AT7519, a Cyclin-Dependent Kinase Inhibitor, Exerts Its Effects by Transcriptional Inhibition in Leukemia Cell Lines and Patient Samples

Abstract
AT7519 is a potent inhibitor of multiple cyclin-dependent kinases and is currently in early-phase clinical development. Recent research has identified cyclin-dependent kinases 7, 8, and 9 as key regulators of transcription through phosphorylation of RNA polymerase II. B-cell lymphoproliferative disorders, including chronic lymphocytic leukemia (CLL), rely on short-lived transcripts such as Mcl-1, Bcl-2, and XIAP for survival. This study evaluates the activity of AT7519 in leukemia cell lines and compares responses with those observed in cell lines from solid tumors. The analysis is extended to primary peripheral blood mononuclear cells isolated from sixteen CLL patients.

AT7519 induced apoptosis in leukemia cells at concentrations between 100 and 700 nanomolar and showed similar effectiveness regardless of Rai stage or established prognostic indicators. Short-term exposure (four to six hours) inhibited phosphorylation of RNA polymerase II and caused a significant reduction in Mcl-1 protein levels without affecting XIAP or Bcl-2. The decline in Mcl-1 was associated with increased levels of cleaved poly(ADP-ribose) polymerase, indicating activation of apoptotic pathways. These findings suggest AT7519 has therapeutic potential for patients with advanced B-cell leukemia.

Introduction
AT7519 is a novel multitargeted inhibitor designed to selectively inhibit cyclin-dependent kinases 1, 2, 4, 5, and 9, with minimal off-target effects except partial inhibition of glycogen synthase kinase-3β. Developed through structure-based drug design using high-throughput X-ray crystallography, its efficacy has been demonstrated in preclinical models and phase I clinical trials involving patients with refractory solid tumors. More recently, cyclin-dependent kinases 7, 8, and 9 have gained attention for their role in transcription regulation via phosphorylation of the COOH-terminal domain of RNA polymerase II, which facilitates transcription initiation and elongation. Inhibiting these kinases selectively reduces levels of short-lived transcripts encoding proteins involved in cell cycle control, mitosis, NF-κB signaling, and apoptosis, including Mcl-1 and XIAP. Reducing these transcripts and their proteins may produce antitumor effects or increase cell susceptibility to apoptosis.

AT7519 shows potent activity against solid tumor cell survival mainly through cell cycle inhibition but also appears to impair transcriptional processes. Based on this transcriptional effect, it was hypothesized that AT7519 would induce apoptosis in leukemia cells, including those from CLL patients. Consistent with this, AT7519 inhibited phosphorylation of RNA polymerase II and rapidly decreased Mcl-1 protein levels in leukemia cells. In animal models, AT7519 treatment of mice bearing HL60 tumors reduced Mcl-1 protein expression. The most effective dosing in leukemia-bearing mice was once daily at 15 mg/kg, differing from the twice-daily dosing needed in solid tumor models. These distinctions suggest AT7519 acts via different mechanisms in leukemia versus solid tumors.

Chronic lymphocytic leukemia is marked by malignant B cells with dysregulated proliferation and apoptotic pathways. Despite treatments including chlorambucil, fludarabine, alemtuzumab, rituximab, bendamustine, lenalidomide, lumiliximab, flavopiridol, and oblimerson, significant improvements in progression-free and overall survival remain limited. Cyclin-dependent kinases and their associated regulators are central to cell cycle control and are often deregulated in cancer, making them attractive therapeutic targets. Several cdk inhibitors are in development, including flavopiridol, which has shown promising early clinical results in refractory CLL. Other agents such as seliciclib and SNS-032 have demonstrated preclinical efficacy and are under clinical investigation.

In this study, primary CLL cells were obtained from patients across different disease stages, including low and high Rai classifications. Samples were characterized for cytogenetic abnormalities and ZAP70 expression. AT7519 effectively induced apoptosis irrespective of these prognostic markers. ZAP70 overexpression is generally linked with poorer progression-free survival, while cytogenetic changes such as 13q deletion and trisomy 12 have variable prognostic impact. More severe p53 pathway abnormalities like deletions at 17p or 11q are less frequent. The 13q deletion affects regions encoding microRNAs, though their precise functional targets remain unclear.

Since transcriptional cdks regulate short-lived antiapoptotic transcripts, intermittent inhibition could selectively reduce antiapoptotic proteins in tumor cells while sparing normal cells. The data support the hypothesis that AT7519 induces apoptosis in CLL primarily through transcriptional inhibition, acting independently of cytogenetic background. These promising results warrant further clinical evaluation. Phase I pharmacokinetic data indicate that clinically tolerated doses of AT7519 achieve plasma levels sufficient to induce apoptosis in ex vivo CLL samples.

Materials and Methods
Materials
AT7519 is a small-molecule inhibitor synthesized by Astex Therapeutics Ltd. with a molecular weight of 382 daltons.

Cells
HL60 and HCT116 cell lines were obtained from certified biological resource centers and cultured in RPMI 1640 and DMEM media, respectively, each supplemented with 10% fetal bovine serum. Cells were incubated at 37 degrees Celsius in a humidified atmosphere containing 5% carbon dioxide. All cell culture reagents were commercially sourced. Cell line authenticity was confirmed through DNA profiling and isozyme analysis. Experiments were conducted using cultures maintained for less than six months post-thaw from original stocks.

Patient Samples and Preparation
Peripheral blood samples were collected from sixteen patients diagnosed with chronic lymphocytic leukemia across Rai stages 0/I, II, and III/IV. Samples were obtained with informed consent according to an approved institutional protocol. Whole blood was processed to isolate cells and serum. Mononuclear cells were separated by density gradient centrifugation and either cryopreserved at minus 80 degrees Celsius or maintained in RPMI 1640 medium supplemented with 10% human serum at 37 degrees Celsius in a 5% carbon dioxide atmosphere. Serum samples were aliquoted and stored at minus 80 degrees Celsius for future analysis. Isolated CLL cells were cultured under standard conditions for subsequent experiments.

Tritiated Uridine Assay
mRNA synthesis was assessed by measuring incorporation of tritiated uridine into mRNA transcripts. HCT116 cells were seeded in 96-well plates at 2 × 10⁴ cells per well in 72 microliters of complete medium and allowed to recover overnight. Compounds were diluted in medium and added to non-edge wells, maintaining a final concentration of 0.1% DMSO. Plates were incubated at 37 degrees Celsius for 30 minutes. Tritiated uridine (0.1 μCi) was then added to each well, followed by incubation for 3.5 hours. Media were removed and cells fixed with 5% ice-cold trichloroacetic acid. Plates were washed with trichloroacetic acid and distilled water, then air-dried. Subsequently, 50 microliters of 0.1 mol/L sodium hydroxide was added to each well, followed by 200 microliters of scintillation fluid. Samples were mixed and counted using a scintillation counter.

MTS Cell Viability Assays
B cells were seeded at 300,000 cells per well in 96-well plates and allowed to recover overnight. AT7519 or vehicle control (0.5% DMSO) was added and incubated for 72 hours. Afterwards, 20 microliters of MTS reagent was added to each well. Plates were incubated for 6 hours before absorbance was measured at 490 nm. Background values were subtracted, and IC50 values were calculated using GraphPad Prism software.

Apoptosis Assays
B cells were seeded at 4 × 10⁶ cells per well in 12-well plates and allowed to recover overnight. AT7519 or vehicle control (0.5% DMSO) was added for 4 to 6 hours. A 20-microliter sample of culture was mixed with ethidium bromide and acridine orange, covered with a coverslip, and examined under a UV microscope. Cells were classified morphologically as normal, apoptotic, or necrotic. Two hundred cells were counted per slide, with all experiments performed in triplicate.

Real-Time Quantitative PCR for ZAP70 Expression
B cells were isolated by Ficoll gradient centrifugation. Fifteen milliliters of blood were layered on 12.5 milliliters of Ficoll and centrifuged at 500 g for 35 minutes. The B-cell layer was collected and washed three times with sterile Dulbecco’s phosphate-buffered saline. One hundred nanograms of total RNA was reverse transcribed in a 20-microliter reaction using SuperScript III Platinum Reverse Transcriptase, incubated at 42 degrees Celsius for 50 minutes, followed by RNase H treatment at 37 degrees Celsius for 20 minutes. Real-time PCR was performed using an Opticon DNA Engine. One microliter of cDNA was added to 12.5 microliters of Platinum SYBR Green qPCR SuperMix-UDG, 1 microliter of gene-specific or β-actin primer pair, and 10.5 microliters of distilled water, for a total volume of 25 microliters. Amplification consisted of 44 cycles of 15 seconds at 95 degrees Celsius, 30 seconds at 55 degrees Celsius, and 30 seconds at 72 degrees Celsius. Threshold cycle values represented fluorescence detection points. Results were normalized to β-actin expression and compared with normal B-cell controls to calculate relative expression ratios. All reactions were performed in triplicate.

PCR primers were designed using MacVector software to generate amplicons between 80 and 250 base pairs to optimize qPCR efficiency. β-actin primers were obtained from QuantumRNA β-Actin Internal Standards for normalization.

Cytogenetics
A standard fluorescence in situ hybridization panel for chronic lymphocytic leukemia was performed by a reference laboratory.

Immunoblotting
HL60 and chronic lymphocytic leukemia (CLL) cells were seeded at concentrations of 1 × 10⁶ and 4 × 10⁶ cells per well, respectively, in six-well plates and allowed to recover for 16 hours. After recovery, AT7519 was added at various concentrations or vehicle control (0.1% DMSO) for specified durations. Following treatment, cells were harvested and lysed in 100 microliters of ice-cold Triton lysis buffer. The lysates were clarified by centrifugation to remove debris, and protein concentrations were measured. Equal amounts of protein from each sample were mixed with SDS sample buffer and boiled for 5 minutes to denature the proteins. The samples were then separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes for protein detection. Membranes were incubated with primary antibodies targeting Mcl-1, Bcl-2, cleaved PARP, total retinoblastoma protein (Rb), phosphorylated Rb at threonine 821, RNA polymerase II, and β-actin as a loading control. After washing, membranes were probed with infrared dye-labeled secondary antibodies, and the signal was detected using an infrared imaging system.

Pharmacodynamic Studies
In vivo pharmacodynamic studies were conducted using subcutaneous xenograft tumors excised from nude mice at predetermined time points after a single intraperitoneal injection of AT7519 at a dose of 10 mg/kg. Tumor tissues were flash-frozen in liquid nitrogen and pulverized to a fine powder. Proteins were extracted from the pulverized tumor tissue using Triton lysis buffer, and protein concentrations were determined. Western blotting was performed following the same procedure as described for the cell lysates to analyze relevant pharmacodynamic markers.

Xenograft Studies
Animal experiments were carried out using male BALB/c nu/nu mice, aged six to eight weeks, housed under pathogen-free conditions in compliance with regulatory guidelines. Mice were implanted subcutaneously in the right flank with 1 × 10⁷ HL60 cells. After tumors were allowed to establish over 11 to 14 days, mice were grouped based on tumor volume, which averaged around 75 mm³. Treatment was administered according to a predetermined dosing schedule. Tumor volumes were measured regularly every two to three days throughout the study. Treatment efficacy was assessed by comparing tumor volume reduction or growth inhibition against control groups. Complete tumor regression was defined as a tumor volume below detectable levels, specifically less than 3 mm in any dimension. Tolerability of the treatment was monitored by tracking body weight, clinical signs, and overall survival of the animals.

Results

AT7519 Inhibits Transcription in Human Tumor Cell Lines
AT7519 is a potent inhibitor of several cyclin-dependent kinases, including CDK9, a critical regulator of transcriptional activity. In the HL60 leukemia cell line, AT7519 completely inhibited phosphorylation of RNA polymerase II at serine residues 2 and 5 within the carboxy-terminal domain at concentrations exceeding 400 nanomolar. This inhibition of phosphorylation correlated with a rapid and significant decrease in the levels of proteins characterized by short half-lives, such as the anti-apoptotic protein Mcl-1. The reduction in Mcl-1 protein levels was observed within four hours of treatment with AT7519 at concentrations greater than 400 nanomolar.

The transcriptional inhibitory activity of AT7519 was further confirmed in HCT116 cells by a decrease in the incorporation of tritiated uridine into the mRNA fraction. The mean inhibitory concentration (IC50) for this effect was approximately 54 nanomolar, demonstrating the compound’s potency in suppressing transcription, thereby affecting cell survival.

In a subcutaneous xenograft model using HL60 cells, a single intraperitoneal dose of 10 mg/kg AT7519 led to inhibition of markers associated with cyclin-dependent kinase 2 activity, including phosphorylated nucleophosmin and phosphorylated retinoblastoma protein. This inhibition persisted for eight to sixteen hours post-dose. Mcl-1 protein levels decreased within one hour of dosing and remained suppressed for at least twenty-four hours. These findings support the hypothesis that transcriptional inhibition is the primary mechanism by which AT7519 exerts its effect in leukemia cells and suggest that once-daily dosing may be more effective than twice-daily dosing in this context.

Although inhibition of cell cycle markers was transient, previous studies with solid tumor cell lines indicated that more frequent dosing was required to maintain efficacy. This suggests that in leukemia cells, which depend heavily on short-lived transcripts such as Mcl-1 for survival, AT7519’s main effect is transcriptional inhibition. Nonetheless, inhibition of cell cycle targets was also observed and may contribute to apoptosis induction.

AT7519 Is Cytotoxic to Chronic Lymphocytic Leukemia Patient-Derived Cells
Chronic lymphocytic leukemia cells from sixteen patients representing a range of disease stages, including low and high Rai stages, were isolated and studied. These patient samples were well characterized clinically, including information on prior treatments, expression of the prognostic marker ZAP70, and cytogenetic abnormalities relevant to prognosis. After seventy-two hours of exposure to AT7519, cell viability was assessed using an MTS assay. The half-maximal effective concentration (EC50) values ranged between 100 and 700 nanomolar across patient samples, with an average of approximately 264 nanomolar. This demonstrates that AT7519 is active against CLL cells regardless of disease stage, treatment history, ZAP70 expression, or cytogenetic profile.

Kinetic studies of cytotoxicity showed that the effective concentration required to reduce cell viability decreased with longer exposure times, reaching minimal values at approximately twenty-four hours. Notably, significant cytotoxic effects were observed after only four hours of treatment, with effective concentrations below one micromolar in most samples. This suggests that short-term exposure to AT7519 at micromolar concentrations is sufficient to induce substantial cell death in CLL cells.

AT7519 Induces Apoptosis in Chronic Lymphocytic Leukemia Patient-Derived Cells
The cytotoxic mechanism of AT7519 was investigated by examining nuclear morphology after acridine orange staining. Apoptotic cells were quantified as a percentage of the total cell population. A significant increase in apoptosis was detected after six hours of treatment with various concentrations of AT7519 across multiple patient samples. A smaller, yet notable, increase in apoptosis was also observed after four hours of exposure. These findings indicate that even brief exposure to AT7519 can commit CLL cells to apoptosis.

AT7519 Inhibits RNA Polymerase II Phosphorylation and Reduces Levels of Anti-Apoptotic Proteins
CLL cells treated with AT7519 for six hours showed inhibition of RNA polymerase II phosphorylation on the carboxy-terminal domain repeats at concentrations of 300 nanomolar or higher. This was accompanied by a reduction in the anti-apoptotic protein Mcl-1, consistent with AT7519’s role in suppressing Mcl-1 mRNA transcription. Additionally, an increase in cleaved poly(ADP-ribose) polymerase (PARP), an apoptosis marker, was observed following treatment. In some patient samples, apoptosis occurred despite only modest Mcl-1 reduction, suggesting either heightened sensitivity to Mcl-1 depletion or involvement of other prosurvival proteins. Levels of the anti-apoptotic protein Bcl-2 and phosphorylation of retinoblastoma protein remained unchanged, reflecting the typically non-proliferative state of circulating CLL cells. The temporal sequence showed rapid inhibition of RNA polymerase II phosphorylation between four and six hours of treatment, followed by a decline in Mcl-1 protein levels and peak apoptosis markers at twenty-four hours. These results support that AT7519 primarily induces cytotoxicity in CLL cells through transcriptional inhibition.

Discussion
AT7519 is a potent inhibitor of multiple cyclin-dependent kinases, including those regulating transcription via phosphorylation of RNA polymerase II carboxy-terminal domain repeats. This study shows that AT7519 inhibits RNA polymerase II phosphorylation in human tumor cell lines, consistent with global transcriptional suppression as indicated by reduced tritiated uridine incorporation. In leukemia models, this transcriptional inhibition leads to decreased levels of short-lived proteins, including the anti-apoptotic protein Mcl-1. Mcl-1 reduction occurs rapidly and precedes increased apoptosis in tumor cells. These effects were observed in vitro at concentrations of 400 nanomolar and in vivo after a 10 mg/kg intraperitoneal dose. Optimal in vivo dosing suggested a dominant anti-transcriptional effect, with greater efficacy achieved after a once-daily dose in leukemia models. This contrasts with solid tumor models, where maximal efficacy required dividing the daily dose into twice-daily administrations to better target the cell cycle. Solid tumor xenografts responded mainly through cell cycle inhibition and depend less on short-lived anti-apoptotic proteins, making them less sensitive to their loss. Chronic lymphocytic leukemia cells rely heavily on short-lived anti-apoptotic proteins like Mcl-1 and are generally quiescent in peripheral blood, so AT7519’s cell cycle effects in ex vivo CLL cells are minimal. Based on these observations, it was hypothesized that AT7519 would effectively inhibit CLL cell survival.

CLL is classified by Rai stage and prognostic markers such as ZAP70 expression, where high ZAP70 correlates with more aggressive disease, as well as by cytogenetic abnormalities predicting disease progression, therapy response, or time to treatment initiation. Patients were selected to represent the heterogeneity of CLL. The cytotoxic effects of AT7519 were characterized ex vivo with respect to concentration and exposure duration. Data showed that exposure to AT7519 for four to six hours was sufficient to commit CLL cells to apoptosis at concentrations below one micromolar. Rai stage, ZAP70 expression, and cytogenetic abnormalities did not significantly affect sensitivity; AT7519 demonstrated similar activity across all samples, including those from high-risk patients predicted to respond poorly to standard therapies. Cytotoxicity correlated with inhibition of RNA polymerase II phosphorylation and decreased Mcl-1 protein levels, while Bcl-2 levels remained unchanged. These changes occurred rapidly within four hours of exposure at concentrations below 400 nanomolar.

Similar pan-cyclin-dependent kinase inhibitors have been studied in CLL. Flavopiridol, the most clinically advanced, exerts comparable effects on transcription and Mcl-1 turnover in CLL cells ex vivo and has shown promising clinical responses. Flavopiridol is highly protein-bound in human plasma, has low volume of distribution, and rapid clearance, requiring a dosing schedule involving a short loading infusion followed by extended maintenance. Its main dose-limiting toxicity is tumor lysis syndrome due to rapid apoptosis of CLL blasts in patients with high white blood cell counts. SNS-032 is another pan-CDK inhibitor under clinical investigation in CLL with a similar two-stage dosing regimen. CYC-202 also shows activity in CLL samples but with lower potency than AT7519 or the other compounds.

Phase I clinical trials of AT7519 demonstrated a favorable pharmacokinetic profile, achieving plasma concentrations at tolerated doses predicted to be effective against CLL blasts based on preclinical data. These doses maintain pharmacologically active levels up to 500 nanomolar for over twelve hours. AT7519 shows moderate protein binding of 57% in human plasma and a high volume of distribution of 1.4 L/kg in mice. This pharmacokinetic profile supports efficacy in CLL patients with intermittent dosing schedules. AT7519 is expected to be effective across a broad CLL patient population, including those refractory to purine analogue-based therapies. A single-agent phase II clinical trial is planned.