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The Signal Transducer and Activator of Transcription 3 (STAT3) oncogene is a master regulator of many human cancers, and a well-recognized target for therapeutic intervention. A well known STAT3 inhibitor, S3I-201 (NSC 74859), is hypothesized to block STAT3 function in cancer cells by binding the STAT3 SH2 domain and disrupt STAT3 protein complexation events. In this study, liquid chromatography tandem mass spectrometry analysis revealed that STAT3, in the presence of S3I-201, showed a minimum of five specific sites of modification, cysteine's 108, 259, 367, 542, and 687. Moreover, a prepared fluorescently labeled chemical probe of S3I-201 (DB-6-055) revealed that S3I-201 non-specifically and globally alkylated intracellular proteins at concentrations consistent with S3I-201's reported IC50. These data are consistent with the hypothesis that S3I-201 is a sub-optimal probe for interrogating STAT3-related cell biology.

In this report, the interaction of the signal transducer and activator of transcription (STAT3 and STAT5) proteins with a green tea polyphenol epigallocatechin-3-gallate (EGCG) was investigated using differential pulse voltammetry (DPV) at carbon paste electrodes (CPEs). Superparamagnetic agarose nickel beads were modified with His-tagged STAT proteins and exposed to EGCG in solution. After magnetic separation of the beads, the electrochemical oxidation of the remaining EGCG in the supernatant was monitored at ∼0.18 V (vs. Ag/AgCl). The changes in the peak current signal displayed the interaction of EGCG with STAT proteins. Our electrochemical results were in agreement with the surface plasmon resonance (SPR) method that indicated the KD values of EGCG for STAT3 and STAT5 proteins were 19.33 ± 2.11 μM and 19.53 ± 2.37 μM, respectively. To the best of our knowledge, the electrochemical detection of interaction between STAT proteins and EGCG is reported here for the first time. The voltammetric method described here provides a promising platform for the rapid and cost-effective screening of small electro-active molecules that interact with STAT proteins and other clinically important proteins.

Membrane-embedded negatively charged phospholipids (MENCP) can be used as biomarkers for a range of biological processes, including early detection of apoptosis in animal cells, drug-induced phospholipidosis, and selective detection of bacterial over animal cells. Currently, several technologies for the detection of apoptosis and bacterial cells are based on the recognition of MENCPs, including the AnnexinV stain and PSVue™ probes. As probes, these technologies have limitations, the most significant of which is the need for washing the unbound probe away to achieve optimal signal. In contrast, a turn-on chemosensor selective for MENCP would address this shortcoming, and allow for a more rapid protocol for the detection of apoptosis, bacteria and for other relevant applications. In this work, the aim was to explore whether ProxyPhos chemosensors, previously reported by our group for the detection of proximally phosphorylated peptides and proteins, could be re-purposed for the detection of MENCPs. Six lead ProxyPhos sensors were screened against synthetic vesicles containing biologically relevant negatively charged phospholipids including phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS). Through these screens, ProxyPhos sensors exhibiting high selectivity for the detection of MENCPs over zwitterionic lipids were identified. Particular selectivity was observed for PA and CL. Sensitivity of the lead sensors for MENCPs was suitable for the detection of apoptosis: ProxyPhos detected vesicles containing as little as 2.5% PS and detected camptothecin-induced apoptosis in mammalian cells in flow cytometry experiments. The results suggest that ProxyPhos sensors can be used for the detection of MENCPs in synthetic vesicles and live mammalian cells.

Chemosensors for the detection of phosphate-containing biological species are in high need. Detection of proximally phosphorylated sites of PPi and those found in peptides and proteins has been demonstrated using chemosensors containing pyrene, as a fluorescent reporter, and a Zn2+-chelate, as a phosphate-binding group. Using these sensors, detection of proximal phosphate groups is afforded by binding of at least two of the sensor molecules to the adjacent phosphates, via the Zn2+ centres, leading to excimer formation between the pyrene groups and the corresponding shift in emission from 376 to 476 nm. Although several reports of this chemosensor class have been made, no detailed studies of selectivity of these sensors among major phosphate targets have been reported. In this study, a library of this class of chemosensors, termed ProxyPhos, which contained various linkers and Zn2+-chelating groups (i.e. DPA, cyclen and cyclam), was prepared and the effects of structural variation on the sensing efficiency and selectivity were evaluated among proximally phosphorylated peptides, proteins, nucleotides, Pi and PPi. As a result of this study, we have identified ProxyPhos library members that are most suitable for the detection of proximally phosphorylated peptides, PPi, UTP, and a DpYD peptide motif, and have generally provided a foundation for the selection of ProxyPhos chemosensors for further development of specific biologically relevant assays. The broad utility of ProxyPhos is further supported by the demonstrated lack of these sensors' cytotoxicity, ability to rapidly permeate into live and fixed cells and compatibility with gel staining methods.

Proximal phosphorylation on proteins appears to have functional significance and has been associated with several diseases, including Alzheimer's and cancer. While much remains to be learned about the role of proximal phosphorylation in biological systems, no simple and/or affordable technique is available for its detection. To this end, we have previously developed a ProxyPhos chemosensor, which detects proximally phosphorylated peptides and proteins over mono- and non-phosphorylated motifs in aqueous solutions. In this follow-up work, we performed extensive characterization of peptide and protein ProxyPhos assay conditions to achieve enhanced detection, and further explored the selectivity of ProxyPhos, and its potential off-targets. As a result of characterization studies, selective sensing of proximally phosphorylated over mono-phosphorylated peptides and proteins was achieved. Moreover, studies demonstrated that ProxyPhos was compatible with the detection of all commonly phosphorylated residues (i.e. tyrosine, serine and threonine residues). Under optimized conditions, ProxyPhos efficiently discriminated between peptides derived from the activated (proximally phosphorylated, disease-relevant) and inactive (mono-phosphorylated) forms of JAK2, SYK and MAPK1 kinases. In addition, ProxyPhos can be used to probe phosphatase activity on peptides and proteins via detecting changes in proximal phosphorylation, demonstrating immediate utility of this chemosensing system.

It was previously demonstrated that differentiation of some established breast epithelial cell lines requires confluence and stimulation with hydrocortisone, insulin and prolactin inducers. We and others previously demonstrated that E-cadherin engagement, which is favored under conditions of confluence, increases the levels and activity of the Rac small GTPase. To investigate the functional relationship between the transforming ability of Rac and its role as an integral component of the differentiative E-cadherin signaling pathway, we introduced a mutationally activated form of Rac, RacV12, into the mouse breast epithelium-derived cell line, HC11. Our results demonstrate that the strength of the Rac signal is key for the outcome of the differentiation process; cRac1 is critically required for differentiation, and at low levels, mutationally activated RacV12 is able to increase differentiation, presumably reinforcing the E-cadherin/Rac differentiative signal. However, high RacV12 expression blocked differentiation concomitant with E-cadherin downregulation, while inducing neoplastic transformation. Therefore, the intensity of the Rac signal is a central determinant in the balance between cell proliferation vs differentiation, two fundamentally opposed processes, a finding which could also have important therapeutic implications.

The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization.

In order to survive and proliferate, cancer cells adapt to high levels of oxidative stress by countering the accumulation of reactive oxygen species (ROS) with an increased production of intracellular antioxidant molecules such as glutathione. The cell surface transport system Xc- is a cystine/glutamate antiporter that exports glutamate while importing cystine, thereby mediating levels of cysteine required for glutathione synthesis and the maintenance of cellular redox balance. Transcription factors that regulate key antioxidant defense mechanisms, including system Xc-, may therefore be of therapeutic interest. Recently, signal transducer and activator of transcription (STAT) proteins have emerged as potential targets for the development of novel anti-cancer therapies. In particular, inhibitors of STAT3 and STAT5 may become clinically relevant as anti-cancer agents for breast and brain cancer, as well as leukemia. Interestingly, suppression of STAT3 has been linked with increases in ROS and the induction of apoptosis. Upon activation by phosphorylation, dimerization, and nuclear translocation, STAT proteins transcriptionally regulate diverse target genes by binding to promoter regions containing gamma-activated site (GAS) motifs. The human xCT (SLC7A11) gene encodes the functional subunit of system Xc-. We provide evidence that expression of xCT is regulated by STAT3, and potentially also STAT5, affecting antiporter function in both MCF-7 and MDA-MB-231 human breast cancer cells. Computational analysis of the xCT promoter region revealed the presence of a distal GAS site. Its truncation significantly increased luciferase activity in a reporter assay, with similar increases obtained after treating cells transfected with the full-length xCT promoter construct with various STAT3/5 pharmacologic inhibitors. Knock-down of STAT3 or STAT5A using specific siRNAs produced similar results, suggesting that these STAT proteins act in a transcriptionally repressive manner. We also demonstrated binding of STAT3 and STAT5A to the xCT promoter in MDA-MB-231 cells, which was disrupted by preincubating the cells with specific inhibitors. xCT mRNA and protein levels increased significantly following treatment with STAT3/5 inhibitors. Pharmacologically suppressing STAT3/5 activation also significantly increased glutamate release and total levels of intracellular glutathione. We hypothesize that blocking STAT3/5-mediated signaling induces an adaptive, compensatory mechanism that protects breast cancer cells from ROS by up-regulating Xc- antiporter expression and function. Our findings suggest that targeting system Xc- may synergize with STAT3/5 inhibitors, heightening their therapeutic anti-cancer effects, particularly in conjunction with traditional chemotherapy treatments. This work is supported by the Canadian Breast Cancer Foundation (CBCF). Citation Format: Katja Linher-Melville, Jennifer Fazzari, Patrick Gunning, Gurmit Singh. Regulation of system Xc- by signal transducer and activator of transcription 3 and 5 in human breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-07-05.

The STAT3 gene is abnormally active in glioblastoma (GBM) and is a critically important mediator of tumor growth and therapeutic resistance in GBM. Thus, for poorly treated brain cancers such as gliomas, astrocytomas, and glioblastomas, which harbor constitutively activated STAT3, a STAT3-targeting therapeutic will be of significant importance. Herein, we report a most potent, small molecule, nonphosphorylated STAT3 inhibitor, 31 (SH-4-54) that strongly binds to STAT3 protein (K D = 300 nM). Inhibitor 31 potently kills glioblastoma brain cancer stem cells (BTSCs) and effectively suppresses STAT3 phosphorylation and its downstream transcriptional targets at low nM concentrations. Moreover, in vivo, 31 exhibited blood-brain barrier permeability, potently controlled glioma tumor growth, and inhibited pSTAT3 in vivo. This work, for the first time, demonstrates the power of STAT3 inhibitors for the treatment of BTSCs and validates the therapeutic efficacy of a STAT3 inhibitor for GBM clinical application.

The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes. In particular, Stat3 protein has attracted attention as it regulates the expression of genes involved in a variety of malignant processes, including proliferation, survival, migration, and drug resistance. Multiple myeloma (MM) is an incurable hematologic malignancy that often exhibits abnormally high levels of Stat3 activity. Although current treatment strategies can improve the clinical management of MM, it remains uniformly incurable with a dismal median survival time post-treatment of 3-4 years. Thus, novel targeted therapeutics are critically needed to improve MM patient outcomes. We herein report the development of a series of small molecule Stat3 inhibitors with potent anti-MM activity in vitro. These compounds showed high-affinity binding to Stat3's SH2 domain, inhibited intracellular Stat3 phosphorylation, and induced apoptosis in MM cell lines at low micromolar concentrations.

Myocarditis, often triggered by viral infection, may lead to heart auto-immunity and dilated cardiomyopathy. What determines the switch between disease resolution and progression is however incompletely understood. We show that pharmacological inhibition of STAT3, the main mediator of IL-6 signalling and of Th17-cell differentiation, protects mice from the development of Experimental Auto-immune Myocarditis reducing liver production of the complement component C3, and can act therapeutically when administered at disease peak. Further, we demonstrate that STAT3 is sufficient when constitutively active for triggering the onset of immune-mediated myocarditis, involving enhanced complement C3 production and IL-6 signalling amplification in the liver. Disease development can be prevented by C3 depletion and IL-6 receptor neutralization. This appears to be relevant to disease pathogenesis in humans, since acute myocarditis patients display significantly elevated circulating IL-6 and C3 levels and activated heart STAT3. Thus, aberrant IL-6/STAT3-mediated induction of liver acute phase response genes including C3, which occurs as a consequence of pre-existing inflammatory conditions, might represent an important factor determining the degree of myocarditis and its clinical outcome.

The labeling of proteins with ubiquitin/ubiquitin-like (Ubl) proteins is crucial for several physiological processes and in the onset of various diseases. Recently, targeting ubiquitin protein labeling has shifted toward the use of allosteric mechanisms over classical activity-based approaches. Allosteric enzyme regulation offers the potential for greater selectivity and has demonstrated less susceptibility to acquired resistance often associated with active site inhibitors. Furthermore, the isoform diversity among E1 activating, E2 conjugating, E3 ligase, and deubiquitinating (DUB) enzymes offers an ideal platform for modulating activity via allostery. Herein, we have reviewed allosteric inhibitors of the ubiquitin E1-E2-E3 and DUB enzymatic cascade developed over the past decade with a focus on their mechanisms of action. We have highlighted the advantages as well as the challenges associated with designing allosteric modulators of the ubiquitin labeling machinery, and the future promise in targeting these systems using allosteric approaches.

Phosphate is ubiquitously found in biological systems either in its inorganic form or incorporated into biomolecules such as DNA, lipids, and proteins. The essential roles of phosphates in biological processes sparked significant interest in the development of phosphate detection methods. Of these, fluorescence-based chemosensors offer particular advantages. In this article, readers are introduced to the progress in the development of chemosensors for inorganic phosphates, nucleotides, phosphopeptides, phosphoproteins, and phospholipids. The principles of designing a chemosensor for phosphate detection in water are introduced, including the factors that contribute to the association with a phosphate target and the resultant change in a fluorescence signal. Specific focus is placed on the challenges associated with deriving selectivity among various phosphate species and some approaches discovered to overcome this challenge.

A screen of a library of diverse small-molecules against a subset of phosphatases identified 7b and 7c, which potently inhibit TC-PTP, PTPσ and PTP1B with no inhibition of PTP-LAR, PRL2 A/S, MKPX or papain. In CHO-IR cells these inhibitors effectively suppress PTP1B activity restoring the insulin receptor phosphorylation levels.

We herein explore the structural determinants of selective phosphopeptide recognition by employing novel bivalent, bis-(Zn2+-dipicolylamine)-based coordination complexes against a select library of model phosphoserine-containing peptides of varying amino acid composition. The results demonstrated that Lewis acidic coordination complexes equipped with cationic binding groups might be best utilized as selective receptors for binding phosphopeptides with anionic side chain residues proximal to the phosphoserine residue.

Transcriptional networks control the differentiation of the hepatocyte and cholangiocyte lineages from embryonic liver progenitor cells, and their subsequent maturation to the adult phenotype. However, how accurate levels of hepatocyte and cholangiocyte gene expression are determined during differentiation remains poorly understood. Here we identify miR-337-3p as a novel regulator of liver development. MiR-337-3p stimulates expression of cholangiocyte genes and represses hepatocyte genes in undifferentiated progenitor cells in vitro and in embryonic mouse livers. Beyond the stage of lineage segregation, miR-337-3p controls the transcriptional network dynamics of developing hepatocytes, and balances both cholangiocyte populations that constitute the ductal plate. MiR-337-3p requires Notch and Transforming Growth Factor-β signaling, and exerts a biphasic control on the hepatocyte transcription factor Hepatocyte Nuclear Factor (HNF) 4α, by modulating its activation and repression. With help of an experimentally validated mathematical model we show that this biphasic control results from an incoherent feedforward loop between miR-337-3p and HNF4α. Conclusion: Our results identify miR-337-3p as a novel regulator of liver development, and highlight how tight quantitative control of hepatic cell differentiation is exerted via specific gene regulatory network motifs. This article is protected by copyright. All rights reserved.

Pharmacologically targeting activated STAT3 and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter, system xc-, contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This "ying-yang" effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient's breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers.

At the interface: Guest editors Patrick Gunning and Andrew Wilson summarize the collection of ChemMedChem articles that are part of this joint special issue with ChemBioChem focused on protein-protein interactions as targets for therapeutic intervention.

A procedure for determining force constants that is independent of the internal redundant coordinate choice is presented. The procedure is based on solving each bond and angle term separately, using the Wilson B matrix. The method only requires a single ab initio frequency calculation at the minimum energy structure and is made available in the software "parafreq". The methodology is validated with a set of small molecules, by showing it can reproduce ab initio frequencies better than other methods such as taking the diagonal terms of the Hessian in internal coordinates or by using standard AMBER force fields. Finally, the utility of the method is demonstrated by parametrizing the dizinc scaffold of bis-dipicolylamine (BDPA) bound to phosphotyrosine, which is then functionalized into promising antitumor drug proteomimetics.

A growing body of evidence shows that Signal Transducer and Activator of Transcription 5 (STAT5) protein, a key member of the STAT family of signaling proteins, plays a pivotal role in the progression of many human cancers, including acute myeloid leukemia and prostate cancer. Unlike STAT3, where significant medicinal effort has been expended to identify potent direct inhibitors, Stat5 has been poorly investigated as a molecular therapeutic target. Thus, in an effort to identify direct inhibitors of STAT5 protein, we conducted an in vitro screen of a focused library of SH2 domain binding salicylic acid-containing inhibitors (∼150) against STAT5, as well as against STAT3 and STAT1 proteins for SH2 domain selectivity. We herein report the identification of several potent (K(i)5 μM) and STAT5 selective (3-fold specificity for STAT5 cf. STAT1 and STAT3) inhibitors, BP-1-107, BP-1-108, SF-1-087, and SF-1-088. Lead agents, evaluated in K562 and MV-4-11 human leukemia cells, showed potent induction of apoptosis (IC(50)'s ∼ 20 μM) which correlated with potent and selective suppression of STAT5 phosphorylation, as well as inhibition of STAT5 target genes cyclin D1, cyclin D2, C-MYC, and MCL-1. Moreover, lead agent BP-1-108 showed negligible cytotoxic effects in normal bone marrow cells not expressing activated STAT5 protein. Inhibitors identified in this study represent some of the most potent direct small molecule, nonphosphorylated inhibitors of STAT5 to date.

Evidence shows that signal transducer and activator of transcription 5 (Stat5) protein, a member of the STAT family of signalling proteins, plays a pivotal role in the progression of many human cancers including acute myeloid leukemias and prostate cancer. This mini-review outlines progress made towards identifying agents capable of silencing aberrant Stat5 signalling.

We herein report the first application of Tb3+ for the selective detection of an important subset of the phosphoproteome, namely, proximally di-phosphorylated peptide motifs where at least one phosphorylated residue is tyrosine.

Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen–deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.

Signal transducer and activator of transcription 3 (Stat3) protein is a cytosolic transcription factor that is aberrantly activated in numerous human cancers. Inhibitors of activated Stat3–Stat3 protein complexes have been shown to hold therapeutic promise for the treatment of human cancers harboring activated Stat3. Herein, we report the design and synthesis of a focused library of salicylic acid containing Stat3 SH2 domain binders. The most potent inhibitor, 17o , effectively disrupted Stat3–phosphopeptide complexes ( K i = 13 μM), inhibited Stat3–Stat3 protein interactions (IC 50 = 19 μM) and silenced intracellular Stat3 phosphorylation and Stat3-target gene expression profiles. Inhibition of Stat3 function in both breast and multiple myeloma (MM) tumor cells correlated with induced cell death (EC 50 = 10 and 16 μM, respectively).

MLN4924 is a selective inhibitor of the NEDD8-activating enzyme (NAE) and has advanced into clinical trials for the treatment of both solid and hematological malignancies. In contrast, the structurally similar compound 1 (developed by Millennium: The Takeda Oncology Company) is a pan inhibitor of the E1 enzymes NAE, ubiquitin activating enzyme (UAE), and SUMO-activating enzyme (SAE) and is currently viewed as unsuitable for clinical use given its broad spectrum of E1 inhibition. Here, we sought to understand the determinants of NAE selectivity. A series of compound 1 analogues were synthesized through iterative functionalization of the purine C6 position and evaluated for NAE specificity. Optimal NAE specificity was achieved through substitution with primary N-alkyl groups, while bulky or secondary N-alkyl substituents were poorly tolerated. When assessed in vitro, inhibitors reduced the growth and viability of malignant K562 leukemia cells. Through this study, we have successfully identified a series of sub-10 nM NAE-specific inhibitors and thereby highlighted the functionalities that promote NAE selectivity.

Gap junctional, intercellular communication (GJIC) is interrupted in cells transformed by oncogenes such as activated Src. The Src effector, Ras, is required for this effect, so that Ras inhibition restores GJIC in Src-transformed cells. Interestingly, the inhibition of the Src effector phosphatidyl-inositol-3 kinase (PI3k) or Signal Transducer and Activator of Transcription-3 (Stat3) pathways does not restore GJIC. In the contrary, inhibition of PI3k or Stat3 in non-transformed rodent fibroblasts or epithelial cells or certain human lung carcinoma lines with extensive GJIC inhibits communication, while mutational activation of PI3k or Stat3 increases GJIC. Therefore, it appears that oncogenes such as activated Src have a dual role upon GJIC; acting as inhibitors of communication through the Ras pathway, and as activators through activation of PI3k or Stat3. In the presence of high Src activity the inhibitory functions prevail so that the net effect is gap junction closure. PI3k and Stat3 constitute potent survival signals, so that their inhibition in non-transformed cells triggers apoptosis which, in turn, has been independently demonstrated to suppress GJIC. The interruption of gap junctional communication would confine the apoptotic event to single cells and this might be essential for the maintenance of tissue integrity. We hypothesize that the GJIC activation by PI3k or Stat3 may be linked to their survival function.

Historically, Lewis acidic metal coordination complexes have played a pivotal and defining role in the broad field of molecular recognition and more specifically in the sensing and sequestration of biologically relevant anionic species. More recently, with the expanding interest in chemical biology, there has been a resurgence in the use of coordination complexes, specifically, through their application as medicinal therapeutics and chemo/biosensors. From the disruption of oncogenic protein–protein interactions to the fluorescent sensing of PTP1B phosphatase enzyme activity, the powerful binding potency of coordination complexes has been harnessed to great effect. The ingenuity of the rationally designed coordinating ligands has facilitated the diversity of roles played by Lewis metal complexes. Herein, we will review the recent advances in the application of coordination complexes in medicinal and chemo/biosensory roles over the last decade. In particular, this review will focus on Cu(II) and Zn(II) coordination complexes.

Guanine poses several problems to the synthetic chemist owing to its polyfunctional nature and poor solubility. Over the past few decades, synthetic guanines have found applications as anti-cancer and anti-viral agents. Coupled with the ever-growing interest in designer PNAs and G-quartets, simple and efficient synthetic routes to novel guanines would be of significant benefit. We herein report that, upon simple protection and/or activation step(s), the guanine precursor 2-amino-6-chloropurine is rendered an excellent substrate for Mitsunobu chemistry, furnishing, after subsequent hydrolytic dechlorination and appropriate deprotection step(s), the desired N9-mono-, N2-mono- or N2,N9-di-substituted guanines in excellent yields (≥80%). Importantly, we demonstrate that N9-functionalization proceeds with very good N9/N7 regioselectivity and with complete inversion of stereochemistry.

A facile, efficient and mild synthesis of 2,6,9-tri-substituted purines is presented, starting from commercially available 2-amino-6-chloropurine, which employs sequential N9 then N2 Mitsunobu reactions as key steps. Importantly, our synthetic approach to N2-functionalization of the purine nucleus obviates the harsh conditions required by the traditional nucleophilic aromatic substitution of a 2-halo group with primary amines. Benzylic, allylic, propargylic and aliphatic alcohols all coupled in very good to excellent yields in both Mitsunobu reactions. Significantly, excellent chemoselectivity and N9-regioselectivity were observed for the first coupling, and reactions were complete within 15 min at room temperature. Our novel methodology may be readily adapted to furnish N 9 -mono- or N 2 , N 9 -di-functionalized guanine analogues, and the utility of our protocol is further demonstrated by the efficient synthesis of the CDK inhibitor bohemine.

The signal transducers and activators of transcription (STATs) are a class of transcription factor proteins that regulate cell growth and survival by modulating the expression of specific target genes.[1] A total of seven different STAT isoforms, encoded in distinct genes, have been identified in mammalian cells. Stat3, a member of the STAT family, has been identified in an increasing number of tumor cell lines. Stat3 drives malignant progression through the misregulation of key proteins, including cell survival proteins such as Bcl-xL and Mcl-1, cell cycle regulators such as cyclin D1/D2 and c-myc, and inducers of angiogenesis such as vascular endothelial growth factor (VEGF).[2] In contrast to normal cells, where Stat3 activation is rapid and transient, neoplastic cells are found to display constitutive Stat3 activation that, once inhibited, correlates with suppression of both cell transformation and growth, and induction of apoptosis.[3-8] While STAT signaling is just one of many pathways compromised in oncogenesis, interruption of this pathway is sufficient to block cell transformation; this suggests that these cells have an irreversible dependence on constitutively active Stat3 for survival. Numerous reports highlight the relevance of persistent Stat3 activation in human cancers; abnormal levels of Stat3 activation have been observed in breast,[9,10] ovarian,[9] prostate,[11] haematological,[12] and head and neck cancer cell lines.[13] These investigations suggest that agents designed to disrupt Stat3 signaling hold considerable promise for the prevention and treatment of human cancers.

Pharmacologic blockade of the activation of signal transducer and activator of transcription 3 (STAT3) in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) cell lines characterized by kinase-independent resistance was shown to re-sensitize CML cells to TKI therapy, suggesting that STAT3 inhibitors in combination with TKIs are an effective combinatorial therapeutic for the treatment of CML. Benzoic acid- and hydroxamic acid-based STAT3 inhibitors SH-4-054 and SH-5-007, developed previously in our laboratory, demonstrated promising activity against these resistant CML cell lines. However, pharmacokinetic studies in murine models (CD-1 mice) revealed that both SH-4-054 and SH-5-007 are susceptible to glutathione conjugation at the para position of the pentafluorophenyl group via nucleophilic aromatic substitution (SN Ar). To determine whether the electrophilicity of the pentafluorophenyl sulfonamide could be tempered, an in-depth structure-activity relationship (SAR) study of the SH-4-054 scaffold was conducted. These studies revealed that AM-1-124, possessing a 2,3,5,6-tetrafluorophenylsulfonamide group, retained STAT3 protein affinity (Ki =15 μm), as well as selectivity over STAT1 (Ki >250 μm). Moreover, in both hepatocytes and in in vivo pharmacokinetic studies (CD-1 mice), AM-1-124 was found to be dramatically more stable than SH-4-054 (t1/2 =1.42 h cf. 10 min, respectively). AM-1-124 is a promising STAT3-targeting inhibitor with demonstrated bioavailability, suitable for evaluation in preclinical cancer models.

Constitutively activated STAT3 protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. In this study, PG-S3-001, a small molecule derived from the SH-4-54 class of STAT3 inhibitors, was found to inhibit patient-derived pancreatic cancer cell proliferation in vitro and in vivo in the low micromolar range. PG-S3-001 binds the STAT3 protein potently, Kd = 324 nmol/L by surface plasmon resonance, and showed no effect in a kinome screen (>100 cancer-relevant kinases). In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in pancreatic cancer cells. To better model the tumor and its microenvironment, we utilized three-dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAF). In this coculture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. Confocal microscopy was used to verify tumor cell death following treatment of 3D cocultures with PG-S3-001. The 3D model was predictive of in vivo efficacy as significant tumor growth inhibition was observed upon administration of PG-S3-001. These studies showed that the inhibition of STAT3 was able to impact the survival of tumor cells in a relevant 3D model, as well as in a xenograft model using patient-derived cells. Mol Cancer Ther; 15(5); 794–805. ©2016 AACR.

The presence of small phospho-anions, such as PPi and ATP in protein samples often complicates the robust detection of phosphoproteins by metal-based chemosensors and receptors. We herein report the development of a bis(Zn(2+)-cyclen)-triethylbenzene scaffold which can selectively sequester PPi and ATP without affecting the detection of a di-phosphorylated peptide by a ProxyPhos chemosensor.

The clinical utility of effective direct STAT inhibitors, particularly STAT3 and STAT5, for treating cancer and other diseases is well studied and known.This review will highlight the STAT inhibitor patent literature from 2011 to 2015 inclusive. Emphasis will be placed on inhibitors of the STAT3, STAT5a/b, and STAT1 proteins for cancer treatment. The review will, where suitably investigated, describe the mode and the site of inhibition, list indications that were evaluated, and rank the inhibitor's relative potency among compounds in the same class. The reader will gain an understanding of the diverse set of approaches, used both in academia and industry, to target STAT proteins.There is still much work to be done to directly target the STAT3 and STAT5 proteins. As yet, there is still no direct STAT3 inhibitor in the clinic. While the SH2 domain remains a popular target for therapeutic intervention, the DNA-binding domain and N-terminal region are now attracting attention as possible sites for inhibition. Multiple putative STAT3 and STAT5 inhibitors have now been patented across a broad spectrum of chemotypes, each with their own advantages and limitations.

PTP1B is a master regulator in the insulin and leptin metabolic pathways. Hyper-activated PTP1B results in insulin resistance and is viewed as a key factor in the onset of type II diabetes and obesity. Moreover, inhibition of PTP1B expression in cancer cells dramatically inhibits cell growth in vitro and in vivo. Herein, we report the computationally guided optimization of a salicylic acid-based PTP1B inhibitor 6, identifying new and more potent bidentate PTP1B inhibitors, such as 20h, which exhibited a > 4-fold improvement in activity. In CHO-IR cells, 20f, 20h, and 20j suppressed PTP1B activity and restored insulin receptor phosphorylation levels. Notably, 20f, which displayed a 5-fold selectivity for PTP1B over the closely related PTPσ protein, showed no inhibition of PTP-LAR, PRL2 A/S, MKPX, or papain. Finally, 20i and 20j displayed nanomolar inhibition of PTPσ, representing interesting lead compounds for further investigation.

Natural killer (NK) cells are tightly regulated by the JAK–STAT signaling pathway and cannot survive in the absence of STAT5. We now report that STAT5-deficient NK cells can be rescued by overexpression of BCL2. Our experiments define STAT5 as a master regulator of NK-cell proliferation and lytic functions. Although NK cells are generally responsible for killing tumor cells, the rescued STAT5-deficient NK cells promote tumor formation by producing enhanced levels of the angiogenic factor VEGFA. The importance of VEGFA produced by NK cells was verified by experiments with a conditional knockout of VEGFA in NK cells. We show that STAT5 normally represses the transcription of VEGFA in NK cells, in both mice and humans. These findings reveal that STAT5-directed therapies may have negative effects: In addition to impairing NK-cell–mediated tumor surveillance, they may even promote tumor growth by enhancing angiogenesis. Significance: The importance of the immune system in effective cancer treatment is widely recognized. We show that the new signal interceptors targeting the JAK–STAT5 pathway may have dangerous side effects that must be taken into account in clinical trials: inhibiting JAK–STAT5 has the potential to promote tumor growth by enhancing NK-cell–mediated angiogenesis. Cancer Discov; 6(4); 414–29. ©2016 AACR. See related commentary by Ni and Cerwenka, p. 347. This article is highlighted in the In This Issue feature, p. 331

Protein conjugation with ubiquitin and ubiquitin-like small molecules, such as UFM1, is important for promoting cancer cell survival and proliferation. Herein, the development of the first selective micromolar inhibitor of the UBA5 E1 enzyme that initiates UFM1 protein conjugation is described. This organometallic inhibitor incorporates adenosine and zinc(II)cyclen within its core scaffold and inhibits UBA5 noncompetitively and selectively over other E1 enzymes and a panel of human kinases. Furthermore, this compound selectively impedes the cellular proliferation (above 50μM) of cancer cells containing higher levels of UBA5. This inhibitor may be used to further probe the intracellular role of the UFM1 pathway in disease progression.

The coordination chemistry of the tetradentate pyridyl N-donor ligand cis-3,5-bis-[2-pyridinyleneamin]-trans-hydroxycyclohexane (DDOP) has been investigated with zinc(II) nitrate and triflate. The resulting complexes, [Zn(DDOP)(H2O)(NO3)](NO3) (1), and [Zn(DDOP)(H2O)(OTf)](OTf) (2) differ not only in their counterions, but also the arrangement of the axial ligands and their solid state hydrogen bonded networks. Isothermal titration calorimetry was used to assess the difference in binding properties exhibited by the two zinc complexes at physiological pH in an aqueous environment. A series of coordinating amino acids were found to preferentially bind to the mononuclear zinc triflate (1) complex over the corresponding nitrate (2) assembly, with histidine exhibiting a two centre binding mode.

We herein report the first application of Tb(3+) for the selective detection of an important subset of the phosphoproteome, namely, proximally di-phosphorylated peptide motifs where at least one phosphorylated residue is tyrosine.

// Mohini Singh 1, 4 , Neha Garg 1, 6 , Chitra Venugopal 1, 6 , Robin Hallett 2 , Tomas Tokar 7 , Nicole McFarlane 1, 6 , Sujeivan Mahendram 1, 6 , David Bakhshinyan 1, 4 , Branavan Manoranjan 1, 3, 4 , Parvez Vora 1, 6 , Maleeha Qazi 1, 4 , Carolynn C. Arpin 10 , Brent Page 10 , Sina Haftchenary 10 , David A. Rosa 10 , Ping-Shan Lai 10 , Rodolfo F. Gomez-Biagi 10 , Ahmed M. Ali 11 , Andrew Lewis 10 , Mulu Geletu 10 , Naresh K. Murty 6 , John A. Hassell 1, 2, 4, 5 , Igor Jurisica 7, 8, 9 , Patrick T. Gunning 10 , Sheila K. Singh 1, 4, 6 1 McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, Ontario, Canada 2 McMaster Centre for Functional Genomics, McMaster University, Hamilton, Ontario, Canada 3 Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada 4 Departments of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada 5 Departments of Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario, Canada 6 Departments of Surgery, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada 7 Princess Margaret Cancer Centre, University Health Network, IBM Life Sciences Discovery Centre, Toronto Medical Discovery Tower, Toronto, Ontario, Canada 8 TECHNA Institute for the Advancement of Technology for Health, UHN and University of Toronto, Toronto, Ontario, Canada 9 Departments of Medical Biophysics and Computer Science, University of Toronto, Toronto, Ontario, Canada 10 Department of Chemistry, University of Toronto, Mississauga, Ontario, Canada 11 Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt Correspondence to: Sheila K. Singh, e-mail: ssingh@mcmaster.ca Keywords: brain metastases, brain metastasis initiating cell, STAT3, miR-21 Received: February 26, 2015 Accepted: July 13, 2015 Published: July 25, 2015 ABSTRACT Brain metastases (BM) represent the most common tumor to affect the adult central nervous system. Despite the increasing incidence of BM, likely due to consistently improving treatment of primary cancers, BM remain severely understudied. In this study, we utilized patient-derived stem cell lines from lung-to-brain metastases to examine the regulatory role of STAT3 in brain metastasis initiating cells (BMICs). Annotation of our previously described BMIC regulatory genes with protein-protein interaction network mapping identified STAT3 as a novel protein interactor. STAT3 knockdown showed a reduction in BMIC self-renewal and migration, and decreased tumor size in vivo . Screening of BMIC lines with a library of STAT3 inhibitors identified one inhibitor to significantly reduce tumor formation. Meta-analysis identified the oncomir microRNA-21 (miR-21) as a target of STAT3 activity. Inhibition of miR-21 displayed similar reductions in BMIC self-renewal and migration as STAT3 knockdown. Knockdown of STAT3 also reduced expression of known downstream targets of miR-21. Our studies have thus identified STAT3 and miR-21 as cooperative regulators of stemness, migration and tumor initiation in lung-derived BM. Therefore, STAT3 represents a potential therapeutic target in the treatment of lung-to-brain metastases.

Activation of the transcription factor STAT5 is essential for the pathogenesis of acute myeloid leukemia (AML) containing the FLT3 internal tandem duplication (ITD). FLT3 ITD is a constitutively active tyrosine kinase (TK) that drives the activation of STAT5 leading to the growth and survival of AML cells. Although there has been some success in identifying TK inhibitors that block the function of FLT3 ITD, treatment options are still limited. This is mainly due to drug resistance development by mutations that allow for the continued activation of STATs. Since STAT5 represents a critical mediator of malignant cellular behavior and sits at the convergence point of many kinase pathways, the direct targeting of STAT5 may be an effective means of overcoming this resistance to TK inhibitors. First, we screened a library of potential STAT5 inhibitors for specific SH2 domain binders using a fluorescent polarization assay. Thereby, we identified small inhibitory molecules, called AC-3-019 and AC-4-130, that bind to the SH2 domain of STAT5, subsequently resulting in the disruption of the reciprocal STAT5-phosphopeptide interactions. They efficiently blocked kinase-mediated phosphorylation, dimer formation, nuclear translocation, DNA binding and STAT5 mediated target gene expression. Further, we observed a time- and dose-dependent impairment of proliferation, blocked cell cycle progression and increased apoptosis. Studies with human AML patient-derived samples similarly showed an induction of apoptotic cell death and decreased colony forming capabilities. A combinatorial drug screen revealed synergistic effects with TK inhibitors, as well as with drugs standardly used in the treatment of AML patients, e.g. Cytarabine. Finally, AC-3-019 and AC-4-130 significantly suppressed tumor growth in vivo without general toxicity in healthy organs. Overall, our findings indicate that AC-3-019 and AC-4-130 are potent and selective inhibitors of STAT5. These compounds provide lead structures for further chemical modifications and clinical development for the identification of compounds to improve existing therapies. Citation Format: Bettina Wingelhofer, Barbara Maurer, Elizabeth C. Heyes, Patricia Freund, Abbarna A. Cumaraswamy, Jisung Park, Stefan Kubicek, Peter Valent, Patrick T. Gunning, Richard Moriggl. Characterization of novel STAT5 inhibitors to interfere with the oncogenic activities of STAT5 in hematopoietic diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1184. doi:10.1158/1538-7445.AM2017-1184

Chronic myeloid leukemia (CML) is a hematopoietic cell disorder, with an annual incidence of 1–1.5 per 100 000 in the United States. The BCR-ABL1 oncogene is necessary and sufficient to cause the leukemic phenotype. CML is typically diagnosed in the chronic phase, where the myeloid cell compartment is expanded, but cellular differentiation maintained. Without effective therapy, there is inevitable transformation to the blastic phase, which resembles an acute leukemia of myeloid or lymphoid phenotype. Current CML therapy is based on tyrosine kinase inhibitors (TKIs), which block progression from the chronic to the blastic phase in the majority of patients. The first-generation TKI, imatinib mesylate (IM), is generally well tolerated, but has a failure rate of about 25% due to resistance or intolerance. Second-generation TKIs (dasatinib; nilotinib) provide effective salvage options, but some patients will eventually progress to blastic phase and have a poor prognosis. Additionally, even in patients with profound responses (complete molecular response), recurrence is common upon discontinuation of TKI therapy, a situation referred to as persistence. TKI resistance can result from two fundamentally different mechanisms: BCR-ABL1-dependent resistance, in which kinase domain mutations restore kinase activity by disrupting TKI binding,1, 2 and BCR-ABL1-independent resistance, which can be divided into (i) extrinsic resistance, in which contact between CML cells and bone marrow-derived factors protects CML cells from TKIs,3, 4 and (ii) cell-autonomous (intrinsic) resistance, in which CML cells activate alternative signaling pathways in the absence of bone marrow-derived factors.5, 6 Importantly, BCR-ABL1-independent resistance occurs despite continued suppression of BCR-ABL1 activity. It is thought that persistence of residual leukemia in patients responding to TKIs is due to the innate BCR-ABL1 independence of CML stem cells.7 Consistent with this, we have found that primitive CML progenitor cells, including quiescent Lin− CML cells, survive despite TKI inhibition of BCR-ABL1.8 Our recent studies have implicated STAT3 in BCR-ABL1-independent resistance. In line with previous reports, we have found upregulation of pSTAT3Y705 in TKI-naive CML progenitor cells grown in HS-5 bone marrow stromal cell-conditioned medium in the presence of IM, and that pSTAT3Y705 promotes CML cell survival, implicating STAT3 as a critical mediator of extrinsic BCR-ABL1-independent resistance. Interestingly, we have also found pSTAT3Y705 activation in CML CD34+ cells from patients with intrinsic TKI resistance (clinical resistance in the absence of BCR-ABL1 mutations). This suggests that extrinsic and intrinsic BCR-ABL1-independent resistance mechanisms converge on pSTAT3Y705, a notion that was validated with STAT3-specific small hairpin RNA and dominant-negative mutants. Our observations prompted us to evaluate the effects of small-molecule STAT3 inhibitors that block STAT3 dimerization through binding to the SH2 domain. Successive STAT3 inhibitor library screens identified BP-5-087 as a highly potent and selective STAT3 inhibitor. Computational modeling and physicochemical assays confirmed binding of BP-5-087 with STAT3. In biological assays using TKI-resistant progenitors, we found that BP-5-087 combined with imatinib reduces survival and colony formation by CML progenitor and stem cells from patients with BCR-ABL1-independent resistance, consistent with a synthetically lethal pharmacologic interaction. Taken together, our data indicate that activation of pSTAT3Y705 is central to BCR-ABL1 kinase-independent TKI resistance, and that genetic, functional or pharmacologic inhibition of STAT3 reduces TKI-resistant clonogenic potential in vitro. BP-5-087 is a highly potent and selective STAT3 inhibitor. Combinations of BP5-087 with imatinib greatly reduce the survival of primary TKI-resistant stem and progenitor cells, and as such, may be considered a promising combinatorial therapy to overcome BCR-ABL1-independent TKI resistance and eliminate residual leukemia.

© 2014 American Chemical Society. We herein report the design and synthesis of the first nanomolar binding inhibitor of STAT5 protein. Lead compound 13a, possessing a phosphotyrosyl-mimicking salicylic acid group, potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain complexation events in vitro, silences activated STAT5 in leukemic cells, as well as STAT5's downstream transcriptional targets, including MYC and MCL1, and, as a result, leads to apoptosis. We believe 13a represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials.

Objective Nearly 20%–29% of patients with colorectal cancer (CRC) succumb to liver or lung metastasis and there is a dire need for novel targets to improve the survival of patients with metastasis. The long isoform of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-L or CC1-L) is a key regulator of immune surveillance in primary CRC, but its role in metastasis remains largely unexplored. We have examined how CC1-L expression impacts on colon cancer liver metastasis. Design Murine MC38 transfected with CC1-L were evaluated in vitro for proliferation, migration and invasion, and for in vivo experimental liver metastasis. Using shRNA silencing or pharmacological inhibition, we delineated the role in liver metastasis of Chemokine (C-C motif) Ligand 2 (CCL2) and Signal Transducer and Activator of Transcription 3 (STAT3) downstream of CC1-L. We further assessed the clinical relevance of these findings in a cohort of patients with CRC. Results MC38-CC1-L-expressing cells exhibited significantly reduced in vivo liver metastasis and displayed decreased CCL2 chemokine secretion and reduced STAT3 activity. Down-modulation of CCL2 expression and pharmacological inhibition of STAT3 activity in MC38 cells led to reduced cell invasion capacity and decreased liver metastasis. The clinical relevance of our findings is illustrated by the fact that high CC1 expression in patients with CRC combined with some inflammation-regulated and STAT3-regulated genes correlate with improved 10-year survival. Conclusions CC1-L regulates inflammation and STAT3 signalling and contributes to the maintenance of a less-invasive CRC metastatic phenotype of poorly differentiated carcinomas.

The Met receptor tyrosine kinase is activated or genetically amplified in some gastric cancers, but resistance to small-molecule inhibitors of Met often emerges in patients. We found that Met abundance correlated with a proliferation marker in patient gastric tumor sections, and gastric cancer cell lines that have MET amplifications depended on Met for proliferation and anchorage-independent growth in culture. Inhibition of Met induced temporal changes in gene expression in the cell lines, initiated by a rapid decrease in the expression of genes encoding transcription factors, followed by those encoding proteins involved in epithelial-mesenchymal transition, and finally those encoding cell cycle-related proteins. In the gastric cancer cell lines, microarray and chromatin immunoprecipitation analysis revealed considerable overlap between genes regulated in response to Met stimulation and those regulated by signal transducer and activator of transcription 3 (STAT3). The activity of STAT3, extracellular signal-regulated kinase (ERK), and the kinase Akt was decreased by Met inhibition, but only inhibitors of STAT3 were as effective as the Met inhibitor in decreasing tumor cell proliferation in culture and in xenografts, suggesting that STAT3 mediates the pro-proliferative program induced by Met. However, the phosphorylation of ERK increased after prolonged Met inhibition in culture, correlating with decreased abundance of the phosphatases DUSP4 and DUSP6, which inhibit ERK. Combined inhibition of Met and the mitogen-activated protein kinase kinase (MEK)-ERK pathway induced greater cell death in cultured gastric cancer cells than did either inhibitor alone. These findings indicate combination therapies that may counteract resistance to Met inhibitors.

The mild and efficient deblocking of aryl benzyl ethers with TFA is reported. Cleavage was fastest with ortho-electron-withdrawing groups on the phenolic ring, which we have attributed to a proton chelation effect, furnishing the deprotected phenols in excellent yields. The corresponding para-methoxybenzyl, allyl and iso-propyl ethers were also cleanly removed under these conditions. In addition, the selective aryl benzyl ether debenzylation in the presence of benzyl ester, Cbz carbamate and Boc carbamate functionalities was also observed.