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Cyclin-dependent kinases (CDKs) are serine/threonine kinases that regulate cell cycle and have been vigorously pursued as druggable targets for cancer. There are over 20 members of the CDK family. Given their structural similarity, selective inhibition by small molecules has been elusive. In addition, collateral damage to highly proliferative normal cells by CDK inhibitors remains a safety concern. Intestinal epithelial cells are highly proliferative and the impact of individual CDK inhibition on intestinal cell proliferation has not been well studied. Using the rat intestinal epithelial (IEC6) cells as an in vitro model, we found that the selective CDK4/6 inhibitor palbociclib lacked potent anti-proliferative activity in IEC6 relative to the breast cancer cell line MCF7, indicating the absence of intestinal cell reliance on CDK4/6 for cell cycle progression. To further illustrate the role of CDKs in intestinal cells, we chose common targets of CDK inhibitors (CDK 1, 2, 4, 6, and 9) for targeted gene knockdown to evaluate phenotypes. Surprisingly, only CDK1 and CDK9 knockdown demonstrated profound cell death or had moderate growth effects, respectively. CDK2, 4, or 6 knockdowns, whether single, double, or triple combinations, did not have substantial impact. Studies evaluating CDK1 knockdown under various cell seeding densities indicate direct effects on viability independent of proliferation state and imply a potential noncanonical role for CDK1 in intestinal epithelial biology. This research supports the concept that CDK1 and CDK9, but not CDKs 2, 4, or 6, are essential for intestinal cell cycle progression and provides safety confidence for interphase CDK inhibition., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

AG-012986 is a pan-CDK (cyclin-dependent kinase) inhibitor that has in vitro and in vivo antitumor properties but was stopped in development due in part to rapid bone-marrow-independent white blood cell toxicity in preclinical studies and the potential for acute and delayed immunosuppression in humans. Because peripheral lymphocytes are largely nonproliferating, it was hypothesized the toxicity of AG-012986 was due to an off-target mechanism and not driven by the intended pharmacology. We show the toxicity mechanism in primary human immune cells is caspase driven. T-cells treated with AG-012986 and acutely stimulated through the T-cell receptor exhibited decreased toxicity while still maintaining cell division inhibition. This indicated that the pharmacology of AG-012986 functioned as expected but the toxicity had now been decoupled through activation. Induced phosphorylation of p38 and IL-2 production was impaired with AG-012986. Thus, AG-012986 could cause apoptosis of T-cells by targeting upstream kinases in the p38 Mitogen-activated protein kinase (MAPK) pathway and impairing cellular survival., (Copyright © 2012 Wiley Periodicals, Inc.)

Purpose: Cyclin-dependent kinases (CDKs) play a significant role in the control of cell-cycle progression and exhibit aberrant regulation in various neoplastic diseases. AG-024322 is a potent inhibitor of CDK1, CDK2, and CDK4 that produces cell-cycle arrest and antitumor activity in preclinical models. This study evaluated the toxicity of AG-024322 when given by intravenous (IV) infusion to cynomolgus monkeys, including reversibility of effects., Methods: Male and female monkeys received AG-024322 by 30-min IV infusion once daily for 5 days at doses of 2, 6, and 10 mg/kg (24, 72, and 120 mg/m(2), respectively). Controls received vehicle alone which was aqueous 5% dextrose, pH 3.8. Three animals/sex/group were necropsied on day 6, and two animals/sex/group at 6 and 10 mg/kg were necropsied on day 22 (reversal cohort). Doses were based upon the results of a dose range-finding study in monkeys; decreased white blood cells occurred at > or =3 mg/kg and 12 mg/kg produced central nervous system effects and was above the maximum-tolerated dose., Results: No deaths occurred and clinical signs of toxicity, including swelling at the IV administration site, were seen at > or =6 mg/kg. AG-024322 at > or =6 mg/kg produced pancytic bone marrow hypocellularity, lymphoid depletion, and vascular injury at the injection site. Renal tubular degeneration occurred at 10 mg/kg. These changes were either reversible or in a process of repair following the 17-day recovery period. Hematology changes included decreases in reticulocytes and/or granulocytes at > or =6 mg/kg, which were reversible and consistent with changes in the bone marrow. Lymphoid and bone marrow depletion are consistent with pharmacologic inhibition of CDKs by AG-024322 and were expected findings. On day 22, vacuolar degeneration of pancreatic acinar cells with increased serum amylase and lipase levels occurred in one female at 10 mg/kg. Neither sex-related differences in toxicokinetics nor plasma accumulation over 5 days of dosing were seen. Terminal phase overall mean half-life on day 5 ranged from 6.69 to 8.87 h (across dose levels) and was not dose dependent., Conclusion: The no-adverse-effect dose of AG-024322 was 2 mg/kg and associated with overall mean plasma AUC(0-24.5) of 2.11 microg h/mL.

AG-012986 is a multitargeted cyclin-dependent kinase (CDK) inhibitor active against CDK1, CDK2, CDK4/6, CDK5, and CDK9, with selectivity over a diverse panel of non-CDK kinases. Here, we report the potent antitumor efficacies of AG-012986 against multiple tumor lines in vitro and in vivo. AG-012986 showed antiproliferative activities in vitro with IC(50)s of <100 nmol/L in 14 of 18 tumor cell lines. In vivo, significant antitumor efficacy induced by AG-012986 was seen (tumor growth inhibition, >83.1%) in 10 of 11 human xenograft tumor models when administered at or near the maximum tolerated dose for 8 or 12 days. AG-012986 caused dose-dependent hypophosphorylation at Ser(795) of the retinoblastoma protein, cell cycle arrest, and apoptosis in vitro. Colony-forming assays indicated that the potency of AG-012986 substantially decreased with treatment time of <24 h. In vivo, AG-012986 also showed dose-dependent retinoblastoma Ser(795) hypophosphorylation, cell cycle arrest, decreased Ki-67 tumor staining, and apoptosis in conjunction with antitumor activity. Studies comparing i.p. bolus with s.c. implanted minipump dosing regimens revealed that in vivo efficacy correlated with the duration of minimally effective plasma levels rather than maximal drug plasma levels. Dosing optimization of AG-012986 provided guidance for selecting a treatment schedule to achieve the best antitumor efficacy while minimizing the risk of adverse side effects.

Cyclin-dependent kinases (CDKs) have been pursued for more than a decade for the treatment of cancer. CDK inhibitors are expected to slow the rate of cell division and potentially increase the apoptotic fraction of rapidly dividing cells. Although CDK activity is often increased in tumors, normal dividing tissues are also susceptible to the cytostatic and cytotoxic effects of CDK inhibitor action. Therefore the typical toxicity profile associated with cytotoxic anti-cancer therapy, bone marrow suppression and gastrointestinal toxicity, is expected with CDK inhibitors. Bone marrow toxicity and the ensuing delayed peripheral leukocyte suppression often limit the therapeutic application of cytotoxic anticancer drugs. Here we characterize an unusual bone marrow-independent acute toxicity toward leukocytes from broad spectrum CDK inhibitors in monkeys and rodents. The potential combination of both acute and delayed immunosuppression would likely further restrict the application of these particular compounds. Since the cells targeted were non-proliferating, it was assumed that the toxicity was not driven by the intended pharmacological mechanism thereby facilitating the development of a testing strategy to identify compounds with a reduced potential for acute leukocyte toxicity. This testing strategy resulted in a CDK inhibitor void of bone marrow-independent leukocyte toxicity that is currently undergoing clinical testing.

Cyclin-dependent kinases (cdks) play a crucial role in cell cycle regulation and are considered promising targets for cancer therapy. Intravenous administration of AG-012986, a pan-cyclin-dependent kinase inhibitor (cdk(i)), resulted in unexpected retinal and peripheral nerve toxicity in mice. AG-012986 was administered daily to CD-1 or B6C3F1 mice for 5 consecutive days. Mice were euthanized 24 h after the last dose (study day 6) or after a 21-day post-dose period (study day 26). Compound related microscopic findings were seen in the sciatic nerves (axonal degeneration) of both strains and in the retina (retinal degeneration/atrophy) of CD-1 mice only after the post-dose period. Although retinal degeneration/atrophy was not detected by routine histology in mice euthanized on day 6, apoptotic retinal cells were evident at this time using TUNEL assay. To our knowledge retinal or peripheral nerve toxicity secondary to the administration of cdk(i)s has not been previously reported. Although the pathogenesis of these lesions is unclear, the toxicities may reflect the unique profile of cdk inhibition, off-target kinase inhibition or receptor binding, or metabolism/distribution properties of AG-012986. Multi-targeted-inhibitors may interfere with cdks and other kinases involved in a wide range of functions other than cell cycle regulation, which could result in unexpected toxicities that may hinder their clinical applications.