Multiple Cyclin-dependent Kinase Inhibitors Research Articles

Abstract 529: Investigating the synergistic effect of milciclib and PD0325901 in colorectal cancer therapy

Abstract Milciclib, characterized as a potent nanomolar inhibitor of multiple cyclin-dependent kinases, has recently emerged as a promising therapeutic agent in cancer treatment, particularly with its additional role as a spliceosome modulator. Our recent studies employing RNA sequencing technology revealed a comprehensive profile of Milciclib-induced transcriptional changes in colorectal cancer cells, identifying several key regulated pathways. In light of these findings, we explored a novel therapeutic strategy: combining Milciclib with specific inhibitors targeting pathways upregulated by Milciclib treatment. This hypothesis is based on the rationale that such a combination could exert a synergistic effect. We have assessed the efficacy of this combinational therapy and found that the combination of Milciclib and PD0325901 greatly potentiates the killing of colorectal cancer cells versus either treatment alone. PD0325901 has been extensively studied for its efficacy in targeting the MEK/ERK signaling pathway, a critical pathway in many cancers, including colorectal cancer. To further characterize the underlying molecular mechanisms contributing to the observed synergistic effect, we applied quantitative proteomic analysis of 8107 proteins in human colorectal cancer cells with the treatment of different doses of Milciclib and PD0325901 administered alone, or in combination. Our results demonstrate 49 proteins were significantly decreased in a dose-dependent manner, and that many of these proteins are key players regulating pathways associated with the cell cycle, apoptosis, gene expression, metabolism, WNT signaling, and the MEK/ERK signaling pathway. The discovery of the down-regulation of these proteins and pathways after treatment provides a molecular basis to elucidate the synergistic mechanism underlying Milciclib and PD0325901 combination therapy. In conclusion, our study establishes the combinatorial use of Milciclib and PD0325901 as a potential breakthrough in the treatment of colorectal cancer, offering a promising prospect for enhancing future patient outcomes. Citation Format: Xinyu Pei, Anh Nguyen, Hannah Neiger, Wenjia Wang, William Bautista, Lily Wang, Alvan Cai, Valerie A. Gerriets, John K. Cusick, Yihui Shi. Investigating the synergistic effect of milciclib and PD0325901 in colorectal cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 529.

Induction of β-cell replication by a synthetic HNF4α antagonist

Increasing the number of β cells is critical to a definitive therapy for diabetes. Previously, we discovered potent synthetic small molecule antagonists of the nuclear receptor transcription factor HNF4α. The natural ligands of HNF4α are thought to be fatty acids. Because obesity, in which there are high circulating levels of free fatty acids, is one of the few conditions leading to β-cell hyperplasia, we tested the hypothesis that a potent HNF4α antagonist might stimulate β-cell replication. A bioavailable HNF4α antagonist was injected into normal mice and rabbits and β-cell ablated mice and the effect on β-cell replication was measured. In normal mice and rabbits, the compound induced β-cell replication and repressed the expression of multiple cyclin-dependent kinase inhibitors, including p16 that plays a critical role in suppressing β-cell replication. Interestingly, in β-cell ablated mice, the compound induced α- and δ-cell, in addition to β-cell replication, and β-cell number was substantially increased. Overall, the data presented here are consistent with a model in which the well-known effects of obesity and high fat diet on β-cell replication occur by inhibition of HNF4α. The availability of a potent synthetic HNF4α antagonist raises the possibility that this effect might be a viable route to promote significant increases in β-cell replication in diseases with reduced β-cell mass, including type I and type II diabetes.

A phase I pharmacokinetic and pharmacodynamic study of AT7519, a cyclin-dependent kinase inhibitor in patients with refractory solid tumors

BackgroundAT7519 is an inhibitor of multiple cyclin-dependent kinases (CDKs). Based on potent antitumor activity in preclinical models, a first-in-human clinical trial in refractory solid tumors investigated its safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD). Patients and methodsAT7519 was administered in a ‘3 + 3’ dose- escalation scheme on 5 consecutive days every 3 weeks to patients with advanced, refractory solid tumors. Samples to monitor AT7519 PK and PD were obtained. ResultsTwenty-eight patients were treated at seven dose levels (1.8–40 mg/m2/day). At 40 mg/m2/day, one patient developed hypotension and ST segment elevation. At 34 mg/m2/day, dose-limiting toxic effects (DLTs) were QTc prolongation with one death (grade 5), fatigue (grade 4) and mucositis (grade 3). Electrocardiogram review suggested a dose-dependent increase in QTc and recruitment was discontinued without establishing a maximum tolerated dose. Four patients exhibited stable disease for >6 months and one had a prolonged partial response. PK profile revealed modest interpatient variation with linear exposure at increasing doses. Inhibition of markers of CDK activity was observed across the dose range and manifested in antiproliferative activity at a dose of 28 mg/m2. ConclusionAT7519 elicited clinical and PD activity resulting from CDK inhibition at doses below the appearance of DLT of QTc prolongation.

A first in man, phase I dose-escalation study of PHA-793887, an inhibitor of multiple cyclin-dependent kinases (CDK2, 1 and 4) reveals unexpected hepatotoxicity in patients with solid tumors

Background: PHA-793887 is an inhibitor of multiple cyclin dependent kinases (CDK) with activity against CDK2, CDK1 and CDK4. The primary objectives of this first in man study were to determine the dose limiting toxicities (DLTs), maximum tolerated dose (MTD) and recommended phase II dose of PHA-793887. Results: Although toxicity was acceptable at initial dose levels, PHA-793887 was poorly tolerated at doses ≥44 mg/m2. The most frequent events across all dose levels were gastrointestinal or nervous system events. DLTs were experienced by two of three patients at the dose level of 66 mg/m2, and by three of nine patients at the dose level of 44 mg/m2. In all but one patient the DLT was hepatotoxicity; fatal hepatorenal failure was seen in one patient treated at the 44 mg/ m2 dose level. There were no objective responses, but disease stabilization was observed in five patients. Over the dose range investigated, pharmacokinetic studies showed that systemic exposure to PHA-793887 increased with the dose and was time-independent. The study terminated after the enrolment of 19 patients due to the severe hepatic toxicity. Patients and Methods: Cohorts of three to six patients were treated at doses of 11, 22, 44 and 66 mg/m2 of PHA-793887 administered as 1-hour intravenous infusion on days 1, 8 and 15 in a 4-week cycle. Safety and pharmacokinetics were investigated. Conclusion: PHA-793887 induces severe, dose-related hepatic toxicity, which was not predicted by pre-clinical models and currently precludes its further clinical development.

Rosiglitazone sensitizes MDA-MB-231 breast cancer cells to anti-tumour effects of tumour necrosis factor-α, CH11 and CYC202

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear hormone superfamily and has multiple endogenous and pharmacological ligands, including 15-deoxy-Delta (12,14)-prostaglandin J(2) and two thiazolidinediones (TZD), rosiglitazone and pioglitazone, which are used clinically to treat type-2 diabetes mellitus. PPARgamma agonists regulate development, cellular growth and metabolism in various tissues and have been documented to decrease cellular proliferation and to induce apoptosis of various tumour phenotypes, including breast cancer. However, the full spectrum of anti-tumour effects occurs only at suprapharmacological doses. In this study, we investigated the mechanism of rosiglitazone-induced anti-tumour effects of MDA-MB-231 human breast cancer cells, and used that information to predict rosiglitazone-induced sensitization of breast cancer cells to the effects of other compounds. We first confirmed that 100 microM rosiglitazone, but not lower doses, decreases MDA-MB-231 cell viability in vitro. We then used microarray gene expression analysis to determine early rosiglitazone-induced gene expression changes after 4-h exposure, which included 1298 genes that we grouped into functional categories. We selectively confirmed rosiglitazone-mediated effects on expression of key regulators of breast cancer proliferation and apoptosis, including p53, p21 and Bax. Finally, we used this information to predict that rosiglitazone would sensitize MDA-MB-231 cells to the anti-tumour effects of CH11, which trimerizes Fas, as well as tumour necrosis factor-alpha. Moreover, we used the confirmed array data to predict cooperative activity of rosiglitazone and R-roscovitine (CYC202), an inhibitor of multiple cyclin-dependent kinases. We conclude that microarray analysis can determine early TZD-modulated changes in gene expression that help to predict effective in vitro drug combinations.

Alvocidib (Flavopiridol) suppresses tumor growth in SCID mice with human esophageal cancer xenografts without inducing apoptosis

Alvocidib (Flavopiridol, HMR1275) is a potent inhibitor of multiple cyclin-dependent kinases and has been identified recently as an antitumor agent in several cancers. Previous studies have shown that alvocidib could potentially treat esophageal cancer in vitro. This study evaluates alvocidib for its ability to suppress tumor growth in severe combined immunodeficiency (SCID) mice bearing TE8 human esophageal squamous cell carcinoma (SCC) xenografts. Alvocidib treatment of 10mg/kg body weight reduced tumor volume significantly. Immunohistochemistry analysis of alvocidib-treated tumor sections showed significant reductions in cyclin D1, VEGF, and Rb levels. Alvocidib treatment did not cause a marked increase in apoptotic tumor cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis, yet hematoxylin and eosin staining revealed tumor necrosis. In vivo investigation of alvocidib treatment confirmed antitumor activity in TE8 esophageal xenografts. These findings suggest that alvocidib could be a useful anti-cancer agent for esophageal cancer.

Loss of the hSNF5 Gene Concomitantly Inactivates p21CIP/WAF1 and p16INK4a Activity Associated with Replicative Senescence in A204 Rhabdoid Tumor Cells

hSNF5, the smallest member of the SWI/SNF chromatin remodeling complex, is lost in most malignant rhabdoid tumors (MRT). In MRT cell lines, reexpression of hSNF5 induces G1 cell cycle arrest, elevated p16INK4a, and activated replicative senescence markers, such as beta-galactosidase (beta-Gal) and plasminogen activator inhibitor-1. To compare the replicative senescence caused by hSNF5 in A204 cells to normal cellular senescence, we examined the activation of both p16INK4a and p21CIP/WAF1. Analogous to normal cellular senescence, both p16INK4a and p21CIP/WAF1 were up-regulated following hSNF5 restoration. Furthermore, we found that hSNF5 bound the p16INK4a and p21CIP/WAF1 promoters, suggesting that it directly regulates transcription of these genes. Using p16INK4a RNA interference, we showed its requirement for the replicative senescence caused by hSNF5 but not the growth arrest. Instead, p21CIP/WAF1 remained activated by hSNF5 in the absence of high p16INK4a expression, apparently causing the growth arrest in A204. Interestingly, we also found that, in the absence of p16INK4a, reexpression of hSNF5 also increased protein levels of a second cyclin-dependent kinase (CDK) inhibitor, p18INK4c. However, our data show that lack of hSNF5 does not abrogate cellular responsiveness to DNA damage or growth-inhibitory factors. In summary, our studies suggest that hSNF5 loss may influence the regulation of multiple CDK inhibitors involved in replicative senescence.

Alterations in cyclin-dependent kinase 2 function during differentiation of primary human keratinocytes.

Terminal differentiation of epithelial cells is intimately linked to cell-cycle withdrawal. The tight coupling of these two processes is critical to maintenance of epidermal tissue homeostasis and is frequently disrupted in squamous cell carcinoma. To identify possible molecular targets of epithelial carcinogenesis, we investigated the regulatory pathways that couple cellular differentiation and proliferation in primary cultures of human keratinocytes and found that the cyclin-dependent kinase inhibitors (CKIs) p21cip1/waf1 and p27kip1 were induced early during differentiation of human keratinocytes, whereas p15ink4B was induced later in differentiation. The induction of p21c1/waf1 was mediated by both transcriptional and non-transcriptional mechanisms, and the activities of cyclin A/cyclin-dependent kinase (cdk) 2 and cyclin E/cdk2 complexes were specifically inhibited during keratinocyte differentiation. In contrast, p21cip1/wafl did not associate with cdk4, and the activities of cdk4 complexes remained unchanged. Hence, our results support the model that multiple CKIs participate in linking cellular proliferation and differentiation in human keratinocytes by specific modulation of cdk2 activity.

Molecular cloning and characterization of the human p27 Kip1 gene promoter

p27 Kip1 is an inhibitor of multiple cyclin-dependent kinases (cdk), and can arrest the cell-cycle progression by inhibiting the phosphorylation of the retinoblastoma gene family products. Tumor formation in p27 Kip1 knockout mice clearly shows that p27 Kip1 plays an important role in inhibiting tumor formation and progression. To investigate the mechanism of transcriptional p27 Kip1 gene expression, we isolated the genomic DNA fragment of the 5′ flanking region of the human p27 Kip1 gene and characterized its promoter region. The human p27 Kip1 promoter is TATA-less, and the sequence is highly homologous to the murine p27 Kip1 promoter sequence. In the promoter assay, deletion from −774 to −435 relative to the initiating codon resulted in a 15–20-fold reduction of the p27 Kip1 promoter activity, suggesting that the elements for basal promoter activity exist in this highly conserved 340 bp region, where putative CTF and ATF sites are conserved.