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2.  Imbalanced expression of Tif1γ inhibits pancreatic ductal epithelial cell growth 
Transcriptional intermediary factor 1 gamma (Tif1γ) (Ectodermin/PTC7/RFG7/TRIM33) is a transcriptional cofactor with an important role in the regulation of the TGFβ pathway. It has been suggested that it competes with Smad2/Smad3 for binding to Smad4, or alternatively that it may target Smad4 for degradation, although its role in carcinogenesis is unclear. In this study, we showed that Tif1γ interacts with Smad1/Smad4 complex in vivo, using both yeast two-hybrid and coimmunoprecipitation assays. We demonstrated that Tif1γ inhibits transcriptional activity of the Smad1/Smad4 complex through its PHD domain or bromo-domainin pancreatic cells by luciferase assay. Additionally, there is a dynamic inverse relationship between the levels of Tif1γ and Smad4 in benign and malignant pancreatic cell lines. Overexpression of Tif1γ resulted in decreased level of Smad4. Both overexpression and knockdown of Tif1γ resulted in growth inhibition in both benign and cancerous pancreatic cell lines, attributable to a G2-phase cell cycle arrest, but only knockdown of Tif1γ reduces tumor cell invasiveness in vitro. Our study demonstrated that imbalanced expression of Tif1γ results in inhibition of pancreatic ductal epithelial cell growth. In addition, knockdown of Tif1γ may inhibit tumor invasion. These data suggest that Tif1γ might serve as a potential therapeutic target for pancreatic cancer.
PMCID: PMC4065401  PMID: 24959375
Transcriptional intermediary factor 1 gamma (Tif1γ); Smad; pancreatic cancer
3.  Three New Resveratrol Derivatives from the Mangrove Endophytic Fungus Alternaria sp. 
Marine Drugs  2014;12(5):2840-2850.
Three new resveratrol derivatives, namely, resveratrodehydes A–C (1–3), were isolated from the mangrove endophytic fungus Alternaria sp. R6. The structures of these compounds were elucidated by analysis of their MS, 1D and 2D NMR spectroscopic data. All compounds showed broad-spectrum inhibitory activities against three human cancer cell lines including human breast MDA-MB-435, human liver HepG2, and human colon HCT-116 by MTT assay (IC50 < 50 μM). Among them, compounds 1 and 2 both exhibited marked cytotoxic activities against MDA-MB-435 and HCT-116 cell lines (IC50 < 10 μM). Additionally, compounds 1 and 3 showed moderate antioxidant activity by DPPH radical scavenging assay.
doi:10.3390/md12052840
PMCID: PMC4052320  PMID: 24828291
marine fungus; Alternaria sp.; mangrove; stilbenes; resveratrol; antioxidant; antitumor
4.  Discovery of a Potent and Selective DDR1 Receptor Tyrosine Kinase Inhibitor 
ACS Chemical Biology  2013;8(10):2145-2150.
The DDR1 receptor tyrosine kinase is activated by matrix collagens and has been implicated in numerous cellular functions such as proliferation, differentiation, adhesion, migration, and invasion. Here we report the discovery of a potent and selective DDR1 inhibitor, DDR1-IN-1, and present the 2.2 Å DDR1 co-crystal structure. DDR1-IN-1 binds to DDR1 in the ‘DFG-out’ conformation and inhibits DDR1 autophosphorylation in cells at submicromolar concentrations with good selectivity as assessed against a panel of 451 kinases measured using the KinomeScan technology. We identified a mutation in the hinge region of DDR1, G707A, that confers >20-fold resistance to the ability of DDR1-IN-1 to inhibit DDR1 autophosphorylation and can be used to establish what pharmacology is DDR1-dependent. A combinatorial screen of DDR1-IN-1 with a library of annotated kinase inhibitors revealed that inhibitors of PI3K and mTOR such as GSK2126458 potentiate the antiproliferative activity of DDR1-IN-1 in colorectal cancer cell lines. DDR1-IN-1 provides a useful pharmacological probe for DDR1-dependent signal transduction.
doi:10.1021/cb400430t
PMCID: PMC3800496  PMID: 23899692
6.  Development of ATP-competitive mTOR Inhibitors 
The mTOR mediated signaling transduction pathway has been observed to be deregulated in a wide variety of cancer and metabolic diseases. Despite extensive clinical development efforts, the well-known allosteric mTOR inhibitor rapamycin and structurally related rapalogs have failed to show significant single-agent anti-tumor efficacy in most types of cancer. This limited clinical success maybe due to the inability of the rapalogs to maintain a complete blockade mTOR mediated signaling. Therefore numerous efforts have been initiated to develop ATP-competitive mTOR inhibitors that would block both mTORC1 and mTORC2 complex activity. Here we describe our experimental approaches to develop Torin1 using a medium throughput cell-based screening assay and structure-guided drug design.
doi:10.1007/978-1-61779-430-8_29
PMCID: PMC3964610  PMID: 22125084
mTOR; mTORC1; mTORC2; PI3K; PIKK; Akt; Rapamycin; Torin1
7.  FOXC1 regulates the functions of human basal-like breast cancer cells by activating NF-κB signaling 
Oncogene  2012;31(45):4798-4802.
Human basal-like breast cancer (BLBC) is an enigmatic and aggressive malignancy with a poor prognosis. There is an urgent need to identify therapeutic targets for BLBC because current treatment modalities are limited and not effective. The forkhead box transcription factor FOXC1 has recently been identified as a critical functional biomarker for BLBC. However, how it orchestrates BLBC cells was not clear. Here we show that FOXC1 activates the transcription factor NF-κB in BLBC cells by increasing p65/RelA protein stability. High NF-κB activity has been associated with estrogen receptor-negative breast cancer, particularly BLBC. The effect of FOXC1 on p65/RelA protein stability is mediated by increased expression of Pin1, a peptidyl-prolyl isomerase. FOXC1 requires NF-κB for its regulation of cell proliferation, migration, and invasion. Notably, FOXC1 overexpression renders breast cancer cells more susceptible to pharmacologic inhibition of NF-κB. These results suggest that BLBC cells may rely on FOXC1-driven NF-κB signaling. Interventions of this pathway may provide modalities for the treatment of BLBC.
doi:10.1038/onc.2011.635
PMCID: PMC3961006  PMID: 22249250
basal-like breast cancer; FOXC1; NF-κB; p65/RelA; Pin1; protein stability
8.  Discovery and optimization of potent and selective benzonaphthyridinone analogs as small molecule mTOR inhibitors with improved mouse microsome stability 
Starting from small molecule mTOR inhibitor Torin1, replacement of the piperazine ring with a phenyl ring resulted in a new series of mTOR inhibitors (as exemplified by 10) that showed superior potency and selectivity for mTOR, along with significantly improved mouse liver microsome stability and a longer in vivo half-life.
doi:10.1016/j.bmcl.2011.04.129
PMCID: PMC3929239  PMID: 21621413
mTOR; PI3K; Torin1
9.  B7-H3 Associated with Tumor Progression and Epigenetic Regulatory Activity in Cutaneous Melanoma 
B7-H3, a cell surface transmembrane glycoprotein, was assessed for its functional and prognostic role in cutaneous melanoma progression. B7-H3 expression in melanoma cells was shown to be related to specific downstream signal transduction events as well as associated with functional epigenetic activity. B7-H3 expression and prognostic utility was shown by RT-qPCR and IHC analysis on individual melanoma specimens and then verified in clinically annotated melanoma stage III and stage IV metastasis tissue microarrays in a double blind study. B7-H3 mRNA expression was shown to be significantly increased with stage of melanoma(P<0.0001) and significantly associated with melanoma-specific survival(MSS) in both stage III(P<0.0001) and stage IV(P<0.012) melanoma patients. B7-H3 expression was related to migration and invasion; overexpression B7-H3 increased migration and invasion while knockdown of B7-H3 reduced cell migration and invasion. MiR-29c expression was shown to inversely regulate B7-H3 expression. Furthermore, we demonstrated that melanoma B7-H3 expression was correlated to p-STAT3 activity level in melanoma tissues and cell lines. These studies demonstrate that B7-H3 is a significant factor in melanoma progression, and events of metastasis.
doi:10.1038/jid.2013.114
PMCID: PMC3760237  PMID: 23474948
B7-H3; melanoma; metastasis; miR-29c; prognosis; STAT3
10.  Discovery of 1-(4-(4-propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one as a highly potent, selective Mammalian Target of Rapamycin (mTOR) inhibitor for the treatment of cancer 
Journal of medicinal chemistry  2010;53(19):7146-7155.
The mTOR protein is a master regulator of cell growth and proliferation, and inhibitors of its kinase activity have the potential to become new class of anti-cancer drugs. Starting from quinoline 1, which was identified in a biochemical mTOR assay, we developed a tricyclic benzonaphthyridinone inhibitor Torin1(26), which inhibited phosphorylation of mTORC1 and mTORC2 substrates in cells at concentrations of 2 nM and 10 nM, respectively. Moreover, Torin1 exhibits 1000-fold selectivity for mTOR over PI3K (EC50 = 1800 nM) and exhibits 100-fold binding selectivity relative to 450 other protein kinases. Torin1 was efficacious at a dose of 20 mg/kg in a U87MG xenograft model, and demonstrated good pharmacodynamic inhibition of downstream effectors of mTOR in tumor and peripheral tissues. These results demonstrate that Torin1 is a useful probe of mTOR-dependent phenomena and that benzonaphthridinones represent a promising scaffold for the further development of mTOR-specific inhibitors with the potential for clinical utility.
doi:10.1021/jm101144f
PMCID: PMC3893826  PMID: 20860370
11.  Decreased expression of stromal estrogen receptor α and β in prostate cancer 
Background: Recently there has been an increased interest in the role of tumor-associated stroma in prostate tumorigenesis, but little is known about the respective roles of stomal ERα and ERβ in prostate cancer (PCa). This study characterizes the expression patterns of ERα and ERβ in tumor-associated stroma in association with various clinicopathological factors of importance in PCa prognosis and treatment. Design: Immunohistochemistry was performed using antibodies against ERα and ERβ to characterize their expression patterns in PCa tissue. Stromal ER levels (ERα and ERβ) on tissue sections (n=47), were compared between tumor associated stroma and adjacent benign associated stroma. Immunohistochemistry was also performed on a PCa tissue microarray (TMA) (n=177) to correlate stromal expression with various clinicopathological parameters. The levels of ER nuclear expression were scored semi-quantitatively. Results: The expression levels of both ERα and ERβ were significantly lower in tumor-associated stroma than stroma surrounding benign prostatic glands on the same tissue section (ERα: p<0.01; ERβ: p=0.01). When correlated with clinicopathological factors, the level of ERα expression in tumor-associated stroma showed a positive correlation with Gleason score (R2=0.8638). The expression of ERα was higher in PCa with advanced tumor stage (p=0.05) and not significantly different in extraprostatic extension (p>0.05). The level of ERβ expression in tumor-associated stroma was decreased in patients older than 60 years compared to younger patients (p=0.01). Conclusion: This study demonstrates significant down-regulation of ERα and ERβ expression in the tumor-associated stroma of PCa. However, the level of ERα expression in tumor-associated stroma shows a positive correlation with cancer differentiation and tumor stage.
PMCID: PMC3902224  PMID: 24489993
Estrogen receptors; prostate cancer; stromal
12.  Dichlorodiaportinol A – A new chlorine-containing isocoumarin from an endophytic fungus Trichoderma sp. 09 from Myoporum bontioides A. Gray and its cytotoxic activity 
Pharmacognosy Magazine  2014;10(Suppl 1):S153-S158.
Background:
Myoporum bontioides A. Gray, an evergreen shrub from the Myoporaceae family, is a commonly used medicinal plant. Many studies have been conducted on the biologically active constituents of whole parts of M. bontioides. However, the endophytes of M. bontioides have not been intensively investigated. A new chlorine-containing isocoumarin, named dichlorodiaportinol A (1) was isolated from the endophytic fungus Trichoderma sp. 09 isolated from the root of M. bontioides. Its cytotoxic activity against human breast cancer (MCF-7) and human liver cancer (HepG2) cell lines was evaluated.
Materials and Methods:
Different open silica gel column chromatographic techniques with different solvent systems were used for the separation of the constituents of the ethyl acetate extract of the culture broth of the endophytic fungus Trichoderma sp. 09. The structure of compound one was identified by analysis of spectroscopic data [one-dimensional (1D), two-dimensional (2D)-nuclear magnetic resonance (NMR), ultraviolet (UV), infrared (IR) and Mass spectrometry (MS)]. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay method was used for the evaluation of cytotoxic activity of compound one against MCF-7 and HepG2 cell lines.
Results:
Compound one was identified as 3-(3,3-dichloro-2,3-dihydroxy-propyl)-8-hydroxy-6- methoxy-isochromen-1-one. It inhibited MCF-7 and HepG2 cell lines, with half maximal inhibitory concentration (IC50) values of 17.8 and 39.6 μg/mL, respectively.
Conclusions:
Compound one is a new chlorine-containing isocoumarin with moderate cytotoxic activity against MCF-7 and HepG2 cell lines. Thus, endophytes of M. bontioides are worthy of consideration for the development and research of antitumor agents.
doi:10.4103/0973-1296.127367
PMCID: PMC4047575  PMID: 24914297
Cytotoxic activity; dichlorodiaportinol A; isocoumarin; Trichoderma sp.
13.  Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases 
Biochemical Journal  2013;457(Pt 1):215-225.
The related NUAK1 and NUAK2 are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor kinase. Recent work suggests they play important roles in regulating key biological processes including Myc-driven tumorigenesis, senescence, cell adhesion and neuronal polarity. In the present paper we describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms (IC50 for NUAK1 is 20 nM and for NUAK2 is 100 nM), whereas HTH-01-015 inhibits only NUAK1 (IC50 is 100 nM). These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases that were tested including ten AMPK family members. In all cell lines tested, WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterized substrate, MYPT1 (myosin phosphate-targeting subunit 1) that is phosphorylated by NUAK1 at Ser445. We also identify a mutation (A195T) that does not affect basal NUAK1 activity, but renders it ~50-fold resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating the phosphorylation of MYPT1 we find that in cells overexpressing drug-resistant NUAK1[A195T], but not wild-type NUAK1, phosphorylation of MYPT1 at Ser445 is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to MEFs (mouse embryonic fibroblasts) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1-knockout. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knockdown. We find that WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knockdown. The results of the present study indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAK kinases.
We describe the discovery of structurally diverse kinase inhibitors to dissect the physiological roles of the NUAK isoforms. We recommend use of an inhibitor-resistant NUAK1[A195T] mutant to verify that the physiological effects of these compounds is indeed mediated through inhibition of NUAKs
doi:10.1042/BJ20131152
PMCID: PMC3969223  PMID: 24171924
AMP-activated protein kinase (AMPK); AMPK-related kinase 5 (ARK5); kinase inhibitor; kinase profiling; liver kinase B1 (LKB1); myosin phosphate-targeting subunit 1(MYPT1); sucrose-non-fermenting protein kinase/AMPKrelated protein kinase (SNARK); ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; BRSK, brain-specific kinase; DMEM, Dulbecco’s modified Eagle’s medium; HA, haemagglutinin; HEK, human embryonic kidney; LKB1, liver kinase B1; MARK, microtubule-affinity-regulating kinase; MEF, mouse embryonic fibroblast; MYPT1, myosin phosphate-targeting subunit 1; NF-κB, nuclear factor κB; PEI, polyethylenimine; PP1, protein phosphatase 1; SIK, salt-induced kinase
14.  Crosstalk between Wnt/β-Catenin and Estrogen Receptor Signaling Synergistically Promotes Osteogenic Differentiation of Mesenchymal Progenitor Cells 
PLoS ONE  2013;8(12):e82436.
Osteogenic differentiation from mesenchymal progenitor cells (MPCs) are initiated and regulated by a cascade of signaling events. Either Wnt/β-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER) signaling by estradiol (E2) or exogenously expressed ERα in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ERα-mediated synergy can be effectively blocked by ERα antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ERα significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on β-catenin/Tcf reporter activity. However, ERα expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ERβ expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ERα expression and down-regulating ERβ expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.
doi:10.1371/journal.pone.0082436
PMCID: PMC3855436  PMID: 24340027
15.  Structural determinants for ERK5 (MAPK7) and leucine rich repeat kinase 2 activities of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones 
The benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-one core was discovered as a novel ERK5 (also known as MAPK7 and BMK1) inhibitor scaffold, previously. Further structure–activity relationship studies of this scaffold led to the discovery of ERK5-IN-1 (26) as the most selective and potent ERK5 inhibitor reported to date. 26 potently inhibits ERK5 biochemically with an IC50 of 0.162 ± 0.006 μM and in cells with a cellular EC50 for inhibiting epidermal growth factor induced ERK5 autophosphorylation of 0.09 ± 0.03 μM. Furthermore, 26 displays excellent selectivity over other kinases with a KINOMEscan selectivity score (S10) of 0.007, and exhibits exceptional bioavailability (F%) of 90% in mice. 26 will serve as a valuable tool compound to investigate the ERK5 signaling pathway and as a starting point for developing an ERK5 directed therapeutic agent.
Graphical abstract
Highlights
•Structural determinants of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones for ERK5.•Highly selective ERK5 inhibitor with good efficacy both in vitro and in vivo.•Represents a good template for developing ERK5 directed therapeutic agent.
doi:10.1016/j.ejmech.2013.10.052
PMCID: PMC3914206  PMID: 24239623
ERK5 inhibitor; Kinase selectivity; Benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-one; BMK1, big MAP kinase 1; DIEA, N,N-diisopropylethylamine; DCAMKL2, doublecortin and CaM kinase-like 2; DMA, N,N-dimethylacetamide; EGF, epidermal growth factor; ERK5, extracelluar-signal-regulated kinase 5; HCC, hepatocellular carcinoma; LRRK2, leucine rich repeat kinase 2; MAPK, mitogen-activated protein kinase; ERK5, mitogen-activated protein kinase 7; MEK5, MAP kinase kinase 5; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium-(0); PLK, polo-like kinase; PML, promyelocytic leukemia protein; RSK, ribosomal S6 kinase; SAR, structure–activity relationship; X-phos, 2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-biphenyl
16.  Serum Protein Profile at Remission Can Accurately Assess Therapeutic Outcomes and Survival for Serous Ovarian Cancer 
PLoS ONE  2013;8(11):e78393.
Background
Biomarkers play critical roles in early detection, diagnosis and monitoring of therapeutic outcome and recurrence of cancer. Previous biomarker research on ovarian cancer (OC) has mostly focused on the discovery and validation of diagnostic biomarkers. The primary purpose of this study is to identify serum biomarkers for prognosis and therapeutic outcomes of ovarian cancer.
Experimental Design
Forty serum proteins were analyzed in 70 serum samples from healthy controls (HC) and 101 serum samples from serous OC patients at three different disease phases: post diagnosis (PD), remission (RM) and recurrence (RC). The utility of serum proteins as OC biomarkers was evaluated using a variety of statistical methods including survival analysis.
Results
Ten serum proteins (PDGF-AB/BB, PDGF-AA, CRP, sFas, CA125, SAA, sTNFRII, sIL-6R, IGFBP6 and MDC) have individually good area-under-the-curve (AUC) values (AUC = 0.69–0.86) and more than 10 three-marker combinations have excellent AUC values (0.91–0.93) in distinguishing active cancer samples (PD & RC) from HC. The mean serum protein levels for RM samples are usually intermediate between HC and OC patients with active cancer (PD & RC). Most importantly, five proteins (sICAM1, RANTES, sgp130, sTNFR-II and sVCAM1) measured at remission can classify, individually and in combination, serous OC patients into two subsets with significantly different overall survival (best HR = 17, p<10−3).
Conclusion
We identified five serum proteins which, when measured at remission, can accurately predict the overall survival of serous OC patients, suggesting that they may be useful for monitoring the therapeutic outcomes for ovarian cancer.
doi:10.1371/journal.pone.0078393
PMCID: PMC3823861  PMID: 24244307
17.  Blockade of miR-150 maturation by MLL-fusion/MYC/LIN-28 is required for MLL-associated leukemia 
Cancer cell  2012;22(4):524-535.
Summary
Expression of microRNAs (miRNAs) is under stringent regulation at both transcriptional and post-transcriptional levels. Disturbance at either level could cause dysregulation of miRNAs. Here we show that MLL fusion proteins negatively regulate production of miR-150, an miRNA widely repressed in acute leukemia, by blocking miR-150 precursors from being processed to mature miRNAs through MYC/LIN28 functional axis. Forced expression of miR-150 dramatically inhibited leukemic cell growth and delayed MLL-fusion-mediated leukemogenesis, likely through targeting FLT3 and MYB and thereby interfering with the HOXA9/MEIS1/FLT3/MYB signaling network, which in turn caused downregulation of MYC/LIN28. Collectively, we revealed a MLL-fusion/MYC/LIN28⊣miR-150⊣FLT3/MYB/HOXA9/MEIS1 signaling circuit underlying the pathogenesis of leukemia, where miR-150 functions as a pivotal gatekeeper and its repression is required for leukemogenesis.
doi:10.1016/j.ccr.2012.08.028
PMCID: PMC3480215  PMID: 23079661
miR-150; MLL-associated leukemia; MYC; LIN28; FLT3; MYB; HOXA9; MEIS1; microRNA maturation; signaling axis; leukemogenesis
18.  Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM and ATR 
Cancer research  2013;73(8):2574-2586.
mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here we report the characterization of Torin2, a second generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pM with approximately 800-fold selectivity for cellular mTOR versus PI3K. Torin2 also exhibited potent biochemical and cellular activity against PIKK family kinases including ATM (EC50 28 nM), ATR (EC50 35 nM) and DNA-PK (EC50 118 nM) (PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with MEK inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncological settings where mTOR signaling has a pathogenic role.
doi:10.1158/0008-5472.CAN-12-1702
PMCID: PMC3760004  PMID: 23436801
mTOR; ATM; ATR; lung cancer; kinase inhibitors
19.  Long Chain Fatty Acyl-CoA Synthetase 4 Is a Biomarker for and Mediator of Hormone Resistance in Human Breast Cancer 
PLoS ONE  2013;8(10):e77060.
The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4) in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2) in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC), characterized by the absence of androgen receptor (AR) and therefore referred to as quadruple negative breast cancer (QNBC). Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor α (ER)-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed in vivo in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype.
doi:10.1371/journal.pone.0077060
PMCID: PMC3796543  PMID: 24155918
20.  Correction: Epigenetic Regulation of BMP2 by 1,25-dihydroxyvitamin D3 through DNA Methylation and Histone Modification 
PLoS ONE  2013;8(9):10.1371/annotation/1b1eb36e-60a4-4d28-bb3a-ee5a12a3d5d8.
doi:10.1371/annotation/1b1eb36e-60a4-4d28-bb3a-ee5a12a3d5d8
PMCID: PMC3779267
21.  Endoplasmic Reticulum (ER) Stress Inducible Factor Cysteine-Rich with EGF-Like Domains 2 (Creld2) Is an Important Mediator of BMP9-Regulated Osteogenic Differentiation of Mesenchymal Stem Cells 
PLoS ONE  2013;8(9):e73086.
Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.
doi:10.1371/journal.pone.0073086
PMCID: PMC3760886  PMID: 24019898
22.  Relapse specific mutations in NT5C2 in childhood acute lymphoblastic leukemia 
Nature genetics  2013;45(3):290-294.
Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis despite intensive retreatment, due to intrinsic drug resistance1-2. The biological pathways that mediate resistance are unknown. Here we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten pediatric B lymphoblastic leukemia patients using RNA-sequencing. Transcriptome sequencing identified 20 newly acquired novel non-synonymous mutations not present at initial diagnosis, of which two patients harbored relapse specific mutations in the same gene, NT5C2, a 5′-nucleotidase. Full exon sequencing of NT5C2 was completed in 61 additional relapse specimens, identifying five additional cases. Enzymatic analysis of mutant proteins revealed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analogue therapies. Clinically, all patients who harbored NT5C2 mutations relapsed early, or within 36 months of initial diagnosis (p=0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug resistant clones in ALL.
doi:10.1038/ng.2558
PMCID: PMC3681285  PMID: 23377183
23.  An Amino-indazole Scaffold with Spectrum Selective Kinase Inhibition of FLT3, PDGFRα and Kit 
Here we describe the synthesis and characterization of a number of 3-amino-1H-indazol-6-yl-benzamides that were designed to target the “DFG-out” conformation of the kinase activation loop. Several compounds such as 4 and 11 exhibit single-digit nanomolar EC50s against FLT3, c-Kit and the gatekeeper T674M mutant of PDGFRα.
doi:10.1016/j.bmcl.2012.05.107
PMCID: PMC3547541  PMID: 22727638
24.  Synthesis and Structure-Activity Relationship Studies of HIV-1 Virion Infectivity Factor (Vif) Inhibitors that Block Viral Replication 
ChemMedChem  2012;7(7):1217-1229.
The HIV-1 Vif protein, essential for in vivo viral replication, protects the virus from innate antiviral cellular factor APOBEC3G (A3G), and is an attractive target for developing antiviral therapeutics. Here we have evaluated the structure-activity relationships of RN18, a small molecule recently identified as an inhibitor of Vif function that blocks viral replication only in non-permissive cells expressing A3G, by inhibiting Vif-A3G interactions. Microwave-assisted cross-coupling reactions were developed to prepare a series of RN18 analogues with diverse linkages and substitutions on the phenyl rings. A dual cell-based assay system was used to assess antiviral activity against wild-type HIV-1 in both non-permissive (H9) and permissive (MT-4) cells that also allowed evaluation of specificity. In general, variations of phenyl substitutions were detrimental for antiviral potency and specificity, but isosteric replacements of amide and ether linkages were relatively well tolerated. These SAR data define structural requirements for Vif-specific activity, identify new compounds with improved antiviral potency and specificity, and provide leads for further exploration to develop new antiviral therapeutics.
doi:10.1002/cmdc.201200079
PMCID: PMC3517065  PMID: 22555953
APOBEC3G; HIV-1 Vif; inhibitors; structure-activity relationships; drug discovery; antiviral agents
25.  Synthesis and characterization of a perchlorotriphenylmethyl (trityl) triester radical: a potential sensor for superoxide and oxygen in biological systems 
Synthesis and characterization of an inert perchlorotriphenylmethyl triester radical, PTM-TE, are reported. PTM-TE was prepared by a facile 3-step synthesis using Friedal-Crafts reaction of tetrachlorobenzene with chloroform followed by ethoxycarbonylation and subsequent oxidation. PTM-TE is paramagnetic, and detected as a single sharp peak by EPR spectroscopy. In solution, the EPR linewidth of PTM-TE is highly sensitive to the dissolved oxygen content, thus enabling accurate measurement of oxygen concentration (oximetry). In addition, the radical also shows high reactivity to superoxide. The ester radical has the potential for use as a high-sensitive probe for determination of oxygen concentration and superoxide in biological systems.
doi:10.1016/j.bmcl.2007.04.070
PMCID: PMC3685182  PMID: 17499990
Perchlorotriphenylmethyl radical; perchlorotriphenylmethyl triester; superoxide; free radicals; EPR; oximetry; spectrophotometry

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