Sabina Cosulich

AZD8055 Is a Potent, Selective, and Orally Bioavailable ATP-Competitive Mammalian Target of Rapamycin Kinase Inhibitor with In vitro and In vivo Antitumor Activity

The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, with an IC50 of 0.8 nmol/L. AZD8055 showed excellent selectivity (approximately 1,000-fold) against all class I phosphatidylinositol 3-kinase (PI3K) isoforms and other members of the PI3K-like kinase family. Furthermore, there was no significant activity against a panel of 260 kinases at concentrations up to 10 micromol/L. AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. The rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1 were fully inhibited by AZD8055, resulting in significant inhibition of cap-dependent translation. In vitro, AZD8055 potently inhibits proliferation and induces autophagy in H838 and A549 cells. In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials.

Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)

mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of ∼10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.

Preclinical Pharmacology of AZD5363, an Inhibitor of AKT: Pharmacodynamics, Antitumor Activity, and Correlation of Monotherapy Activity with Genetic Background

AKT is a key node in the most frequently deregulated signaling network in human cancer. AZD5363, a novel pyrrolopyrimidine-derived compound, inhibited all AKT isoforms with a potency of 10 nmol/L or less and inhibited phosphorylation of AKT substrates in cells with a potency of approximately 0.3 to 0.8 μmol/L. AZD5363 monotherapy inhibited the proliferation of 41 of 182 solid and hematologic tumor cell lines with a potency of 3 μmol/L or less. Cell lines derived from breast cancers showed the highest frequency of sensitivity. There was a significant relationship between the presence of PIK3CA and/or PTEN mutations and sensitivity to AZD5363 and between RAS mutations and resistance. Oral dosing of AZD5363 to nude mice caused dose- and time-dependent reduction of PRAS40, GSK3β, and S6 phosphorylation in BT474c xenografts (PRAS40 phosphorylation EC50 ∼ 0.1 μmol/L total plasma exposure), reversible increases in blood glucose concentrations, and dose-dependent decreases in 2[18F]fluoro-2-deoxy-d-glucose (18F-FDG) uptake in U87-MG xenografts. Chronic oral dosing of AZD5363 caused dose-dependent growth inhibition of xenografts derived from various tumor types, including HER2+ breast cancer models that are resistant to trastuzumab. AZD5363 also significantly enhanced the antitumor activity of docetaxel, lapatinib, and trastuzumab in breast cancer xenografts. It is concluded that AZD5363 is a potent inhibitor of AKT with pharmacodynamic activity in vivo, has potential to treat a range of solid and hematologic tumors as monotherapy or a combinatorial agent, and has potential for personalized medicine based on the genetic status of PIK3CA, PTEN, and RAS. AZD5363 is currently in phase I clinical trials. Mol Cancer Ther; 11(4); 873–87. ©2012 AACR.

Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor

Precision medicines exert selective pressure on tumor cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next generation therapies to treat the evolving cancer. The PIK3CA/AKT/mTOR pathway is one of the most commonly activated pathways in human cancers1, which has led to the development of small molecule inhibitors that target various nodes in the pathway. Among these agents, first generation mTOR inhibitors (rapalogs) have caused responses in so-called “N-of-1” cases and second generation mTOR kinase inhibitors (TORKi) are currently in clinical trials2–4. We sought to delineate the likely resistance mechanisms to existing mTOR inhibitors as a guide for next generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active site mutation interfering with drug binding. Indeed, the identical drug resistant mutations have been also identified in drug-naïve patients4, suggesting that tumors with activating mTOR mutations will be intrinsically resistant to second generation mTOR inhibitors. Here, we report the development of a new class of mTOR inhibitors which overcomes resistance to existing first and second generation inhibitors. The third generation mTOR inhibitor exploits the unique juxtaposition of two drug binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.

Kinase-Substrate Enrichment Analysis Provides Insights into the Heterogeneity of Signaling Pathway Activation in Leukemia Cells

Computational analysis of phosphoproteomics data predicts the sensitivity of leukemia cells to kinase inhibitors. Therapeutic Targeting with Phosphoproteomics Because oncogenic mutations frequently occur in genes encoding kinases, kinase inhibitors are often used as cancer therapeutics. However, because of the complexity of kinase signaling networks and the heterogeneous nature of oncogenic mutations, it is difficult to predict how cancer cells will respond to a given kinase inhibitor. Casado et al. performed phosphoproteomic analysis of human acute myeloid leukemia (AML) cell lines and used a computational approach of “signal averaging” to reduce the noise in the phosphoproteomics data, thus monitoring global kinase activation patterns as well as the effects of select inhibitors. In addition to identifying kinase networks associated with AML, this approach accurately predicted the relative sensitivities of patient-derived AML cells to inhibitors. Such profiling of kinase networks could be applied to stratify cancers based on their predicted responses to kinase inhibition. Kinases determine the phenotypes of many cancer cells, but the frequency with which individual kinases are activated in primary tumors remains largely unknown. We used a computational approach, termed kinase-substrate enrichment analysis (KSEA), to systematically infer the activation of given kinase pathways from mass spectrometry–based phosphoproteomic analysis of acute myeloid leukemia (AML) cells. Experiments conducted in cell lines validated the approach and, furthermore, revealed that DNA-dependent protein kinase (DNA-PK) was activated as a result of inhibiting the phosphoinositide 3-kinase (PI3K)–mammalian target of rapamycin (mTOR) signaling pathway. Application of KSEA to primary AML cells identified PI3K, casein kinases (CKs), cyclin-dependent kinases (CDKs), and p21-activated kinases (PAKs) as the kinase substrate groups most frequently enriched in this cancer type. Substrates phosphorylated by extracellular signal–regulated kinase (ERK) and cell division cycle 7 (CDC7) were enriched in primary AML cells that were resistant to inhibition of PI3K-mTOR signaling, whereas substrates of the kinases Abl, Lck, Src, and CDK1 were increased in abundance in inhibitor-sensitive cells. Modeling based on the abundances of these substrate groups accurately predicted sensitivity to a dual PI3K and mTOR inhibitor in two independent sets of primary AML cells isolated from patients. Thus, our study demonstrates KSEA as an untargeted method for the systematic profiling of kinase pathway activities and for increasing our understanding of diseases caused by the dysregulation of signaling pathways.

PPARα and the regulation of cell division and apoptosis

Peroxisome proliferators (PPs) such as the hypolipidaemic drug, nafenopin and the phthalate plasticiser 2-diethylhexylphthalate induce rodent hepatocyte cell proliferation and suppress apoptosis leading to tumours. PPs act via the nuclear hormone receptor peroxisome proliferator activated receptor α (PPARα) which directly regulates genes implicated in the response to PPs such as the peroxisomal gene acyl CoA oxidase. As expected for xenobiotics that perturb proliferation, PPs alter expression of cell cycle regulatory proteins. However, the ability to alter expression of cyclins and cyclin-dependent kinases is shared by physiological hepatic mitogens such as epidermal growth factor and is thus unlikely to be specific to the PP-induced aberrant growth associated with hepatocarcinogenesis. Recent evidence suggests that the response of hepatocytes to PPs is not only dependent upon PPARα but also on the trophic environment provided by nonparenchymal cells and by cytokines such as tumour necrosis factor α. Additionally, the ability of PPs to suppress apoptosis and induce proliferation depends upon survival signalling mediated by p38 mitogen activated protein kinase. The cross talk between PPARα-mediated transcription, survival signalling and cell cycle will be discussed with particular emphasis on relevance to toxicology.

Diverse heterocyclic scaffolds as allosteric inhibitors of AKT.

Wide-ranging exploration of potential replacements for a quinoline-based inhibitor of activation of AKT kinase led to number of alternative, novel scaffolds with potentially improved potency and physicochemical properties. Examples showed predictable DMPK properties, and one such compound demonstrated pharmacodynamic knockdown of phosphorylation of AKT and downstream biomarkers in vivo and inhibition of tumor growth in a breast cancer xenograft model.

Suppression of apoptosis and induction of DNA synthesis in vitro by the phthalate plasticizers monoethylhexylphthalate (MEHP) and diisononylphthalate (DINP): a comparison of rat and human hepatocytes in vitro

Abstract Diethylhexylphthalate (DEHP) and diisononylphthalate (DINP) are plasticizers with many important commercial, industrial and medical applications. However, both DEHP and DINP are rodent peroxisome proliferators (PPs), a class of compounds that cause rodent liver tumours associated with peroxisome proliferation, induction of hepatic DNA synthesis and the suppression of apoptosis. Despite these effects in the rodent, humans appear to be nonresponsive to the adverse effects of PPs. Previously, we have shown that the fibrate hypolipidaemic peroxisome proliferator, nafenopin, induced DNA synthesis and suppressed apoptosis in rat but not in human hepatocytes. In this work, we have examined species differences in the response of rat and human hepatocytes to DEHP and DINP in vitro. In rat hepatocytes in vitro, both DINP and MEHP (a principle metabolite of DEHP and the proximal peroxisome proliferator) caused a concentration-dependent induction of DNA synthesis and suppression of both spontaneous and transforming growth factor β1 (TGFβ1)-induced apoptosis. Similarly, both MEHP and DINP caused a concentration-dependent induction of peroxisomal β-oxidation although the response to DINP was less robust. In contrast to the pleiotropic response noted in rat hepatocytes, neither DINP nor MEHP caused an induction of β-oxidation, stimulation of DNA synthesis and suppression of apoptosis in human hepatocytes cultured from three separate donors. These data provide evidence for species differences in the hepatic response to the phthalates DEHP and DINP, confirming that human hepatocytes appear to be refractory to the hepatocarcinogenic effects of PPs first noted in rodents.

AZD2014, an Inhibitor of mTORC1 and mTORC2, Is Highly Effective in ER+ Breast Cancer When Administered Using Intermittent or Continuous Schedules

mTOR is an atypical serine threonine kinase involved in regulating major cellular functions, such as nutrients sensing, growth, and proliferation. mTOR is part of the multiprotein complexes mTORC1 and mTORC2, which have been shown to play critical yet functionally distinct roles in the regulation of cellular processes. Current clinical mTOR inhibitors only inhibit the mTORC1 complex and are derivatives of the macrolide rapamycin (rapalogs). Encouraging effects have been observed with rapalogs in estrogen receptor–positive (ER+) breast cancer patients in combination with endocrine therapy, such as aromatase inhibitors. AZD2014 is a small-molecule ATP competitive inhibitor of mTOR that inhibits both mTORC1 and mTORC2 complexes and has a greater inhibitory function against mTORC1 than the clinically approved rapalogs. Here, we demonstrate that AZD2014 has broad antiproliferative effects across multiple cell lines, including ER+ breast models with acquired resistance to hormonal therapy and cell lines with acquired resistance to rapalogs. In vivo, AZD2014 induces dose-dependent tumor growth inhibition in several xenograft and primary explant models. The antitumor activity of AZD2014 is associated with modulation of both mTORC1 and mTORC2 substrates, consistent with its mechanism of action. In combination with fulvestrant, AZD2014 induces tumor regressions when dosed continuously or using intermittent dosing schedules. The ability to dose AZD2014 intermittently, together with its ability to block signaling from both mTORC1 and mTORC2 complexes, makes this compound an ideal candidate for combining with endocrine therapies in the clinic. AZD2014 is currently in phase II clinical trials. Mol Cancer Ther; 14(11); 2508–18. ©2015 AACR.

Inhibition of PI3Kβ Signaling with AZD8186 Inhibits Growth of PTEN-Deficient Breast and Prostate Tumors Alone and in Combination with Docetaxel

Loss of PTEN protein results in upregulation of the PI3K/AKT pathway, which appears dependent on the PI3Kβ isoform. Inhibitors of PI3Kβ have potential to reduce growth of tumors in which loss of PTEN drives tumor progression. We have developed a small-molecule inhibitor of PI3Kβ and PI3Kδ (AZD8186) and assessed its antitumor activity across a panel of cell lines. We have then explored the antitumor effects as single agent and in combination with docetaxel in triple-negative breast (TNBC) and prostate cancer models. In vitro, AZD8186 inhibited growth of a range of cell lines. Sensitivity was associated with inhibition of the AKT pathway. Cells sensitive to AZD8186 (GI50 < 1 μmol/L) are enriched for, but not exclusively associated with, PTEN deficiency. In vivo, AZD8186 inhibits PI3K pathway biomarkers in prostate and TNBC tumors. Scheduling treatment with AZD8186 shows antitumor activity required only intermittent exposure, and that increased tumor control is achieved when AZD8186 is used in combination with docetaxel. AZD8186 is a potent inhibitor of PI3Kβ with activity against PI3Kδ signaling, and has potential to reduce growth of tumors dependent on dysregulated PTEN for growth. Moreover, AZD8186 can be combined with docetaxel, a chemotherapy commonly used to treat advanced TBNC and prostate tumors. The ability to schedule AZD8186 and maintain efficacy offers opportunity to combine AZD8186 more effectively with other drugs. Mol Cancer Ther; 14(1); 48–58. ©2014 AACR.