Michelle D. Garrett

First-in-Man Clinical Trial of the Oral Pan-AKT Inhibitor MK-2206 in Patients With Advanced Solid Tumors

PURPOSE AKT signaling is frequently deregulated in human cancers. MK-2206 is a potent, oral allosteric inhibitor of all AKT isoforms with antitumor activity in preclinical models. A phase I study of MK-2206 was conducted to investigate its safety, maximum-tolerated dose (MTD), pharmacokinetics (PKs), pharmacodynamics (PDs), and preliminary antitumor efficacy. PATIENTS AND METHODS Patients with advanced solid tumors received MK-2206 on alternate days. Paired tumor biopsies were mandated at the MTD for biomarker studies. PD studies incorporated tumor and hair follicle analyses, and putative predictive biomarker studies included tumor somatic mutation analyses and immunohistochemistry for phosphatase and tensin homolog (PTEN) loss. RESULTS Thirty-three patients received MK-2206 at 30, 60, 75, or 90 mg on alternate days. Dose-limiting toxicities included skin rash and stomatitis, establishing the MTD at 60 mg. Drug-related toxicities included skin rash (51.5%), nausea (36.4%), pruritus (24.2%), hyperglycemia (21.2%), and diarrhea (21.2%). PKs (area under the concentration-time curve from 0 to 48 hours and maximum measured plasma concentration) were dose proportional. Phosphorylated serine 473 AKT declined in all tumor biopsies assessed (P = .015), and phosphorylated threonine 246 proline-rich AKT substrate 40 was suppressed in hair follicles at 6 hours (P = .008), on days 7 (P = .028) and 15 (P = .025) with MK-2206; reversible hyperglycemia and increases in insulin c-peptide were also observed, confirming target modulation. A patient with pancreatic adenocarcinoma (PTEN loss; KRAS G12D mutation) treated at 60 mg on alternate days experienced a decrease of approximately 60% in cancer antigen 19-9 levels and 23% shrinkage in tumor measurements. Two patients with pancreatic neuroendocrine tumors had minor tumor responses. CONCLUSION MK-2206 was well tolerated, with evidence of AKT signaling blockade. Rational combination trials are ongoing to maximize clinical benefit with this therapeutic strategy.

The ZO-1-associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density.

Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1–associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction–associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1–based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4.

CHK2 kinase: cancer susceptibility and cancer therapy – two sides of the same coin?

In the past decade, CHK2 has emerged as an important multifunctional player in the DNA-damage response signalling pathway. Parallel studies of the human CHEK2 gene have also highlighted its role as a candidate multiorgan tumour susceptibility gene rather than a highly penetrant predisposition gene for Li–Fraumeni syndrome. As discussed here, our current understanding of CHK2 function in tumour cells, in both a biological and genetic context, suggests that targeted modulation of the active kinase or exploitation of its loss in tumours could prove to be effective anti-cancer strategies.

The Cyclin-dependent Kinase Inhibitor CYC202 (R-Roscovitine) Inhibits Retinoblastoma Protein Phosphorylation, Causes Loss of Cyclin D1, and Activates the Mitogen-activated Protein Kinase Pathway

Deregulation of the cell cycle commonly occurs during tumorigenesis, resulting in unrestricted cell proliferation and independence from mitogens. Cyclin-dependent kinase inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells. CYC202 (R-roscovitine) is a potent inhibitor of CDK2/cyclin E that is undergoing clinical trials. Drugs selected to act on a particular molecular target may exert additional or alternative effects in intact cells. We therefore studied the molecular pharmacology of CYC202 in human colon cancer cells. Treatment of HT29 and KM12 colon carcinoma cell lines with CYC202 decreased both retinoblastoma protein phosphorylation and total retinoblastoma protein. In addition, an increase in the phosphorylation of extracellular signal-regulated kinases 1/2 was observed. As a result, downstream activation of the mitogen-activated protein kinase pathway occurred, as demonstrated by an increase in ELK-1 phosphorylation and in c-FOS expression. Use of mitogen-activated protein kinase kinases 1/2 inhibitors showed that the CYC202-induced extracellular signal-regulated kinases 1/2 phosphorylation was mitogen-activated protein kinase kinases 1/2 dependent but did not contribute to the cell cycle effects of the drug, which included a reduction of cells in G1, inhibition of bromodeoxyuridine incorporation during S-phase, and a moderate increase in G2-M phase. Despite activation of the mitogen-activated protein kinase pathway, cyclin D1 protein levels were decreased by CYC202, an effect that occurred simultaneously with loss of retinoblastoma protein phosphorylation and inhibition of cell cycle progression. The reduced expression of cyclin D1 protein was independent of the p38SAPK and phosphatidylinositol 3-kinase pathways, which are known regulators of cyclin D1 protein. Interestingly, CYC202 caused a clear reduction in cyclins D1, A, and B1 mRNA, whereas c-FOS mRNA increased by 2-fold. This was accompanied by a loss of RNA polymerase II phosphorylation and total RNA polymerase II protein, suggesting that CYC202 was inhibiting transcription, possibly via inhibition of CDK7 and CDK9 complexes. It can be concluded that although CYC202 can act as a CDK2 inhibitor, it also has the potential to inhibit CDK4 and CDK1 activities in cancer cells through the down-regulation of the corresponding cyclin partners. This provides a possible mechanism by which CYC202 can cause a reduction in retinoblastoma protein phosphorylation at multiple sites and cell cycle arrest in G1, S, and G2-M phases. In addition to providing useful insights into the molecular pharmacology of CYC202 in human cancer cells, the results also suggest potential pharmacodynamic end points for use in clinical trials with the drug.

Regulation of NF‐κB and p53 through activation of ATR and Chk1 by the ARF tumour suppressor

The ARF tumour suppressor is a central component of the cellular defence against oncogene activation. In addition to activating p53 through binding Mdm2, ARF possesses other functions, including an ability to repress the transcriptional activity of the antiapoptotic RelA(p65) NF‐κB subunit. Here we demonstrate that ARF induces the ATR‐ and Chk1‐dependent phosphorylation of the RelA transactivation domain at threonine 505, a site required for ARF‐dependent repression of RelA transcriptional activity. Consistent with this effect, ATR and Chk1 are required for ARF‐induced sensitivity to tumour necrosis factor α‐induced cell death. Significantly, ATR activity is also required for ARF‐induced p53 activity and inhibition of proliferation. ARF achieves these effects by activating ATR and Chk1. Furthermore, ATR and its scaffold protein BRCA1, but not Chk1, relocalise to specific nucleolar sites. These results reveal novel functions for ARF, ATR and Chk1 together with a new pathway regulating RelA NF‐κB function. Moreover, this pathway provides a mechanism through which ARF can remodel the cellular response to an oncogenic challenge and execute its function as a tumour suppressor.

Biomarker-driven Early Clinical Trials in Oncology: A Paradigm Shift in Drug Development

Early clinical trials represent a crucial bridge between preclinical drug discovery and the especially resource-intense randomized phase III trial—the definitive regulatory hurdle for drug approval. High attrition rates and rising costs, when coupled with the extraordinary opportunities opened up by cancer genomics and the promise of personalized medicine call for new approaches in the conduct and design of phase I/II trials. The key challenge lies in increasing the odds for successful and efficient transition of a compound through the drug development pipeline. The incorporation of scientifically and analytically validated biomarkers into rationally designed hypothesis-testing clinical trials offers a promising way forward to achieving this objective. In this article, we provide an overview of biomarkers in early clinical trials, including examples where they have been particularly successful, and the caveats and pitfalls associated with indiscriminate application. We describe the use of pharmacodynamic end points to demonstrate the proof of modulation of target, pathway, and biologic effect, as well as predictive biomarkers for patient selection and trial enrichment. Establishing the pharmacologic audit trail provides a means to assess and manage risk in a drug development program and thus increases the rationality of the decision-making process. Accurate preclinical models are important for pharmacokinetic-pharmacodynamic-efficacy modeling and biomarker validation. The degree of scientific and analytical validation should ensure that biomarkers are fit-for purpose, according to the stage of development and the impact on the trial; specifically they are either exploratory or used to make decisions within the trial. To be maximally useful at an early stage, these must be in place before the commencement of phase I trials. Validation and qualification of biomarkers then continues through clinical development. We highlight the impact of modern technology platforms, such as genomics, proteomics, circulating tumor cells, and minimally invasive functional and molecular imaging, with respect to their potential role in improving the success rate and speed of drug development and in interrogating the consequences of therapeutic intervention and providing a unique insight into human disease biology. With these technologies already having an impact in the clinic today, we predict that further future advances will come from the application of network analysis to clinical trials, leading to individualized systems-based medicine for cancer.

Anticancer therapy with checkpoint inhibitors: what, where and when?

Research into inhibitors of the protein kinases controlling the cellular response to DNA damage has reached an exciting stage, particularly for the checkpoint kinases CHK1 and CHK2. Selective inhibitors are now being tested in clinical trials in cancer patients. In this review, we highlight recent data from cellular and in vivo preclinical models that provide insight into the clinical contexts for checkpoint kinase inhibition (e.g. the timing of treatment and what type of inhibitor would be most appropriate). Although it has been shown that CHK1 inhibition potentiates the efficacy of various DNA-damaging therapies, the context for selective CHK2 inhibition is not yet as well defined. Distinct effects of selective CHK1 or CHK2 inhibition are observed when combined with DNA-damaging agents. It has also been shown that both CHK1 and CHK2 inhibitors potentiate the effects of other molecular targeted therapeutics [e.g. poly(ADP-ribose) polymerase inhibitors]. We also consider the single-agent activity of checkpoint kinase inhibitors for tumours with defined genetic backgrounds.

Discovering novel chemotherapeutic drugs for the third millennium

There is enormous potential for the discovery of innovative cancer drugs with improved efficacy and selectivity for the third millennium. In this review we show how novel mechanism-based agents are being discovered by focusing on the molecular targets and pathways that are causally involved in cancer formation, maintenance and progression. We also show how new technologies, from genomics through high through-put bioscience, combinatorial chemistry, rational drug design and molecular pharmacodynamic and imaging techniques, are accelerating the pace of cancer drug discovery. The process of contemporary small molecule drug discovery is described and progress and current issues are reviewed. New and potential targets and pathways for therapeutic intervention are illustrated. The first examples of a new generation of molecular therapeutics are now entering hypothesis-testing clinical trials and showing activity. The early years of the new millennium will see a range of exciting new agents moving from bench to bedside and beginning to impact on the management and cure of cancer.

Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange

The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine–glutamine/threonine–glutamine cluster domain (SCD), by ATM. The phosphorylated SCD‐segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T‐loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small‐molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T‐loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM‐phosphorylation, and also suggests a mechanism for dimerisation‐driven activation of Chk2 by trans‐phosphorylation.

In vitro and In vivo Pharmacokinetic-Pharmacodynamic Relationships for the Trisubstituted Aminopurine Cyclin-Dependent Kinase Inhibitors Olomoucine, Bohemine and CYC202

Purpose: To investigate pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine, and CYC202 (R-roscovitine; seliciclib) in the HCT116 human colon carcinoma model. Experimental Design: The in vitro activity of the agents was determined in a human tumor panel using the sulforhodamine B assay. The concentration and time dependence was established in HCT116 cells. Molecular biomarkers, including RB phosphorylation and cyclin expression, were assessed by Western blotting. Pharmacokinetic properties were characterized in mice following analysis by liquid chromatography-tandem mass spectrometry. Based on these studies, a dosing regimen was developed for CYC202 that allowed therapeutic exposures in the HCT116 tumor xenograft. Results: The antitumor potency of the agents in vitro was in the order olomoucine (IC50, 56 μmol/L) < bohemine (IC50, 27 μmol/L) < CYC202 (IC50, 15 μmol/L), corresponding to their activities as cyclin-dependent kinase inhibitors. Antitumor activity increased with exposure time up to 16 hours. The agents caused inhibition of RB and RNA polymerase II phosphorylation and depletion of cyclins. They exhibited relatively rapid clearance following administration to mice. CYC202 displayed the slowest clearance from plasma and the highest tumor uptake, with oral bioavailability of 86%. Oral dosing of CYC202 gave active concentrations in the tumor, modulation of pharmacodynamic markers, and inhibition of tumor growth. Conclusions: CYC202 showed therapeutic activity on human cancer cell lines in vitro and on xenografts. Pharmacodynamic markers are altered in vitro and in vivo, consistent with the inhibition of cyclin-dependent kinases. Such markers may be potentially useful in the clinical development of CYC202 and other cyclin-dependent kinase inhibitors.