Publications by authors named "Patrick T Gunning"

80 Publications

Unique Molecular Interaction with the Histone Deacetylase 6 Catalytic Tunnel: Crystallographic and Biological Characterization of a Model Chemotype.

J Med Chem 2021 Mar 12;64(5):2691-2704. Epub 2021 Feb 12.

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd N., Mississauga, Ontario L5L 1C6, Canada.

Histone deacetylase 6 (HDAC6) is involved in multiple regulatory processes, ranging from cellular stress to intracellular transport. Inhibition of aberrant HDAC6 activity in several cancers and neurological diseases has been shown to be efficacious in both preclinical and clinical studies. While selective HDAC6 targeting has been pursued as an alternative to pan-HDAC drugs, identifying truly selective molecular templates has not been trivial. Herein, we report a structure-activity relationship study yielding , which potently binds HDAC6 catalytic domain 2 ( = 0.7 nM) and inhibits the enzyme function (IC = 2 nM). exhibits 158-fold selectivity for HDAC6 over other HDAC isozymes by binding the catalytic Zn and, uniquely, making a never seen before direct hydrogen bond with the Zn coordinating residue, His614. This novel structural motif targeting the second-sphere His614 interaction, observed in a 1.84 Å resolution crystal structure with HDAC6 from zebrafish, can provide new pharmacophores for identifying enthalpically driven, high-affinity, HDAC6-selective inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063965PMC
March 2021

Sensitive Detection of Broad-Spectrum Bacteria with Small-Molecule Fluorescent Excimer Chemosensors.

ACS Sens 2020 09 6;5(9):2753-2762. Epub 2020 Aug 6.

Department of Chemical & Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada.

Antibiotic resistance is a major problem for world health, triggered by the unnecessary usage of broad-spectrum antibiotics on purportedly infected patients. Current clinical standards require lengthy protocols for the detection of bacterial species in sterile physiological fluids. In this work, a class of small-molecule fluorescent chemosensors termed was shown to be capable of rapid, sensitive, and facile detection of broad-spectrum bacteria. The sensors act via a turn-on fluorescent excimer mechanism, where close-proximity binding of multiple sensor units amplifies a red shift emission signal. sensors were able to detect down to 10 CFUs of model strains by flow cytometry assays and showed selectivity over mammalian cells in a bacterial coculture through fluorescence microscopy. The studies reveal that the zinc(II)-chelates cyclen and cyclam are novel and effective binding units for the detection of both Gram-negative and Gram-positive bacterial strains. Mode of action studies revealed that the chemosensors detect Gram-negative and Gram-positive strains with two distinct mechanisms. Preliminary studies applying sensors to sterile physiological fluids (cerebrospinal fluid) in flow cytometry assays were successful. The results suggest that sensors can be developed as a rapid, inexpensive, and robust tool for the "yes-no" detection of broad-spectrum bacteria in sterile fluids.
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http://dx.doi.org/10.1021/acssensors.9b02490DOI Listing
September 2020

Targeting STAT3 and STAT5 in Cancer.

Cancers (Basel) 2020 Jul 22;12(8). Epub 2020 Jul 22.

Institute of Animal Breeding and Genetics, University of Veterinary Medicine, A-1210 Vienna, Austria.

Insights into the mutational landscape of the human cancer genome coding regions defined about 140 distinct cancer driver genes in 2013, which approximately doubled to 300 in 2018 following advances in systems cancer biology studies [...].
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http://dx.doi.org/10.3390/cancers12082002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465272PMC
July 2020

Characterization of Conformationally Constrained Benzanilide Scaffolds for Potent and Selective HDAC8 Targeting.

J Med Chem 2020 08 31;63(15):8634-8648. Epub 2020 Jul 31.

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga ON L5L 1C6, Canada.

Histone deacetylases (HDACs) are an attractive therapeutic target for a variety of human diseases. Currently, all four FDA-approved HDAC-targeting drugs are nonselective, pan-HDAC inhibitors, exhibiting adverse side effects at therapeutic doses. Although selective HDAC inhibition has been proposed to mitigate toxicity, the targeted catalytic domains are highly conserved. Herein, we describe a series of rationally designed, conformationally constrained, benzanilide foldamers which selectively bind the catalytic tunnel of HDAC8. The series includes benzanilides, , , and , which exhibit potent HDAC8 activity (IC = 66, 23, and 66 nM, respectively) and up to 410-fold selectivity for HDAC8 over the next targeted HDAC. Experimental and computational analyses of the benzanilide structure docked with human HDAC8 enzyme showed the adoption of a low-energy L-shaped conformer that favors HDAC8 selectivity. The conformationally constrained HDAC8 inhibitors present an alternative biological probe for further determining the clinical utility and safety of pharmacological knockdown of HDAC8 in diseased cells.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01025DOI Listing
August 2020

PTG-0861: A novel HDAC6-selective inhibitor as a therapeutic strategy in acute myeloid leukaemia.

Eur J Med Chem 2020 Sep 6;201:112411. Epub 2020 Jun 6.

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359, Mississauga Road, Mississauga, Ontario, L5L 1C6, Canada; Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, M5S 3H6, Canada. Electronic address:

Dysregulated Histone Deacetylase (HDAC) activity across multiple human pathologies have highlighted this family of epigenetic enzymes as critical druggable targets, amenable to small molecule intervention. While efficacious, current approaches using non-selective HDAC inhibitors (HDACi) have been shown to cause a range of undesirable clinical toxicities. To circumvent this, recent efforts have focused on the design of highly selective HDACi as a novel therapeutic strategy. Beyond roles in regulating transcription, the unique HDAC6 (with two catalytic domains) regulates the deacetylation of α-tubulin; promoting growth factor-controlled cell motility, cell division, and metastatic hallmarks. Recent studies have linked aberrant HDAC6 function in various hematological cancers including acute myeloid leukaemia and multiple myeloma. Herein, we report the discovery, in vitro characterization, and biological evaluation of PTG-0861 (JG-265), a novel HDAC6-selective inhibitor with strong isozyme-selectivity (∼36× ) and low nanomolar potency (IC = 5.92 nM) against HDAC6. This selectivity profile was rationalized via in silico docking studies and also observed in cellulo through cellular target engagement. Moreover, PTG-0861 achieved relevant potency against several blood cancer cell lines (e.g. MV4-11, MM1S), whilst showing limited cytotoxicity against non-malignant cells (e.g. NHF, HUVEC) and CD-1 mice. In examining compound stability and cellular permeability, PTG-0861 revealed a promising in vitro pharmacokinetic (PK) profile. Altogether, in this study we identified a novel and potent HDAC6-selective inhibitor (∼4× more selective than current clinical standards - citarinostat, ricolinostat), which achieves cellular target engagement, efficacy in hematological cancer cells with a promising safety profile and in vitro PK.
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http://dx.doi.org/10.1016/j.ejmech.2020.112411DOI Listing
September 2020

STAT5 is Expressed in CD34/CD38 Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms.

Cancers (Basel) 2020 Apr 21;12(4). Epub 2020 Apr 21.

Ludwig Boltzmann Institute for Hematology and Oncology, Medical University of Vienna, 1090 Vienna, Austria.

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, V617F or mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, = 10), essential thrombocythemia (ET, = 15) and primary myelofibrosis (PMF, = 9), and in the V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34/CD38 MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34/CD38 MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.
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http://dx.doi.org/10.3390/cancers12041021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225958PMC
April 2020

Advances in covalent kinase inhibitors.

Chem Soc Rev 2020 May 30;49(9):2617-2687. Epub 2020 Mar 30.

Department of Chemical & Physical Sciences, University of Toronto, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada.

Over the past decade, covalent kinase inhibitors (CKI) have seen a resurgence in drug discovery. Covalency affords a unique set of advantages as well as challenges relative to their non-covalent counterpart. After reversible protein target recognition and binding, covalent inhibitors irreversibly modify a proximal nucleophilic residue on the protein via reaction with an electrophile. To date, the acrylamide group remains the predominantly employed electrophile in CKI development, with its incorporation in the majority of clinical candidates and FDA approved covalent therapies. Nonetheless, in recent years considerable efforts have ensued to characterize alternative electrophiles that exhibit irreversible or reversibly covalent binding mechanisms towards cysteine thiols and other amino acids. This review article provides a comprehensive overview of CKIs reported in the literature over a decade period, 2007-2018. Emphasis is placed on the rationale behind warhead choice, optimization approach, and inhibitor design. Current FDA approved CKIs are also highlighted, in addition to a detailed analysis of the common trends and themes observed within the listed data set.
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http://dx.doi.org/10.1039/c9cs00720bDOI Listing
May 2020

Optimization of a high-throughput fluorescence polarization assay for STAT5B DNA binding domain-targeting inhibitors.

J Pharm Biomed Anal 2020 May 19;184:113182. Epub 2020 Feb 19.

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd N., Mississauga, Ontario L5L 1C6, Canada; Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada. Electronic address:

Signal transducer and activator of transcription 5B (STAT5B) is constitutively activated in multiple cancers as a result of hyperactivating mutations or dysregulation of upstream effectors. Therapeutic strategies have predominantly targeted the Src homology 2 (SH2) domain to inhibit STAT phosphorylation, a prerequisite for STAT5B transcriptional activation. An alternative approach for STAT5B pharmacologic inhibition involves targeting the DNA-binding domain (DBD). However, this strategy remains relatively unexplored and is further hindered by the lack of a high-throughput in vitro engagement assay. Herein, we present the development and optimization of a STAT5B DBD fluorescence polarization (FP) assay, which facilitates rapid screening of small molecules targeting the STAT5B DBD though displacement of a fluorescently labelled oligonucleotide. The assay can generate a complete DNA-binding profile in 10 min, with signal stability up to 2 h, and minimal changes under a range of conditions including 10 % (v/v) glycerol, 15 % (v/v) DMSO, 1 mM NaCl, 0.02 % (w/v) BSA, and 1 mM EDTA. This assay is compatible with both unphosphorylated and phosphorylated STAT5B and demonstrates suitability for high-throughput screening with a Z' factor of 0.68 ± 0.07 and a signal to noise ratio of 6.7 ± 0.84.
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http://dx.doi.org/10.1016/j.jpba.2020.113182DOI Listing
May 2020

Class I/IIb-Selective HDAC Inhibitor Exhibits Oral Bioavailability and Therapeutic Efficacy in Acute Myeloid Leukemia.

ACS Med Chem Lett 2020 Jan 13;11(1):56-64. Epub 2019 Dec 13.

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario L5L 1C6, Canada.

The HDAC inhibitor 4--butyl--(4-(hydroxycarbamoyl)phenyl)benzamide (, ) was identified as a promising preclinical candidate for the treatment of acute myeloid leukemia (AML), an aggressive malignancy with a meagre 24% 5-year survival rate. Through screening of low-molecular-weight analogues derived from the previously discovered novel HDAC inhibitor, , compound demonstrated greater HDAC isoform selectivity, higher cytotoxicity in MV4-11 cells, an improved therapeutic window, and more efficient absorption through cellular and lipid membranes. Compound also demonstrated improved oral bioavailability compared to SAHA in mouse models. A broad spectrum of experiments, including FACS, ELISA, and Western blotting, were performed to support our hypothesis that dose-dependently triggers apoptosis in AML cells through HDAC inhibition.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956385PMC
January 2020

Targeting prenylation inhibition through the mevalonate pathway.

RSC Med Chem 2020 Jan 23;11(1):51-71. Epub 2019 Dec 23.

Department of Chemical and Physical Sciences , University of Toronto Mississauga , 3359 Mississauga Rd N. , Mississauga , Ontario L5L 1C6 , Canada . Email:

Protein prenylation is a critical mediator in several diseases including cancer and acquired immunodeficiency syndrome (AIDS). Therapeutic intervention has focused primarily on directly targeting the prenyltransferase enzymes, FTase and GGTase I and II. To date, several drugs have advanced to clinical trials and while promising, they have yet to gain approval in a medical setting due to off-target effects and compensatory mechanisms activated by the body which results in drug resistance. While the development of dual inhibitors has mitigated undesirable side effects, potency remains sub-optimal for clinical development. An alternative approach involves antagonizing the upstream mevalonate pathway enzymes, FPPS and GGPPS, which mediate prenylation as well as cholesterol synthesis. The development of these inhibitors presents novel opportunities for dual inhibition of cancer-driven prenylation as well as cholesterol accumulation. Herein, we highlight progress towards the development of inhibitors against the prenylation machinery.
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http://dx.doi.org/10.1039/c9md00442dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485146PMC
January 2020

Direct Targeting Options for STAT3 and STAT5 in Cancer.

Cancers (Basel) 2019 Dec 3;11(12). Epub 2019 Dec 3.

Institute of Animal Breeding and Genetics, University of Veterinary Medicine, 1210 Vienna, Austria.

Signal transducer and activator of transcription (STAT)3 and STAT5 are important transcription factors that are able to mediate or even drive cancer progression through hyperactivation or gain-of-function mutations. Mutated STAT3 is mainly associated with large granular lymphocytic T-cell leukemia, whereas mutated STAT5B is associated with T-cell prolymphocytic leukemia, T-cell acute lymphoblastic leukemia and γδ T-cell-derived lymphomas. Hyperactive STAT3 and STAT5 are also implicated in various hematopoietic and solid malignancies, such as chronic and acute myeloid leukemia, melanoma or prostate cancer. Classical understanding of STAT functions is linked to their phosphorylated parallel dimer conformation, in which they induce gene transcription. However, the functions of STAT proteins are not limited to their phosphorylated dimerization form. In this review, we discuss the functions and the roles of unphosphorylated STAT3/5 in the context of chromatin remodeling, as well as the impact of STAT5 oligomerization on differential gene expression in hematopoietic neoplasms. The central involvement of STAT3/5 in cancer has made these molecules attractive targets for small-molecule drug development, but currently there are no direct STAT3/5 inhibitors of clinical grade available. We summarize the development of inhibitors against the SH2 domains of STAT3/5 and discuss their applicability as cancer therapeutics.
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http://dx.doi.org/10.3390/cancers11121930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966570PMC
December 2019

Structural Implications of STAT3 and STAT5 SH2 Domain Mutations.

Cancers (Basel) 2019 Nov 8;11(11). Epub 2019 Nov 8.

Centre for Medicinal Chemistry, University of Toronto at Mississauga, Mississauga, ON L5L 1C6, Canada.

Src Homology 2 (SH2) domains arose within metazoan signaling pathways and are involved in protein regulation of multiple pleiotropic cascades. In signal transducer and activator of transcription (STAT) proteins, SH2 domain interactions are critical for molecular activation and nuclear accumulation of phosphorylated STAT dimers to drive transcription. Sequencing analysis of patient samples has revealed the SH2 domain as a hotspot in the mutational landscape of STAT proteins although the functional impact for the vast majority of these mutations remains poorly characterized. Despite several well resolved structures for SH2 domain-containing proteins, structural data regarding the distinctive STAT-type SH2 domain is limited. Here, we review the unique features of STAT-type SH2 domains in the context of all currently reported STAT3 and STAT5 SH2 domain clinical mutations. The genetic volatility of specific regions in the SH2 domain can result in either activating or deactivating mutations at the same site in the domain, underscoring the delicate evolutionary balance of wild type STAT structural motifs in maintaining precise levels of cellular activity. Understanding the molecular and biophysical impact of these disease-associated mutations can uncover convergent mechanisms of action for mutations localized within the STAT SH2 domain to facilitate the development of targeted therapeutic interventions.
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http://dx.doi.org/10.3390/cancers11111757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895964PMC
November 2019

Structural and functional consequences of the STAT5B driver mutation.

Nat Commun 2019 06 7;10(1):2517. Epub 2019 Jun 7.

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON, L5L 1C6, Canada.

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5B, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5B in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5B-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5B patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5B crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5B can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5B, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5B activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.
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http://dx.doi.org/10.1038/s41467-019-10422-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555848PMC
June 2019

High activation of STAT5A drives peripheral T-cell lymphoma and leukemia.

Haematologica 2020 31;105(2):435-447. Epub 2020 Jan 31.

Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria.

Recurrent gain-of-function mutations in the transcription factors and much more in were found in hematopoietic malignancies with the highest proportion in mature T- and natural killer-cell neoplasms (peripheral T-cell lymphoma, PTCL). No targeted therapy exists for these heterogeneous and often aggressive diseases. Given the shortage of models for PTCL, we mimicked graded STAT5A or STAT5B activity by expressing hyperactive or variants at low or high levels in the hematopoietic system of transgenic mice. Only mice with high activity levels developed a lethal disease resembling human PTCL. Neoplasia displayed massive expansion of CD8 T cells and destructive organ infiltration. T cells were cytokine-hypersensitive with activated memory CD8 T-lymphocyte characteristics. Histopathology and mRNA expression profiles revealed close correlation with distinct subtypes of PTCL. Pronounced STAT5 expression and activity in samples from patients with different subsets underline the relevance of JAK/STAT as a therapeutic target. JAK inhibitors or a selective STAT5 SH domain inhibitor induced cell death and ruxolitinib blocked T-cell neoplasia We conclude that enhanced STAT5A or STAT5B action both drive PTCL development, defining both STAT5 molecules as targets for therapeutic intervention.
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http://dx.doi.org/10.3324/haematol.2019.216986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012494PMC
April 2021

Effect of caveolin-1 on Stat3-ptyr705 levels in breast and lung carcinoma cells.

Biochem Cell Biol 2019 10 15;97(5):638-646. Epub 2019 Apr 15.

Department of Biomedical and Molecular Sciences, Pathology and Molecular Medicine, and Queen's University Cancer Research Institute, Queen's University, Kingston, ON K7L 3N6, Canada.

We recently demonstrated that Cav1 (caveolin-1) is a negative regulator of Stat3 (signal transducer and activator of transcription-3) activity in mouse fibroblasts and human lung carcinoma SHP77 cells. We now examined whether the cellular context may affect their levels as well as the relationship between them, by assessing Cav1 and Stat3-ptyr705 amounts in different cell lines. In MDA-MB-231, A549, and HaCat cells, Cav1 levels were high and Stat3-ptyr705 levels were low, consistent with the notion of a negative effect of endogenous Cav1 on Stat3-ptyr705 levels in these lines. In addition, manipulation of Cav1 levels revealed a negative effect in MCF7 and mouse fibroblast cells, while Cav1 upregulation induced apoptosis in MCF7 cells. In contrast, however, line MRC9 had high Cav1 and high Stat3-ptyr705 levels, indicating that high Cav1 is insufficient to reduce Stat3-ptyr705 levels in this line. MCF7 and LuCi6 cells had very low Cav1 and Stat3-ptyr705 levels, indicating that the low Stat3-ptyr705 can be independent from Cav1 levels altogether. Our results reveal a further level of complexity in the relationship between Cav1 and Stat3-ptyr705 than previously thought. In addition, we demonstrate that in a feedback loop, Stat3 inhibition upregulates Cav1 in HeLa cells but not in other lines tested.
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http://dx.doi.org/10.1139/bcb-2018-0367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038897PMC
October 2019

Identification and Characterization of AES-135, a Hydroxamic Acid-Based HDAC Inhibitor That Prolongs Survival in an Orthotopic Mouse Model of Pancreatic Cancer.

J Med Chem 2019 03 6;62(5):2651-2665. Epub 2019 Mar 6.

Department of Chemical and Physical Sciences , University of Toronto Mississauga , 3359 Mississauga Road , Mississauga , Ontario L5L 1C6 , Canada.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, incurable cancer with a 20% 1 year survival rate. While standard-of-care therapy can prolong life in a small fraction of cases, PDAC is inherently resistant to current treatments, and novel therapies are urgently required. Histone deacetylase (HDAC) inhibitors are effective in killing pancreatic cancer cells in in vitro PDAC studies, and although there are a few clinical studies investigating combination therapy including HDAC inhibitors, no HDAC drug or combination therapy with an HDAC drug has been approved for the treatment of PDAC. We developed an inhibitor of HDACs, AES-135, that exhibits nanomolar inhibitory activity against HDAC3, HDAC6, and HDAC11 in biochemical assays. In a three-dimensional coculture model, AES-135 kills low-passage patient-derived tumor spheroids selectively over surrounding cancer-associated fibroblasts and has excellent pharmacokinetic properties in vivo. In an orthotopic murine model of pancreatic cancer, AES-135 prolongs survival significantly, therefore representing a candidate for further preclinical testing.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190078PMC
March 2019

PI3k and Stat3: Oncogenes that are Required for Gap Junctional, Intercellular Communication.

Cancers (Basel) 2019 Feb 1;11(2). Epub 2019 Feb 1.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada.

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.
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http://dx.doi.org/10.3390/cancers11020167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406562PMC
February 2019

The ERBB-STAT3 Axis Drives Tasmanian Devil Facial Tumor Disease.

Cancer Cell 2019 01;35(1):125-139.e9

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria. Electronic address:

The marsupial Tasmanian devil (Sarcophilus harrisii) faces extinction due to transmissible devil facial tumor disease (DFTD). To unveil the molecular underpinnings of this transmissible cancer, we combined pharmacological screens with an integrated systems-biology characterization. Sensitivity to inhibitors of ERBB tyrosine kinases correlated with their overexpression. Proteomic and DNA methylation analyses revealed tumor-specific signatures linked to the evolutionary conserved oncogenic STAT3. ERBB inhibition blocked phosphorylation of STAT3 and arrested cancer cells. Pharmacological blockade of ERBB or STAT3 prevented tumor growth in xenograft models and restored MHC class I expression. This link between the hyperactive ERBB-STAT3 axis and major histocompatibility complex class I-mediated tumor immunosurveillance provides mechanistic insights into horizontal transmissibility and puts forward a dual chemo-immunotherapeutic strategy to save Tasmanian devils from DFTD. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.ccell.2018.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335503PMC
January 2019

A functional in vitro assay for screening inhibitors of STAT5B phosphorylation.

J Pharm Biomed Anal 2019 Jan 18;162:60-65. Epub 2018 Aug 18.

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd N., Mississauga, Ontario L5L 1C6, Canada; Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada. Electronic address:

Inhibition of STAT phosphorylation is recognized as a viable therapeutic strategy for disrupting tumorigenesis. Constitutive STAT phosphorylation is found with high frequency in a number of primary tumor types, while non-cancer cells exhibit low basal activity, providing an exploitable therapeutic window. STAT activation involves phosphorylation of the SH domain by a number of tyrosine kinases followed by STAT dimerization and translocation to the nucleus. By blocking the cognate binding site, STAT SH-domain inhibitors can impede kinase-mediated de novo STAT phosphorylation. Assessing for inhibitors of STAT phosphorylation has previously been conducted exclusively in cellulo using Western blot analysis. However, while providing useful in cellulo efficacy, it is not possible to conclude that inhibition is due to a direct blockade of STAT protein. Here we developed a functional assay that directly reports the blockade of phosphorylation as a result of inhibitor interaction with STAT proteins. We have optimized reaction conditions for the functional assay and validated the assay against known STAT5B ligands, including peptides and small molecule inhibitors. As part of the study, we have also identified several sites of STAT5B phosphorylation by Abl kinase. This assay will serve to delineate the functional mechanism of STAT binders in vitro and deconvolute the mechanism of phospho-STAT inhibition observed in Western blot analysis.
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http://dx.doi.org/10.1016/j.jpba.2018.08.036DOI Listing
January 2019

Implications of STAT3 and STAT5 signaling on gene regulation and chromatin remodeling in hematopoietic cancer.

Leukemia 2018 08 27;32(8):1713-1726. Epub 2018 Mar 27.

Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria.

STAT3 and STAT5 proteins are oncogenic downstream mediators of the JAK-STAT pathway. Deregulated STAT3 and STAT5 signaling promotes cancer cell proliferation and survival in conjunction with other core cancer pathways. Nuclear phosphorylated STAT3 and STAT5 regulate cell-type-specific transcription profiles via binding to promoter elements and exert more complex functions involving interaction with various transcriptional coactivators or corepressors and chromatin remodeling proteins. The JAK-STAT pathway can rapidly reshape the chromatin landscape upon cytokine, hormone, or growth factor stimulation and unphosphorylated STAT proteins also appear to be functional with respect to regulating chromatin accessibility. Notably, cancer genome landscape studies have implicated mutations in various epigenetic modifiers as well as the JAK-STAT pathway as underlying causes of many cancers, particularly acute leukemia and lymphomas. However, it is incompletely understood how mutations within these pathways can interact and synergize to promote cancer. We summarize the current knowledge of oncogenic STAT3 and STAT5 functions downstream of cytokine signaling and provide details on prerequisites for DNA binding and gene transcription. We also discuss key interactions of STAT3 and STAT5 with chromatin remodeling factors such as DNA methyltransferases, histone modifiers, cofactors, corepressors, and other transcription factors.
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http://dx.doi.org/10.1038/s41375-018-0117-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6087715PMC
August 2018

Pharmacologic inhibition of STAT5 in acute myeloid leukemia.

Leukemia 2018 05 2;32(5):1135-1146. Epub 2018 Feb 2.

Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria.

The transcription factor STAT5 is an essential downstream mediator of many tyrosine kinases (TKs), particularly in hematopoietic cancers. STAT5 is activated by FLT3-ITD, which is a constitutively active TK driving the pathogenesis of acute myeloid leukemia (AML). Since STAT5 is a critical mediator of diverse malignant properties of AML cells, direct targeting of STAT5 is of significant clinical value. Here, we describe the development and preclinical evaluation of a novel, potent STAT5 SH2 domain inhibitor, AC-4-130, which can efficiently block pathological levels of STAT5 activity in AML. AC-4-130 directly binds to STAT5 and disrupts STAT5 activation, dimerization, nuclear translocation, and STAT5-dependent gene transcription. Notably, AC-4-130 substantially impaired the proliferation and clonogenic growth of human AML cell lines and primary FLT3-ITD AML patient cells in vitro and in vivo. Furthermore, AC-4-130 synergistically increased the cytotoxicity of the JAK1/2 inhibitor Ruxolitinib and the p300/pCAF inhibitor Garcinol. Overall, the synergistic effects of AC-4-130 with TK inhibitors (TKIs) as well as emerging treatment strategies provide new therapeutic opportunities for leukemia and potentially other cancers.
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http://dx.doi.org/10.1038/s41375-017-0005-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940656PMC
May 2018

Emerging therapeutic targets in myeloproliferative neoplasms and peripheral T-cell leukemia and lymphomas.

Expert Opin Ther Targets 2018 01 24;22(1):45-57. Epub 2017 Nov 24.

a Institute of Animal Breeding and Genetics , University of Veterinary Medicine Vienna , Vienna , Austria.

Introduction: Hematopoietic neoplasms are often driven by gain-of-function mutations of the JAK-STAT pathway together with mutations in chromatin remodeling and DNA damage control pathways. The interconnection between the JAK-STAT pathway, epigenetic regulation or DNA damage control is still poorly understood in cancer cell biology. Areas covered: Here, we focus on a broader description of mutational insights into myeloproliferative neoplasms and peripheral T-cell leukemia and lymphomas, since sequencing efforts have identified similar combinations of driver mutations in these diseases covering different lineages. We summarize how these pathways might be interconnected in normal or cancer cells, which have lost differentiation capacity and drive oncogene transcription. Expert opinion: Due to similarities in driver mutations including epigenetic enzymes, JAK-STAT pathway activation and mutated checkpoint control through TP53, we hypothesize that similar therapeutic approaches could be of benefit in these diseases. We give an overview of how driver mutations in these malignancies contribute to hematopoietic cancer initiation or progression, and how these pathways can be targeted with currently available tools.
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http://dx.doi.org/10.1080/14728222.2018.1406924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743003PMC
January 2018

ProxyPhos sensors for the detection of negatively charged membranes.

Analyst 2017 Nov;142(23):4511-4521

Department of Chemistry and Department of Chemical & Physical Sciences, University of Toronto, Mississauga, 3359 Mississauga Road North, Mississauga, Ontario, Canada L5L 1C6.

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.
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http://dx.doi.org/10.1039/c7an00568gDOI Listing
November 2017

MicroRNA-337-3p controls hepatobiliary gene expression and transcriptional dynamics during hepatic cell differentiation.

Hepatology 2018 01 16;67(1):313-327. Epub 2017 Nov 16.

Université catholique de Louvain, de Duve Institute, Brussels, Belgium.

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 relative levels of hepatocyte and cholangiocyte gene expression are determined during differentiation remains poorly understood. Here, we identify microRNA (miR)-337-3p as a 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 4α by modulating its activation and repression. With the help of an experimentally validated mathematical model, we show that this biphasic control results from an incoherent feedforward loop between miR-337-3p and hepatocyte nuclear factor 4α.

Conclusion: Our results identify miR-337-3p as a regulator of liver development and highlight how tight quantitative control of hepatic cell differentiation is exerted through specific gene regulatory network motifs. (Hepatology 2018;67:313-327).
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http://dx.doi.org/10.1002/hep.29475DOI Listing
January 2018

High-throughput thermofluor-based assays for inhibitor screening of STAT SH2 domains.

J Pharm Biomed Anal 2017 Sep 1;143:159-167. Epub 2017 May 1.

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd N., Mississauga, Ontario L5L 1C6, Canada; Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada. Electronic address:

The development of STAT protein-specific inhibitors has been the focus of a number of drug discovery programs. STAT activation occurs through phosphorylation at the STAT SH2 domain, resulting in dimerization, translocation to the nucleus, and transcription of proliferative genes. Due to the functional significance of the SH2 domain in mediating multiple components of the STAT signalling cascade, many libraries of inhibitors have been designed to target the SH2 domain. This has triggered the requirement for effective high-throughput screening platforms for analyzing binding by larger chemical libraries to STAT proteins. Herein, we present strategies for the development of a high-throughput thermal denaturation-based assay for identifying STAT inhibitors as well as high-yielding recombinant expression and purification of untagged STAT1, STAT3, and STAT5 proteins. This assay reports changes in the fluorescence of a labelled peptide bound to the STAT protein as a function of increasing temperature. STAT inhibitors which displace the labelled peptide elicit a change in the melt profile, which is quantitatively determined as a change in the area under the curve. This assay offers an alternative, but complimentary, high-throughput screening strategy for identifying new inhibitors of STAT proteins as well as characterizing further, the mode of inhibition by existing libraries of compounds.
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http://dx.doi.org/10.1016/j.jpba.2017.04.052DOI Listing
September 2017

Combined targeting of STAT3 and STAT5: a novel approach to overcome drug resistance in chronic myeloid leukemia.

Haematologica 2017 09 8;102(9):1519-1529. Epub 2017 Jun 8.

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Austria.

In chronic myeloid leukemia, resistance against BCR-ABL1 tyrosine kinase inhibitors can develop because of mutations, activation of additional pro-oncogenic pathways, and stem cell resistance. Drug combinations covering a broad range of targets may overcome resistance. CDDO-Me (bardoxolone methyl) is a drug that inhibits the survival of leukemic cells by targeting different pro-survival molecules, including STAT3. We found that CDDO-Me inhibits proliferation and survival of tyrosine kinase inhibitor-resistant cell lines and primary leukemic cells, including cells harboring or T315I compound mutations. Furthermore, CDDO-Me was found to block growth and survival of CD34/CD38 leukemic stem cells (LSC). Moreover, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with BCR-ABL1 tyrosine kinase inhibitors. These drug-combinations were found to block multiple signaling cascades and molecules, including STAT3 and STAT5. Furthermore, combined targeting of STAT3 and STAT5 by shRNA and STAT5-targeting drugs also resulted in synergistic growth-inhibition, pointing to a new efficient concept of combinatorial STAT3 and STAT5 inhibition. However, CDDO-Me was also found to increase the expression of heme-oxygenase-1, a heat-shock-protein that triggers drug resistance and cell survival. We therefore combined CDDO-Me with the heme-oxygenase-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects. Moreover, SMA-ZnPP was found to sensitize cells against the combination 'CDDO-Me+ tyrosine kinase inhibitor'. Together, combined targeting of STAT3, STAT5, and heme-oxygenase-1 overcomes resistance in cells, including stem cells and highly resistant sub-clones expressing BCR-ABL1 or T315I-compound mutations. Whether such drug-combinations are effective in tyrosine kinase inhibitor-resistant patients with chronic myeloid leukemia remains to be elucidated.
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http://dx.doi.org/10.3324/haematol.2016.163436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685220PMC
September 2017

Regulating the Master Regulator: Controlling Ubiquitination by Thinking Outside the Active Site.

J Med Chem 2018 01 28;61(2):405-421. Epub 2017 Feb 28.

Department of Chemistry, University of Toronto , Toronto, ON M5S 3H6, Canada.

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.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01346DOI Listing
January 2018

Strategies for over-expression and purification of recombinant full length STAT5B in Escherichia coli.

Protein Expr Purif 2017 01 31;129:1-8. Epub 2016 Aug 31.

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd N., Mississauga, Ontario L5L 1C6, Canada; Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada. Electronic address:

STAT5B, a ubiquitious transcription factor, has been implicated in the onset and progression of several cancers. Since the inhibition of STAT activity holds significant therapeutic potential, there is a need to develop high-throughput biophysical screening platforms to rapidly identify high affinity binders of STATs. Biophysical assays would benefit from the efficient and cost-effective production of high purity, full-length STAT proteins. Herein, we have sampled a large region of protein expression and purification space that has substantially increased recombinant STAT5B protein yields from Escherichia coli. The identity of STAT5B was confirmed by Western blotting analysis, while the results of a fluorescence polarization assay indicated that the purified protein is correctly folded and functional. A thermal shift assay was employed to assess the effect of various osmolytes on the stability of the protein. The protein expression conditions identified in this study allowed for more efficient and higher recovery of soluble STAT5B protein, which will enable a broad range of biophysical studies and facilitate high-throughput STAT5B drug screening.
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http://dx.doi.org/10.1016/j.pep.2016.08.017DOI Listing
January 2017

A selective inhibitor of the UFM1-activating enzyme, UBA5.

Bioorg Med Chem Lett 2016 09 8;26(18):4542-4547. Epub 2015 Oct 8.

Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada; Department of Chemical and Physical Sciences, University of Toronto at Mississauga, Mississauga, ON L5L 1C6, Canada. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmcl.2015.10.015DOI Listing
September 2016

Chronic Inhibition of STAT3/STAT5 in Treatment-Resistant Human Breast Cancer Cell Subtypes: Convergence on the ROS/SUMO Pathway and Its Effects on xCT Expression and System xc- Activity.

PLoS One 2016 11;11(8):e0161202. Epub 2016 Aug 11.

Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, L8S 4L8, Canada.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161202PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981357PMC
August 2017