin complex plays a key role in organizing mitotic chromosomes. In vertebrates, there are two condensin complexes that have independent and cooperative roles in folding mitotic chromosomes. In this study, we dissect the role of a putative Cdk1 site on the condensin II subunit CAP-D3 in chicken DT40 cells. This conserved site has been shown to activate condensin II during prophase in human cells, and facilitate further phosphorylation by polo-like kinase I. We examined the functional significance of this phosphorylation mark by mutating the orthologous site of CAP-D3 (CAP-D3(T1403A)) in chicken DT40 cells. We show that this mutation is a gain of function mutant in chicken cells; it disrupts prophase, results in a dramatic shortening of the mitotic chromosome axis, and leads to abnormal INCENP localization. Our results imply phosphorylation of CAP-D3 acts to limit condensin II binding onto mitotic chromosomes. We present the first in vivo example that alters the ratio of condensin I:II on mitotic chromosomes. Our results demonstrate this ratio is a critical determinant in shaping mitotic chromosomes.

Focal adhesion kinase antagonizes doxorubicin cardiotoxicity via p21(Cip1.).

Clinical application of potent anthracycline anticancer drugs, especially doxorubicin (DOX), is limited by a toxic cardiac side effect that is not fully understood and preventive strategies are yet to be established. Studies in genetically modified mice have demonstrated that focal adhesion kinase (FAK) plays a key role in regulating adaptive responses of the adult myocardium to pathological stimuli through activation of intracellular signaling cascades that facilitate cardiomyocyte growth and survival. The objective of this study was to determine if targeted myocardial FAK activation could protect the heart from DOX-induced de-compensation and to characterize the underlying mechanisms. To this end, mice with myocyte-restricted FAK knock-out (MFKO) or myocyte-specific expression of an active FAK variant (termed SuperFAK) were subjected to DOX treatment. FAK depletion enhanced susceptibility to DOX-induced myocyte apoptosis and cardiac dysfunction, while elevated FAK activity provided remarkable cardioprotection. Our mec6hanistic studies reveal a heretofore unappreciated role for the protective cyclin-dependent kinase inhibitor p21 in the repression of the pro-apoptotic BH3-only protein Bim and the maintenance of mitochondrial integrity and myocyte survival. DOX treatment induced proteasomal degradation of p21, which exacerbated mitochondrial dysfunction and cardiomyocyte apoptosis. FAK was both necessary and sufficient for maintaining p21 levels following DOX treatment and depletion of p21 compromised FAK-dependent protection from DOX. These findings identify p21 as a key determinant of DOX resistance downstream of FAK in cardiomyocytes and indicate that cardiac-restricted enhancement of the FAK/p21 signaling axis might be an effective strategy to preserve myocardial function in patients receiving anthracycline chemotherapy.

Polycomb repressive complex 1 modulates granulosa cell proliferation in early folliculogenesis to support female reproduction.

Rationale: Premature ovarian insufficiency (POI) is an accelerated reduction in ovarian function inducing infertility. Folliculogenesis defects have been reported to trigger POI as a consequence of ovulation failure. However, the underlying mechanisms remain unclear due to the genetic complexity and heterogeneity of POI. Methods: We used whole genome sequencing (WGS), conditional knockout mouse models combined with laser capture microdissection (LCM), and RNA/ChIP sequencing to analyze the crucial roles of polycomb repressive complex 1 (PRC1) in clinical POI and mammalian folliculogenesis. Results: A deletion mutation of MEL18, the key component of PRC1, was identified in a 17-year-old patient. However, deleting Mel18 in granulosa cells (GCs) did not induce infertility until its homolog, Bmi1, was deleted simultaneously. Double deficiency of BMI1/MEL18 eliminated PRC1 catalytic activity, upregulating cyclin-dependent kinase inhibitors (CDKIs) and thus blocking GC proliferation during primary-to-secondary follicle transition. This defect led to damaged intercellular crosstalk, eventually resulting in gonadotropin response failure and infertility. Conclusions: Our findings highlighted the pivotal role of PRC1 as an epigenetic regulator of gene transcription networks in GC proliferation during early folliculogenesis. In the future, a better understanding of molecular details of PRC1 structural and functional abnormalities may contribute to POI diagnosis and therapeutic options.

Pharmacological inhibition of CDK4/6 impairs diffuse pleural mesothelioma 3D spheroid growth and reduces viability of cisplatin-resistant cells.

Introduction: Diffuse pleural mesothelioma (DPM) of the pleura is a highly aggressive and treatment-resistant cancer linked to asbestos exposure. Despite multimodal treatment, the prognosis for DPM patients remains very poor, with an average survival of 2 years from diagnosis. Cisplatin, a platinum-based chemotherapy drug, is commonly used in the treatment of DPM. However, the development of resistance to cisplatin significantly limits its effectiveness, highlighting the urgent need for alternative therapeutic strategies. New selective inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) have shown promise in various malignancies by inhibiting cell cycle progression and suppressing tumor growth. Recent studies have indicated the potential of abemaciclib for DPM therapy, and a phase II clinical trial has shown preliminary encouraging results. Methods: Here, we tested abemaciclib, palbociclib, and ribociclib on a panel of DPM cell lines and non-tumor mesothelial(MET-5A) cells. Results: Specifically, we focused on abemaciclib, which was the mosteffective cytotoxic agent on all the DPM cell lines tested. Abemaciclib reduced DPM cell viability, clonogenic potential, and ability to grow as three-dimensional (3D) spheroids. In addition, abemaciclib induced prolonged effects, thereby impairing second-generation sphere formation and inducing G0/G1 arrest and apoptosis/ necrosis. Interestingly, single silencing of RB family members did not impair cell response to abemaciclib, suggesting that they likely complement each other in triggering abemaciclib's cytostatic effect. Interestingly, abemaciclib reduced the phosphorylation of AKT, which is hyperactive in DPM and synergized with the pharmacological AKT inhibitor (AKTi VIII). Abemaciclib also synergized with cisplatin and reduced the viability of DPM cells with acquired resistance to cisplatin. Discussion: Overall, our results suggest that CDK4/6 inhibitors alone or in combination with standard of care should be further explored for DPM therapy.

Cyclin-dependent kinase 2 (CDK2) inhibitors and others novel CDK inhibitors (CDKi) in breast cancer: clinical trials, current impact, and future directions.

Aberrant cyclin-dependent kinase 2 (CDK2) activation has been identified as a main resistance mechanism to CDK4/6 inhibition in hormone-receptor positive (HR+) breast cancer. Additionally, consistent preclinical evidence states its crucial role in MYC and CCNE1 overexpressed cancer survival, such as triple-negative breast cancers (TNBC), thus representing an appealing and relatively unexplored target treatment opportunity. Despite emerging initial results of novel CDK2 inhibitors (CDK2i) activity, a comprehensive outcomes collection is currently absent from the scientific literature. We aim to provide an overview of ongoing clinical trials involving CDK2i in the context of metastatic breast cancer (mBC), either as monotherapy or in combination with other agents. The review extends beyond CDK2i to encompass novel emerging CDK4 inhibitors, combined CDK2/4/6 inhibitors, and the well-known pan-CDK inhibitors including those specifically directed at CDK2. Delving into the results, we critically appraise the observed clinical efficacy and offer valuable insights into their potential impact and future applications.

Coumaric acid from M. polymorphum extracts reverses the activated state of hepatic stellate cells (GRX) and inhibits their proliferation by decreasing the p53/p21 pathway.

Coumaric acid is a phenolic compound found in medicinal plants. Its use has been reported in the treatment of inflammatory diseases, prevention of alterations induced by oxidative stress, as well as acetaminophen-induced hepatotoxicity. Thus, this study evaluated coumaric acid as a potential treatment for liver fibrosis. Cell proliferation was assessed by the trypan blue exclusion technique and the cytotoxicity of coumaric acid was performed using an LDH assay. Mechanisms of cell apoptosis were evaluated by flow cytometry. The expression of genes associated with apoptosis, cell cycle control, and fibrosis was assessed by qPCR. The production of lipid droplets was quantified by oil red staining. The experiments performed showed that the treatment with coumaric acid was able to reduce cell proliferation without causing cell cytotoxicity or apoptosis. Coumaric acid was able to inhibit the expression of cyclin D1 and CDK's (CDK2, CDK4, and CDK6), increasing p53 and p21, which could lead to cell cycle arrest. Treatment with coumaric acid was also able to revert the activated phenotype of GRX cells to their quiescent state. Thus, our results suggest that coumaric acid has a potential therapeutic effect against liver fibrosis.

The host-targeting compound peruvoside has a broad-spectrum antiviral activity against positive-sense RNA viruses.

Positive-sense RNA viruses modify intracellular calcium stores, endoplasmic reticulum and Golgi apparatus (Golgi) to generate membranous replication organelles known as viral factories. Viral factories provide a conducive and substantial enclave for essential virus replication via concentrating necessary cellular factors and viral proteins in proximity. Here, we identified the vital role of a broad-spectrum antiviral, peruvoside in limiting the formation of viral factories. Mechanistically, we revealed the pleiotropic cellular effect of Src and PLC kinase signaling via cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) phosphorylation and Golgi vesiculation by peruvoside treatment. The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation. Further investigation showed signaling of ERK1/2 pathway via cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337 (T1337). We also showed 100% of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum. These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside, host-directed antivirals for positive-sense RNA virus-mediated disease, in the interim where no vaccine is available.

Tissue-specific expression of senescence biomarkers in spontaneously hypertensive rats: evidence of premature aging in hypertension.

Background: Cellular senescence is an age-related physiological process that contributes to tissue dysfunction and accelerated onset of chronic metabolic diseases including hypertension. Indeed, elevation of blood pressure in hypertension coincides with premature vascular aging and dysfunction. In addition, onsets of metabolic disturbance and osteopenia in patients with hypertension have also been reported. It is possible that hypertension enhances premature aging and causes progressive loss of function in multiple organs. However, the landscape of cellular senescence in critical tissues affected by hypertension remains elusive. Materials and Methods: Heart, liver, bone, hypothalamus, and kidney were collected from spontaneously hypertensive rats (SHR) and age- and sex-matched normotensive Wistar rats (WT) at 6, 12, 24 and 36 weeks of age (n = 10 animals/group). Changes in mRNA levels of senescence biomarkers namely cyclin-dependent kinase (CDK) inhibitors (CDKIs), i.e., Cdkn2a (encoding p16Ink4a) and Cdkn1a (encoding p21cip1) as well as senescence-associated secretory phenotypes (SASPs), i.e., Timp1, Mmp12, Il6 and Cxcl1, were determined. Additionally, bone collagen alignment and hydroxy apatite crystal dimensions were determined by synchrotron radiation small- and wide-angle X-ray scattering (SAXS/WAXS) techniques. Results: Real-time PCR revealed that transcript levels of genes encoding CDKIs and SASPs in the heart and liver were upregulated in SHR from 6 to 36 weeks of age. Expression of Timp1 and Cxcl1 was increased in bone tissues isolated from 36-week-old SHR. In contrast, we found that expression levels of Timp1 and Il6 mRNA were decreased in hypothalamus and kidney of SHR in all age groups. Simultaneous SAXS/WAXS analysis also revealed misalignment of bone collagen fibers in SHR as compared to WT. Conclusion: Premature aging was identified in an organ directly affected by high blood pressure (i.e., heart) and those with known functional defects in SHR (i.e., liver and bone). Cellular senescence was not evident in organs with autoregulation of blood pressure (i.e., brain and kidney). Our study suggested that cellular senescence is induced by persistently elevated blood pressure and in part, leading to organ dysfunction. Therefore, interventions that can both lower blood pressure and prevent cellular senescence should provide therapeutic benefits for treatment of cardiovascular and metabolic consequences.

pH-Responsive Poly(ethylene glycol)-b-poly(2-vinylpyridine) Micelles for the Triggered Release of Therapeutics.

The use of pH-responsive polymeric micelles is a promising approach to afford the targeted, pH-mediated delivery of hydrophobic drugs within the low-pH tumour milieu and intracellular organelles of cancer cells. However, even for a common pH-responsive polymeric micelle system-e.g., those utilising poly(ethylene glycol)-b-poly(2-vinylpyridine) (PEG-b-PVP) diblock copolymers-there is a lack of available data describing the compatibility of hydrophobic drugs, as well as the relationships between copolymer microstructure and drug compatibility. Furthermore, synthesis of the constituent pH-responsive copolymers generally requires complex temperature control or degassing procedures that limit their accessibility. Herein we report the facile synthesis of a series of diblock copolymers via visible-light-mediated photocontrolled reversible addition-fragmentation chain-transfer polymerisation, with a constant PEG block length (90 repeat units (RUs)) and varying PVP block lengths (46-235 RUs). All copolymers exhibited narrow dispersity values (D ≤ 1.23) and formed polymeric micelles with low polydispersity index (PDI) values (typically <0.20) at physiological pH (7.4), within a suitable size range for passive tumour targeting (<130 nm). The encapsulation and release of three hydrophobic drugs (cyclin-dependent kinase inhibitor (CDKI)-73, gossypol, and doxorubicin) were investigated in vitro at pH 7.4-4.5 to simulate drug release within the tumour milieu and cancer cell endosome. Marked differences in drug encapsulation and release were observed when the PVP block length was increased from 86 to 235 RUs. With a PVP block length of 235 RUs, the micelles exhibited differing encapsulation and release properties for each drug. Minimal release was observed for doxorubicin (10%, pH 4.5) and CDKI-73 exhibited moderate release (77%, pH 4.5), whereas gossypol exhibited the best combination of encapsulation efficiency (83%) and release (91% pH 4.5) overall. These data demonstrate the drug selectivity of the PVP core, where both the block molecular weight and hydrophobicity of the core (and accordingly the hydrophobicity of the drug) have a significant effect on drug encapsulation and release. These systems remain a promising means of achieving targeted, pH-responsive drug delivery-albeit for select, compatible hydrophobic drugs-which warrants their further investigation to develop and evaluate clinically relevant micelle systems.

Cost-effectiveness of palbociclib in early breast cancer patients with a high risk of relapse: Results from the PENELOPE-B trial.

Background: Patients with hormone receptor-positive, HER2-negative breast cancer who have residual invasive disease after neoadjuvant chemotherapy (NACT) are at a high risk of relapse. PENELOPE-B was a double-blind, placebo-controlled, phase III trial that investigated adding palbociclib (PAL) for thirteen 28-day cycles to adjuvant endocrine therapy (ET) in these patients. Clinical results showed no significant improvement in invasive disease-free survival with PAL. Methods: We performed a pre-planned cost-effectiveness analysis of PAL within PENELOPE-B from the perspective of the German statutory health insurance. Health-related quality of life scores, collected in the trial using the EQ-5D-3L instrument, were converted to utilities based on the German valuation algorithm. Resource use was valued using German price weights. Outcomes were discounted at 3% and modeled with mixed-level linear models to adjust for attrition, repeated measurements, and residual baseline imbalances. Subgroup analyses were performed for key prognostic risk factors. Scenario analyses addressed data limitations and evaluated the robustness of the estimated cost-effectiveness of PAL to methodological choices. Results: The effects of PAL on quality-adjusted life years (QALYs) were marginal during the active treatment phase, increasing thereafter to 0.088 (95% confidence interval: -0.001; 0.177) QALYs gained over the 4 years of follow-up. The incremental costs were dominated by PAL averaging EUR 33,000 per patient; costs were higher in the PAL arm but not significantly different after the second year. At an incremental cost-effectiveness ratio of EUR 380,000 per QALY gained, PAL was not cost-effective compared to the standard-of-care ET. Analyses restricted to Germany and other subgroups were consistent with the main results. Findings were robust in the scenarios evaluated. Conclusions: One year of PAL added to ET is not cost-effective in women with residual invasive disease after NACT in Germany.

The Renaissance of Cyclin Dependent Kinase Inhibitors.

Cyclin-dependent kinases (CDK) regulate cell cycle progression. During tumor development, altered expression and availability of CDKs strongly contribute to impaired cell proliferation, a hallmark of cancer. In recent years, targeted inhibition of CDKs has shown considerable therapeutic benefit in a variety of tumor entities. Their success is reflected in clinical approvals of specific CDK4/6 inhibitors for breast cancer. This review provides a detailed insight into the molecular mechanisms of CDKs as well as a general overview of CDK inhibition. It also summarizes the latest research approaches and current advances in the treatment of head and neck cancer with CDK inhibitors. Instead of monotherapies, combination therapies with CDK inhibitors may especially provide promising results in tumor therapy. Indeed, recent studies have shown a synergistic effect of CDK inhibition together with chemo- and radio- and immunotherapy in cancer treatment to overcome tumor evasion, which may lead to a renaissance of CDK inhibitors.

Role of the Combination of Cyclin-Dependent Kinase Inhibitors (CDKI) and Radiotherapy (RT) in the Treatment of Metastatic Breast Cancer (MBC): Advantages and Risks in Clinical Practice.

Targeting cell cycle has become the gold standard for metastatic breast cancer (MBC), being cyclin-dependent kinase inhibitors (CDKIs) cornerstones of its treatment, alongside radiotherapy (RT). To date, no definite evidence regarding safety and efficacy of the combination of CDKIs plus radiotherapy (RT) is currently available. Purpose of this review is to collect data in favor or against the feasibility of the association of CDKIs + RT, describing its potential adverse events. Our review shows how CDKI + RT allows an overall satisfying disease control, proving to be effective and causing a grade of toxicity mainly influenced by the site of irradiation, leaning to favourable outcomes for sites as liver, spine or brain and to poorer outcomes for thoracic lesions or sites close to viscera; controversial evidence is instead for bone treatment. Toxicity also varies from patient to patient. To sum up, our contribution enriches and enlightens a still indefinite field regarding the feasibility of CDKIs + RT, giving cues for innovative clinical management of hormone-responsive MBC.

Effect of Zebularine on p16INK4a, p14ARF, p15INK4b, and DNA Methyltransferase 1 Gene Expression, Cell Growth Inhibition, and Apoptosis Induction in Human Hepatocellular Carcinoma PLC/PRF5 and Pancreatic Cancer PA-TU-8902 Cell Lines.

Tumorigenesis must be understood as a summary of altered genetic and genomic changes resulting in the inactivation of tumor suppressor genes (TSGs). One of the characterizations of epigenetic alterations is DNA methylation. Epigenetic alteration of the p16INK4a, p14ARF, p15INK4b, and DNA methyltransferase 1 gene (DNMT1) expression occurs in hepatocellular carcinoma (HCC) and pancreatic cancer frequently. DNA methyltransferase inhibitors (DNMTIs), such as zebularine, play a significant effect on the demethylation and reactivation of TSGs. This study aimed to investigate the effect of zebularine on p16INK4a, p14ARF, p15INK4b, and DNA methyltransferase 1 gene expression, cell growth inhibition, and apoptosis induction in HCC PLC/PRF5 and pancreatic cancer PA-TU-8902 cell lines. Both cell lines were cultured and treated with zebularine at different times. The MTT assay, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and flow cytometry were used to determine cell viability, gene expression, and apoptotic cells, respectively. The result indicated that zebularine inhibited cell growth of both cell lines significantly as time- and dose-dependent manner (P < 0.007). The agent induced significant down-regulation of DNMT1 and up-regulation of p16INK4a, p14ARF, p15INK4b (P < 0.028). Besides, it had a significant apoptosis effect on both cell lines (P < 0.001). This compound had a strong significant effect on PLC/PRF5 in comparison to PA-TU-8902 cells. Concluding, zebularine inhibited PLC/PRF5 and PA-TU-8902 cell growth and induced apoptosis in these cell lines. The most likely mechanism underlying the zebularine played its role involves down-regulation of DNMT1 and up-regulation of p16INK4a, p14ARF, and p15INK4b genes.

Vulnerability of drug-resistant EML4-ALK rearranged lung cancer to transcriptional inhibition.

A subset of lung adenocarcinomas is driven by the EML4-ALK translocation. Even though ALK inhibitors in the clinic lead to excellent initial responses, acquired resistance to these inhibitors due to on-target mutations or parallel pathway alterations is a major clinical challenge. Exploring these mechanisms of resistance, we found that EML4-ALK cells parental or resistant to crizotinib, ceritinib or alectinib are remarkably sensitive to inhibition of CDK7/12 with THZ1 and CDK9 with alvocidib or dinaciclib. These compounds robustly induce apoptosis through transcriptional inhibition and downregulation of anti-apoptotic genes. Importantly, alvocidib reduced tumour progression in xenograft mouse models. In summary, our study takes advantage of the transcriptional addiction hypothesis to propose a new treatment strategy for a subset of patients with acquired resistance to first-, second- and third-generation ALK inhibitors.

CDKI-73 is a Novel Pharmacological Inhibitor of Rab11 Cargo Delivery and Innate Immune Secretion.

Innate immunity is critical for host defence against pathogen and environmental challenge and this involves the production and secretion of immune mediators, such as antimicrobial peptides and pro-inflammatory cytokines. However, when dysregulated, innate immunity can contribute to multifactorial diseases, including inflammatory rheumatic disorders, type 2 diabetes, cancer, neurodegenerative and cardiovascular diseases and even septic shock. During an innate immune response, antimicrobial peptides and cytokines are trafficked via Rab11 multivesicular endosomes, and then sorted into Rab11 vesicles for traffic to the plasma membrane and secretion. In this study, a cyclin-dependent kinase inhibitor CDKI-73 was used to determine its effect on the innate immune response, based on previously identified targets for this compound. Our results showed that CDKI-73 inhibited the delivery of Rab11 vesicles to the plasma membrane, resulting in the accumulation of large multivesicular Rab11 endosomes near the cell periphery. In addition to the effect on endosome delivery, CDKI-73 down-regulated the amount of innate immune cargo, including the antimicrobial peptide Drosomycin and pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFα). We concluded that CDKI-73 has the potential to regulate the delivery and secretion of certain innate immune cargo, which could be used to control inflammation.

p19INK4d: More than Just a Cyclin-Dependent Kinase Inhibitor.

Cyclin-dependent kinase inhibitors (CDKIs) are important cell cycle regulators. The CDKI family is composed of the INK4 family and the CIP/KIP family. p19INK4d belongs to the INK4 gene family and is involved in a series of normal physiological activities and the pathogenesis of diseases. Many factors play regulatory roles in the p19INK4d gene expression at the transcriptional and posttranscriptional levels. p19INK4d not only regulates the cell cycle but also plays regulatory roles in apoptosis, DNA damage repair, cell differentiation of hematopoietic cells, and cellular senescence. In this review, the regulatory network of the p19INK4d gene expression and its biological functions are summarized, which provides a basis for further study of p19INK4d as a drug target for disease treatment.