Kathleen M. Haskell

Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors

We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 microM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 microM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 microM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.

Breast Tumor Cells with PI3K Mutation or HER2 Amplification Are Selectively Addicted to Akt Signaling

Background Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy. Methodology/Principal Findings A selective allosteric inhibitor of Akt kinase was used to interrogate a panel of breast cancer cell lines characterized for genetic lesions that activate PI3K/Akt signaling: HER2 amplification or PI3K or PTEN mutations in order to determine the biochemical and biologic consequences of inhibition of this pathway. A variety of molecular techniques and tissue culture and in vivo xenograft models revealed that tumors with mutational activation of Akt signaling were selectively dependent on the pathway. In sensitive cells, pathway inhibition resulted in D-cyclin loss, G1 arrest and induction of apoptosis, whereas cells without pathway activation were unaffected. Most importantly, the drug effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt. Conclusions/Significance These data demonstrate that breast cancers with PI3K mutation or HER2 amplification are selectively dependent on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast and other cancers that are addicted to the pathway including tumors with resistant to Herceptin.

A peptide–doxorubicin 'prodrug' activated by prostate-specific antigen selectively kills prostate tumor cells positive for prostate-specific antigen in vivo

We covalently linked doxorubicin with a peptide that is hydrolyzable by prostate-specific antigen. In the presence of prostate tumor cells secreting prostate-specific antigen, the peptide moiety of this conjugate, L-377,202, was hydrolyzed, resulting in the release of leucine-doxorubicin and doxorubicin, which are both very cytotoxic to cancer cells. However, L-377,202 was much less cytotoxic than conventional doxorubicin to cells in culture that do not secrete prostate-specific antigen. L-377,202 was approximately 15 times more effective than was conventional doxorubicin at inhibiting the growth of human prostate cancer tumors in nude mice when both drugs were used at their maximally tolerated doses. Nude mice inoculated with human prostate tumor cells secreting prostate-specific antigen showed considerable reductions in tumor burden with minimal total body weight loss when treated with L-377,202. This improvement in therapeutic index correlated with the selective localization of leucine–doxorubicin and free doxorubicin in tissues secreting prostate-specific antigen after exposure to L-377,202.

Allosteric inhibitors of Akt1 and Akt2: a naphthyridinone with efficacy in an A2780 tumor xenograft model.

A series of naphthyridine and naphthyridinone allosteric dual inhibitors of Akt1 and 2 have been developed. These compounds have been optimized to have potent dual activity against the activated kinase as well as the activation of Akt in cells. One molecule in particular, compound 17, has potent inhibitory activity against Akt1 and 2 in vivo in a mouse lung and efficacy in a tumor xenograft model.

An allosteric Akt inhibitor effectively blocks Akt signaling and tumor growth with only transient effects on glucose and insulin levels in vivo

The PI3K-Akt pathway is dysregulated in the majority of solid tumors. Pharmacological inhibition of Akt is a promising strategy for treating tumors resistant to growth factor receptor antagonists due to mutations in PI3K or PTEN. We have developed allosteric, isozyme-specific inhibitors of Akt activity and activation, as well as ex vivo kinase assays to measure inhibition of individual Akt isozymes in tissues. Here we describe the relationship between PK, Akt inhibition, hyperglycemia and tumor efficacy for a selective inhibitor of Akt1 and Akt2 (AKTi). In nude mice, AKTi treatment caused transient insulin resistance and reversible, dose-dependent hyperglycemia and hyperinsulinemia. Akt1 and Akt2 phosphorylation was inhibited in mouse lung with EC50 values of 1.6 and 7 μM, respectively, and with similar potency in other tissues and xenograft tumors. Weekly subcutaneous dosing of AKTi resulted in dose-dependent inhibition of LNCaP prostate cancer xenografts, an AR-dependent tumor with PTEN deletion and constitutively activated Akt. Complete tumor growth inhibition was achieved at 200 mpk, a dose that maintained inhibition of Akt1 and Akt2 of greater than 80% and 50%, respectively, for at least 12 hours in xenograft tumor and mouse lung. Hyperglycemia could be controlled by reducing Cmax, while maintaining efficacy in the LNCaP model, but not by insulin administration. AKTi treatment was well tolerated, without weight loss or gross toxicities. These studies supported the rationale for clinical development of allosteric Akt inhibitors and provide the basis for further refining of pharmacokinetic properties and dosing regimens of this class of inhibitors.

Comparison of the binding of the human papillomavirus type 16 and cottontail rabbit papillomavirus E7 proteins to the retinoblastoma gene product

Binding of the human papillomavirus type 16 (HPV-16) E7 oncoprotein to the retinoblastoma protein (pRb) is thought to be involved in the cellular transformation mediated by HPV-16. Here we show that the E7 protein of the cottontail rabbit papillomavirus (CRPV) binds to the same C-terminal portion of human pRb as HPV-16 E7, and that both the CRPV and HPV-16 E7 proteins bind specifically through similar domains to rabbit pRb. Furthermore, a single amino acid substitution which reduces the binding of HPV-16 E7 to human pRb also abolishes binding of CRPV E7 to both human and rabbit pRb. The biochemical similarities observed between the HPV-16 and CRPV E7 proteins suggest that they are functionally conserved. These results further validate the use of CRPV as an animal model for the study of HPV-mediated disease.

Lovastatin selectively inhibits ras activation of the 12-O-tetradecanoylphorbol-13-acetate response element in mammalian cells.

To evaluate ras-mediated signal transduction, an alkaline phosphatase gene (SEAP) was placed under the control of the ras-inducible phorbol ester response element (TRE) in murine fibroblasts (TRE-SEAP cells). The Kirsten ras gene was placed under the control of the glucocorticoid-inducible mouse mammary tumor virus promoter and introduced into the TRE-SEAP cells. Dexamethasone increased ras expression in the TRE-SEAP cells carrying the Kirsten ras gene and stimulated SEAP activity 25-fold. Lavostatin blocked dexamethasone induction of SEAP activity (50% inhibitory concentration, 0.5 microM) but did not affect phorbol ester-induced SEAP activity in the same cells. Lovastatin also did not block forskolin induction of SEAP activity in cells expressing SEAP under the control of the cyclic AMP response element.

Quantification of Cy-5 siRNA signal in the intra-vital multi-photon microscopy images

Transgenic mice with Tie2- green fluorescent protein (GFP) are used as a model to study the kinetic distribution of the Cy5-siRNA delivered by lipid nanoparticles (LNP) into the liver. After the mouse is injected with the LNP, it undergoes a procedure of intra-vital multi-photon microscopy imaging over a period of two hours, during which the process for the nanoparticle to diffuse into the hepatocytes from the vasculature system is monitored. Since the images are obtained in-vivo, the quantification of Cy5 kinetics suffers from the moving field of view (FOV). A method is proposed to register the sequence of images through template matching. Based on the semi-automatic segmentations of the vessels in the common FOV, the registered images are segmented into three regions of interest (ROI) in which the Cy5 signals are quantified. Computation of the percentage signal strength in the ROIs over time allows for the analysis of the diffusion of Cy5-siRNA into the hepatocytes, and helps demonstrate the effectiveness of the Cy5-siRNA delivery vehicle.

Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling.

Abstract #3061 Background: Activation of the PI3K/Akt pathway is a common event in human cancers and is responsible for key aspects of the transformed phenotype. This pathway is activated in most breast cancers, but by different mechanisms including HER2 amplification, PI3K mutation and PTEN inactivation. Inhibition of this pathway is thus thought to be clinically useful, but the functional output of PI3K/Akt signaling and the corresponding clinical effects of its inhibition may be due to the mechanisms by which it is activated. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy.
 Methodology/Principal Findings: Using a panel of human breast cancer cell lines that harbor HER2 amplification, PI3K or PTEN mutations, or wild-type of PI3K and PTEN, and a selective, PH-domain dependent, allosteric inhibitor of Akt1 and Akt2 (AKTi-1/2), and a variety of biochemical, molecular, and cellular techniques as well as in vivo xenograft models, we show that AKTi-1/2 resulted in the selective inhibition of breast tumor cell lines with either mutant PI3K or amplified HER2, but not those in which PI3K/Akt signaling is not mutationally activated. Cellular sensitivity was due to induction of G1 arrest and of apoptosis and associated with inhibition of Akt signaling, loss of D-cyclin expression, and dephosphorylation of Rb. Most importantly, AKTi-1/2 effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt.
 Conclusions/Significance: Our data demonstrate the exquisite dependence of PI3K-mutated or HER2-amplified breast cancer cells on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast cancers that are addicted to the pathway and in patients with clinical resistance to Herceptin. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3061.