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.

Small molecule binding sites on the Ras:SOS complex can be exploited for inhibition of Ras activation.

Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras.

Colorectal cancer intrinsic subtypes predict chemotherapy benefit, deficient mismatch repair and epithelial-to-mesenchymal transition.

In most colorectal cancer (CRC) patients, outcome cannot be predicted because tumors with similar clinicopathological features can have differences in disease progression and treatment response. Therefore, a better understanding of the CRC biology is required to identify those patients who will benefit from chemotherapy and to find a more tailored therapy plan for other patients. Based on unsupervised classification of whole genome data from 188 stages I-IV CRC patients, a molecular classification was developed that consist of at least three major intrinsic subtypes (A-, B- and C-type). The subtypes were validated in 543 stages II and III patients and were associated with prognosis and benefit from chemotherapy. The heterogeneity of the intrinsic subtypes is largely based on three biological hallmarks of the tumor: epithelial-to-mesenchymal transition, deficiency in mismatch repair genes that result in high mutation frequency associated with microsatellite instability and cellular proliferation. A-type tumors, observed in 22% of the patients, have the best prognosis, have frequent BRAF mutations and a deficient DNA mismatch repair system. C-type patients (16%) have the worst outcome, a mesenchymal gene expression phenotype and show no benefit from adjuvant chemotherapy treatment. Both A-type and B-type tumors have a more proliferative and epithelial phenotype and B-types benefit from adjuvant chemotherapy. B-type tumors (62%) show a low overall mutation frequency consistent with the absence of DNA mismatch repair deficiency. Classification based on molecular subtypes made it possible to expand and improve CRC classification beyond standard molecular and immunohistochemical assessment and might help in the future to guide treatment in CRC patients.

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, GSK3ß, and S6 phosphorylation in BT474c xenografts (PRAS40 phosphorylation EC(50) ~ 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 ((18)F-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.

Enhanced apoptosis and tumor growth suppression elicited by combination of MEK (selumetinib) and mTOR kinase inhibitors (AZD8055).

The mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT signaling pathways interact at multiple nodes in cancer, including at mTOR complexes, suggesting an increased likelihood of redundancy and innate resistance to any therapeutic effects of single pathway inhibition. In this study, we investigated the therapeutic effects of combining the MAPK extracellular signal-regulated kinase (MEK)1/2 inhibitor selumetinib (AZD6244) with the dual mTORC1 and mTORC2 inhibitor (AZD8055). Concurrent dosing in nude mouse xenograft models of human lung adenocarcinoma (non-small cell lung cancers) and colorectal carcinoma was well tolerated and produced increased antitumor efficacy relative to the respective monotherapies. Pharmacodynamic analysis documented reciprocal pathway inhibition associated with increased apoptosis and Bim expression in tumor tissue from the combination group, where key genes such as DUSP6 that are under MEK functional control were also modulated. Our work offers a strong rationale to combine selumetinib and AZD8055 in clinical trials as an attractive therapeutic strategy.

Subtypes of primary colorectal tumors correlate with response to targeted treatment in colorectal cell lines.

BACKGROUND: Colorectal cancer (CRC) is a heterogeneous and biologically poorly understood disease. To tailor CRC treatment, it is essential to first model this heterogeneity by defining subtypes of patients with homogeneous biological and clinical characteristics and second match these subtypes to cell lines for which extensive pharmacological data is available, thus linking targeted therapies to patients most likely to respond to treatment. METHODS: We applied a new unsupervised, iterative approach to stratify CRC tumor samples into subtypes based on genome-wide mRNA expression data. By applying this stratification to several CRC cell line panels and integrating pharmacological response data, we generated hypotheses regarding the targeted treatment of different subtypes. RESULTS: In agreement with earlier studies, the two dominant CRC subtypes are highly correlated with a gene expression signature of epithelial-mesenchymal-transition (EMT). Notably, further dividing these two subtypes using iNMF (iterative Non-negative Matrix Factorization) revealed five subtypes that exhibit activation of specific signaling pathways, and show significant differences in clinical and molecular characteristics. Importantly, we were able to validate the stratification on independent, published datasets comprising over 1600 samples. Application of this stratification to four CRC cell line panels comprising 74 different cell lines, showed that the tumor subtypes are well represented in available CRC cell line panels. Pharmacological response data for targeted inhibitors of SRC, WNT, GSK3b, aurora kinase, PI3 kinase, and mTOR, showed significant differences in sensitivity across cell lines assigned to different subtypes. Importantly, some of these differences in sensitivity were in concordance with high expression of the targets or activation of the corresponding pathways in primary tumor samples of the same subtype. CONCLUSIONS: The stratification presented here is robust, captures important features of CRC, and offers valuable insight into functional differences between CRC subtypes. By matching the identified subtypes to cell line panels that have been pharmacologically characterized, it opens up new possibilities for the development and application of targeted therapies for defined CRC patient sub-populations.

Apoptosis induction by MEK inhibition in human lung cancer cells is mediated by Bim.

AZD6244 (ARRY-142886) is an inhibitor of MEK1/2 and can inhibit cell proliferation or induce apoptosis in a cell-type dependent manner. The precise molecular mechanism of AZD6244-induced apoptosis is not clear. To investigate mechanisms of AZD6244 induced apoptosis in human lung cancer, we determined the molecular changes of two subgroups of human lung cancer cell lines that are either sensitive or resistant to AZD6244 treatment. We found that AZD6244 elicited a large increase of Bim proteins and a smaller increase of PUMA and NOXA proteins, and induced cell death in sensitive lung cancer cell lines, but had no effect on other Bcl-2 related proteins in those cell lines. Knockdown of Bim by siRNA greatly increased the IC(50) and reduced apoptosis for AZD6244 treated cells. We also found that levels of endogenous p-Thr32-FOXO3a and p-Ser253-FOXO3a were lower in AZD6244-sensitive cells than in AZD6244-resistant cells. In the sensitive cells, AZD6244 induced FOXO3a nuclear translocation required for Bim activation. Moreover, the silencing of FOXO3a by siRNA abrogated AZD6244-induced cell apoptosis. In addition, we found that transfection of constitutively active AKT up-regulated p-Thr32-FOXO3a and p-Ser253-FOXO3a expression and inhibited AZD6244-induced Bim expression in sensitive cells. These results show that Bim plays an important role in AZD6244-induced apoptosis in lung cancer cells and that the PI3K/AKT/FOXO3a pathway is involved in Bim regulation and susceptibility of lung cancer cells to AZD6244. These results have implications in the development of strategies to overcome resistance to MEK inhibitors.

Simultaneous inhibition of mTORC1 and mTORC2 by mTOR kinase inhibitor AZD8055 induces autophagy and cell death in cancer cells.

mTOR is a major biological switch, coordinating an adequate response to changes in energy uptake (amino acids, glucose), growth signals (hormones, growth factors) and environmental stress. mTOR kinase is highly conserved through evolution from yeast to man and in both cases, controls autophagy and cellular translation in response to nutrient stress. mTOR kinase is the catalytic component of two distinct multiprotein complexes called mTORC1 and mTORC2. In addition to mTOR, mTORC1 contains Raptor, mLST8 and PRAS40. mTORC2 contains mTOR, Rictor, mSIN1 and Protor-1. mTORC1 activates p70S6K, which in turn phosphorylates the ribosomal protein S6 and 4E-BP1, both involved in protein translation. mTORC2 activates AKT directly by phosphorylating Serine 473. pAKT(S473) phosphorylates TSC2 (tuberin) and inactivates it, preventing its association with TSC1 (hamartin) and the inhibition of Rheb, an activator of mTOR. pAKT also phosphorylates PRAS40, releasing it from the mTORC1 complex, increasing its kinase activity. Finally, AKT regulates FOXO3 phosphorylation, sequestering it in the cytosol in an inactive state.

Transcriptional pathway signatures predict MEK addiction and response to selumetinib (AZD6244).

Selumetinib (AZD6244, ARRY-142886) is a selective, non-ATP-competitive inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR-based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.

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.

High level of AKT activity is associated with resistance to MEK inhibitor AZD6244 (ARRY-142886).

MEK/ERK activities are increased in many primary lung cancers, and MEK inhibitors have been tested clinically for treatment of non-small cell lung cancers. The molecular mechanisms of resistance to MEK inhibitors have not been clearly demonstrated, however, and no molecular biomarker that can predict lung cancer response to MEK inhibitors is available. By determining the dose-responses of 35 human lung cancer cell lines to MEK-specific inhibitor AZD6244, we identified subsets of lung cancer cell lines that are either sensitive or resistant to this agent. Subsequent molecular characterization showed that treatment with AZD6244 suppressed ERK phosphorylation in both sensitive and resistant cells, suggesting that resistance is not mediated by the activities of MEK/ERK themselves. Interestingly, we found that levels of phosphorylated AKT were dramatically higher in the resistant cancer cells than in the sensitive cells. Stable transfection of dominant-negative AKT into resistant cells by retroviral infection restored their susceptibility to AZD6244. These results indicate that phosphorylated AKT may be a biomarker of response to AZD6244 and that modulation of AKT activity may be a useful approach to overcome resistance to MEK inhibitors.

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 approximately 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.

Design and campaign synthesis of piperidine- and thiazole-based histone deacetylase inhibitors.

A lead benzamide, 3, was identified as a potent and low molecular weight histone deacetylase (HDAC) inhibitor. Optimization led to 16d, demonstrating an excellent balance of efficacy and non-efficacy properties, along with very desirable in vivo DMPK. The final compounds presented are >1000-fold more potent than the initial screen hit, an improvement in potency which was achieved with a concomitant significant improvement in all the main non-efficacy properties.

Design and campaign synthesis of pyridine-based histone deacetylase inhibitors.

A lead benzamide, bearing a cyanopyridyl moiety (3), was identified as a potent and low molecular weight histone deacetylase (HDAC) inhibitor. Various replacements of the cyano group were explored at the C3-position, along with the exploration of solubility-enhancing groups at the C5-position. It was determined that cyano substitution at the C3-position of the pyridyl core, along with a methylazetidinyl substituent at the C5-position yielded optimal HDAC1 inhibition and anti-proliferative activity in HCT-116 cells.