D. Lynn Kirkpatrick

Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866 whereas oncogenic ras is a dominant predictor for resistance

The novel phosphatidylinositol-3-kinase (PI3K) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI3K (PIK3CA) and loss of PTEN activity were sufficient, but not necessary, as predictors of sensitivity to the antitumor activity of the PI3K inhibitor PX-866 in the presence of wild-type Ras, whereas mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI3K signaling measured by tumor phosphorylated Ser(473)-Akt was insufficient to predict in vivo antitumor response to PX-866. Reverse-phase protein array revealed that the Ras-dependent downstream targets c-Myc and cyclin B were elevated in cell lines resistant to PX-866 in vivo. Studies using an H-Ras construct to constitutively and preferentially activate the three best-defined downstream targets of Ras, i.e., Raf, RalGDS, and PI3K, showed that mutant Ras mediates resistance through its ability to use multiple pathways for tumorigenesis. The identification of Ras and downstream signaling pathways driving resistance to PI3K inhibition might serve as an important guide for patient selection as inhibitors enter clinical trials and for the development of rational combinations with other molecularly targeted agents.

The phosphatidylinositol-3-kinase inhibitor PX-866 overcomes resistance to the epidermal growth factor receptor inhibitor gefitinib in A-549 human non–small cell lung cancer xenografts

Epidermal growth factor receptor (EGFR) inhibitors such as gefitinib show antitumor activity in a subset of non–small cell lung cancer (NSCLC) patients having mutated EGFR. Recent work shows that phosphatidylinositol-3-kinase (PI3-K) is coupled to the EGFR only in NSCLC cell lines expressing ErbB-3 and that EGFR inhibitors do not inhibit PI3-K signaling in these cells. The central role PI3-K plays in cell survival suggests that a PI3-K inhibitor offers a strategy to increase the antitumor activity of EGFR inhibitors in resistant NSCL tumors that do not express ErbB-3. We show that PX-866, a PI3-K inhibitor with selectivity for p110α, potentiates the antitumor activity of gefitinib against even large A-549 NSCL xenografts giving complete tumor growth control in the early stages of treatment. A-549 xenograft phospho-Akt was inhibited by PX-866 but not by gefitinib. A major toxicity of PX-866 administration was hyperglycemia with decreased glucose tolerance, which was reversed upon cessation of treatment. The decreased glucose tolerance caused by PX-866 was insensitive to the AMP-activated protein kinase inhibitor metformin but reversed by insulin and by the peroxisome proliferator-activated receptor-γ activator pioglitazone. Prolonged PX-866 administration also caused increased neutrophil counts. Thus, PX-866, by inhibiting PI3-K signaling, may have clinical use in increasing the response to EGFR inhibitors such as gefitinib in patients with NSCLC and possibly in other cancers who do not respond to EGFR inhibition.

Mechanisms of Inhibition of the Thioredoxin Growth Factor System by Antitumor 2-Imidazolyl Disulfides

The interactions of a series of 2-imidazolyl disulfide antitumor compounds with the thioredoxin reductase(TR)/thioredoxin (hTrx) redox system have been studied. Disulfides III-2 (n-butyl 2-mercaptoimidazolyl disulfide) and VI-2 (ethyl 2-mercaptoimidazolyl disulfide) were substrates for reduction by TR with Km values of 43 and 48 microM. Disulfides IV-2 (1-methylpropyl 2-mercaptoimidazolyl disulfide) and DLK-36 (benzyl 2-mercaptoimidazolyl disulfide) were competitive inhibitors of the reduction of hTrx by TR with Ki values of 31 microM. None of the disulfides were substrates for reduction by human glutathione reductase. The disulfides caused reversible thioalkylation of hTrx at the redox catalytic site as shown by the fact that there was no thioalkylation of a mutant hTrx where both the catalytic site Cys32 and Cys35 residues were replaced by Ser. In addition, the disulfides caused a slower irreversible inactivation of hTrx as a substrate for reduction by TR, with half-lives for III-2 of 30 min, for IV-2 of 4 hr, and for IX-2 (t-butyl 2-mercaptoimidazolyl disulfide) of 24 hr. This irreversible inactivation of hTrx occurred at concentrations of the disulfides an order of magnitude below those that inhibited TR, and involved the Cys73 of hTrx, which is outside the conserved redox catalytic site, as shown by the resistance to inactivation of a mutant hTrx where Cys73 was replaced by Ser. Electrophoretic and mass spectral analyses of the products of the reaction between the disulfides and hTrx show that modification of 1-3 Cys residues of the protein occurred in a concentration-dependent fashion. The disulfides inhibited the hTrx-dependent proliferation of MCF-7 breast cancer cells with IC50 values for III-2 and IV-2 of 0.2 and 1.2 microM, respectively. The results show that although the catalytic sites of TR and hTrx are reversibly inhibited by the 2-imidazolyl disulfides, it is the irreversible thioalkylation of Cys73 of hTrx by the disulfides that most probably accounts for the inhibition of thioredoxin-dependent cell growth by the disulfides.

Thioredoxin redox control of cell growth and death and the effects of inhibitors.

Thioredoxin is a redox protein found over-expressed in some human tumors. Thioredoxin is secreted by tumor cells and stimulates cancer cell growth. Redox activity is essential for growth stimulation by thioredoxin. Cells transfected with thioredoxin cDNA show increased tumor growth and decreased apoptosis in vivo and decreased sensitivity to apoptosis induced by a variety of agents both in vitro and in vivo. Cells transfected with a redox-inactive mutant thioredoxin show inhibited tumor growth in vivo. Thus, thioredoxin offers an attractive target for anticancer drug development. A class of disulfide inhibitors of thioredoxin has been identified. These disulfides inhibit cancer cell growth in culture and have antitumor activity against some human tumor xenografts in animals.

Cellular and in vivo activity of a novel PI3K inhibitor, PX-866, against human glioblastoma

The phosphatidylinositol-3-kinase (PI3K)/Akt oncogenic pathway is critical in glioblastomas. Loss of PTEN, a negative regulator of the PI3K pathway or activated PI3K/Akt pathway that drive increased proliferation, survival, neovascularization, glycolysis, and invasion is found in 70%-80% of malignant gliomas. Thus, PI3K is an attractive therapeutic target for malignant glioma. We report that a new irreversible PI3K inhibitor, PX-866, shows potent inhibitory effects on the PI3K/Akt signaling pathway in glioblastoma. PX-866 did not induce any apoptosis in glioma cells; however, an increase in autophagy was observed. PX-866 inhibited the invasive and angiogenic capabilities of cultured glioblastoma cells. In vivo, PX-866 inhibited subcutaneous tumor growth and increased the median survival time of animals with intracranial tumors. We also assessed the potential of proton magnetic resonance spectroscopy (MRS) as a noninvasive method to monitor response to PX-866. Our findings show that PX-866 treatment causes a drop in the MRS-detectable choline-to-NAA, ratio and identify this partial normalization of the tumor metabolic profile as a biomarker of molecular drug action. Our studies affirm that the PI3K pathway is a highly specific molecular target for therapies for glioblastoma and other cancers with aberrant PI3K/PTEN expression.

Oxidative inactivation of thioredoxin as a cellular growth factor and protection by a Cys73 → Ser mutation

Thioredoxin (Trx) is a widely distributed redox protein that regulates several intracellular redox-dependent processes and stimulates the proliferation of both normal and tumor cells. We have found that when stored in the absence of reducing agents, human recombinant Trx undergoes spontaneous oxidation, losing its ability to stimulate cell growth, but is still a substrate for NADPH-dependent reduction by human thioredoxin reductase. There is a slower spontaneous conversion of Trx to a homodimer that is not a substrate for reduction by thioredoxin reductase and that does not stimulate cell proliferation. Both conversions can be induced by chemical oxidants and are reversible by treatment with the thiol reducing agent dithiothreitol. SDS-PAGE suggests that Trx undergoes oxidation to monomeric form(s) preceding dimer formation. We have recently shown by X-ray crystallography that Trx forms a dimer that is stabilized by an intermolecular Cys73-Cys73 disulfide bond. A Cys73-->Ser mutant Trx (C73S) was prepared to determine the role of Cys73 in oxidative stability and growth stimulation. C73S was as effective as Trx in stimulating cell growth and was a comparable substrate for thioredoxin reductase. C73S did not show spontaneous or oxidant-induced loss of activity and did not form a dimer. The results suggest that Trx can exist in monomeric forms, some of which are mediated by Cys73 that do not stimulate cell proliferation but can be reduced by thioredoxin reductase. Cys73 is also involved in formation of an enzymatically inactive homodimer, which occurs on long term storage or by chemical oxidation. Thus, although clearly involved in protein inactivation, Cys73 is not necessary for the growth stimulating activity of Trx.

A Phase I Pharmacokinetic and Pharmacodynamic Study of PX-12, a Novel Inhibitor of Thioredoxin-1, in Patients with Advanced Solid Tumors

Purpose: Thioredoxin-1 (Trx-1) is a cellular redox protein that promotes tumor growth, inhibits apoptosis, and up-regulates hypoxia-inducible factor-1α and vascular endothelial growth factor. Objectives of this study were to determine safety, tolerability, pharmacodynamics, and pharmacokinetics of PX-12, a small-molecule inhibitor of Trx-1. Experimental Design: Thirty-eight patients with advanced solid tumors received PX-12 at doses of 9 to 300 mg/m2, as a 1- or 3-h i.v. infusion on days 1 to 5, repeated every 3 weeks. Results: At the 300 mg/m2 dose level, one patient experienced a reversible episode of pneumonitis during the first cycle, and a second patient developed pneumonitis after the second cycle. Doses up to 226 mg/m2 were well tolerated, and grade 3/4 events were uncommon (<3% of patients). The limiting factor on this dosing schedule was pungent odor caused by expired drug metabolite, 2-butanethiol. The best response was stable disease in seven patients (126-332 days). Whereas PX-12 was not detectable following the infusion, the Cmax of its inactive metabolite, 2-mercaptoimidazole, increased linearly with dose. PX-12 treatment lowered plasma Trx-1 concentrations in a dose-dependent manner. Conclusions: PX-12, the first Trx-1 inhibitor to enter clinical trials, was tolerated up to a dose of 226 mg/m2 by a 3-h infusion. Based on pharmacodynamic and pharmacokinetic data, a trial of prolonged infusion schedule of PX-12 has been initiated.

Molecular pharmacology and antitumor activity of palmarumycin-based inhibitors of thioredoxin reductase

The cytosolic thioredoxin redox system composed of thioredoxin-1 and the NADPH-dependent thioredoxin reductase-1 reductase is an important regulator of cell growth and survival. Thioredoxin-1 is overexpressed in many human tumors where it is associated with increased cell proliferation, decreased apoptosis, and decreased patient survival. We hypothesized that thioredoxin reductase-1 provides a target to inhibit the activity of overexpressed thioredoxin-1 for the development of novel anticancer agents. We found that the naphthoquinone spiroketal fungal metabolite palmarumycin CP1 is a potent inhibitor of thioredoxin reductase-1, but attempts to exploit the activity of palmarumycin CP1 analogues as antitumor agents in vivo were hampered by their insolubility. We have therefore developed PX-916, a water-soluble prodrug of a palmarumycin CP1 analogue. PX-916 rapidly releases the parent compound at physiologic pH and in plasma but is stable at acid pH, allowing its i.v. administration. PX-916 is a potent inhibitor of purified human thioredoxin reductase-1 and of thioredoxin reductase-1 activity in cells and tumor xenografts when given to mice and inhibits the downstream targets of thioredoxin-1 signaling, hypoxia-inducible factor-1α, and vascular endothelial growth factor in tumors. PX-916 showed excellent antitumor activity against several animal tumor models with some cures. Thus, the study shows that water-soluble inhibitors of thioredoxin reductase-1, such as PX-916, can block thioredoxin-1 signaling in tumors producing marked inhibition of tumor growth. [Mol Cancer Ther 2006;5(3):630–6]

Chemosensitivity testing of fresh human leukemia cells using both a dye exclusion assay and a tetrazolium dye (MTT) assay

Chemosensitivity testing using a DEA was performed on 91 leukemia samples from 70 patients with leukemia. In 55 patients, comparison of in-vitro results with actual response of the patient was made. There was good correlation in 82% of the cases, the results being highly significant p less than 0.001. The MTT assay was also adapted for the chemosensitivity testing and the usefulness of this assay was evaluated by comparing the results to the DEA for 24 of the samples. Survival results produced by the two assays were very similar. The MTT procedure was also found to be equally effective as the DEA as a predictive assay of chemosensitivity.

Molecular Pharmacology and Antitumor Activity of PHT-427, a Novel Akt/Phosphatidylinositide-Dependent Protein Kinase 1 Pleckstrin Homology Domain Inhibitor

Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non–small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity. Mol Cancer Ther; 9(3); 706–17