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1.  Potent and Selective Small-Molecule Inhibitors of cIAP1/2 Proteins Reveal That the Binding of Smac Mimetics to XIAP BIR3 Is Not Required for Their Effective Induction of Cell Death in Tumor Cells 
ACS Chemical Biology  2014;9(4):994-1002.
Cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2) and X-linked inhibitor of apoptosis protein (XIAP) are key apoptosis regulators and promising new cancer therapeutic targets. This study describes a set of non-peptide, small-molecule Smac (second mitochondria-derived activator of caspases) mimetics that are selective inhibitors of cIAP1/2 over XIAP. The most potent and most selective compounds bind to cIAP1/2 with affinities in the low nanomolar range and show >1,000-fold selectivity for cIAP1 over XIAP. These selective cIAP inhibitors effectively induce degradation of the cIAP1 protein in cancer cells at low nanomolar concentrations and do not antagonize XIAP in a cell-free functional assay. They potently inhibit cell growth and effectively induce apoptosis at low nanomolar concentrations in cancer cells with a mechanism of action similar to that of other known Smac mimetics. Our study shows that binding of Smac mimetics to XIAP BIR3 is not required for effective induction of apoptosis in tumor cells by Smac mimetics. These potent and highly selective cIAP1/2 inhibitors are powerful tools in the investigation of the role of these IAP proteins in the regulation of apoptosis and other cellular processes.
doi:10.1021/cb400889a
PMCID: PMC4324444  PMID: 24521431
2.  Therapeutic Targeting of BET Bromodomain Proteins in Castration-Resistant Prostate Cancer 
Nature  2014;510(7504):278-282.
Men who develop metastatic castration-resistant prostate cancer (CRPC) invariably succumb to the disease. The development and progression to CRPC following androgen ablation therapy is predominantly driven by unregulated androgen receptor (AR) signaling1-3. Despite the success of recently approved therapies targeting AR signaling such as abiraterone4-6 and second generation anti-androgens MDV3100 (enzalutamide)7,8, durable responses are limited, presumably due to acquired resistance. Recently JQ1 and I-BET, two selective small molecule inhibitors that target the amino-terminal bromodomains of BRD4, have been shown to exhibit anti-proliferative effects in a range of malignancies9-12. Here we show that AR signaling-competent CRPC cell lines are preferentially sensitive to BET bromodomain inhibition. BRD4 physically interacts with the N-terminal domain of AR and can be disrupted by JQ111,13. Like the direct AR antagonist, MDV3100, JQ1 disrupted AR recruitment to target gene loci. In contrast to MDV3100, JQ1 functions downstream of AR, and more potently abrogated BRD4 localization to AR target loci and AR-mediated gene transcription including induction of TMPRSS2-ERG and its oncogenic activity. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft models. Taken together, these studies provide a novel epigenetic approach for the concerted blockade of oncogenic drivers in advanced prostate cancer.
doi:10.1038/nature13229
PMCID: PMC4075966  PMID: 24759320
3.  A Sequence Variant in the Phospholipase C Epsilon C2 Domain Is Associated With Esophageal Carcinoma and Esophagitis 
Molecular carcinogenesis  2013;52(0 1):E80-E86.
A single-nucleotide polymorphism (rs2274223: A5780G:His1927Arg) in the phospholipase C epsilon gene (PLCε) was recently identified as a susceptibility locus for esophageal cancer in Chinese subjects. To determine the underlying mechanisms of PLCε and this SNP in esophageal carcinogenesis, we analyzed PLCε genotypes, expression, and their correlation in esophageal cancer cell lines, non-transformed esophageal cells, 58 esophageal squamous cell carcinomas and 10,614 non-cancer subjects from China. We found that the G allele (AG or GG) was associated with increased PLCε mRNA and protein expression in esophageal cancer tissues and in esophageal cancer cell lines. G allele was also associated with higher enzyme activity, which might be associated with increased protein expression. Quantitative analysis of the C2 domain sequences revealed that A:G allelic imbalance was strongly linked to esophageal malignancy. Moreover, the analysis of 10,614 non-cancer subjects demonstrated that the G allele was strongly associated with moderate to severe esophagitis in the subjects from the high-incidence areas of China (OR 6.03, 95% CI 1.59–22.9 in high-incidence area vs. OR 0.74, 95% CI 0.33–1.64 in low-incidence area; P = 0.008). In conclusion, the PLCε gene, particularly the 5780G allele, might play a pivotal role in esophageal carcinogenesis via upregulating PLCε mRNA, protein, and enzyme activity, and augmenting inflammatory process in esophageal epithelium. Thus, 5780G allele may constitute a promising biomarker for esophageal squamous cell carcinoma risk stratification, early detection, and progression prediction.
doi:10.1002/mc.22016
PMCID: PMC3931523  PMID: 23390063
phospholipase C epsilon; esophageal cancer; esophagitis
4.  Anti-oxidant treatment enhances anti-tumor cytotoxicity of (−)-gossypol 
Cancer biology & therapy  2008;7(5):767-776.
We showed that tumor cells with wild-type p53 and high levels of Bcl-xL are cisplatin resistant but are induced to undergo apoptosis by (−)-gossypol, making this a promising agent for overcoming cisplatin resistance. However, some cells in a population with this phenotype are not killed and continue to survive. Conversely, tumor cells with low Bcl-xL expression and either wild type or mutant p53 are relatively cisplatin sensitive and do not exhibit such high levels of apoptosis. However, these do undergo progressive loss of viability after (−)-gossypol that may not be tumor specific. We sought to elucidate the basis for these observations using cDNA microarray analysis of (−)-gossypol treated cisplatin sensitive and resistant cells. Genes in the reactive oxygen species (ROS) pathway were highly up-regulated in response to (−)-gossypol. The up-regulation was of much greater magnitude in cisplatin sensitive than resistant cells. Staining with an oxidation reporter dye confirmed differential induction of ROS in tumor cells with low Bcl-xL. As (−)-gossypol is known to undergo oxidative metabolism in vivo, ROS generation may be responsible for both off-target cytotoxicity and inactivation of the drug. In agreement with this hypothesis, oxidation of (−)-gossypol by pre-treatment with hydrogen peroxide eliminated its activity. Combined treatment with the antioxidant N-acetyl-cysteine (NAC) to block ROS increased (−)-gossypol-induced cytotoxicity to tumor but not normal cells. Furthermore, NAC increased the induction of apoptosis as measured by the sub G1 population, in both cisplatin sensitive and resistant cells. We postulate that concurrent treatment with antioxidant to block ROS prevents oxidative inactivation of (−)-gossypol and limits off-target toxicity allowing more potent (−)-gossypol-induced anti tumor activity.
PMCID: PMC4157391  PMID: 18376141
Cisplatin resistance; (−)-gossypol; Bcl-xL; reactive oxygen species; head and neck squamous cell carcinoma; apoptosis
5.  Apogossypolone, a nonpeptidic small molecule inhibitor targeting Bcl-2 family proteins, effectively inhibits growth of diffuse large cell lymphoma cells in vitro and in vivo 
Cancer biology & therapy  2008;7(9):1418-1426.
Apogossypolone (ApoG2) is a semi-synthesized derivative of gossypol. The principal objective of this study was to compare stability and toxicity between ApoG2 and gossypol, and to evaluate anti-lymphoma activity of ApoG2 in vitro and in vivo. ApoG2 shows better stability when compared with a racemic gossypol and can be better tolerated by mice compared to gossypol. ApoG2 showed significant inhibition of cell proliferation of WSU-DLCL2 and primary cells obtained from lymphoma patients, whereas it displayed no toxicity on normal peripheral blood lymphocytes. For a treatment of 72 h, the IC50 of ApoG2 was determined to be 350 nM against WSU-DLCL2 cells. Treatment with ApoG2 at 600 mg/kg resulted in significant growth inhibition of WSU-DLCL2 xenografts. When combined with CHOP, ApoG2 displayed even more complete inhibition of tumor growth. ApoG2 binds to purified recombinant Bcl-2, Mcl-1 and Bcl-XL proteins with high affinity and is shown to block the formation of heterodimers between Bcl-XL and Bim. For a treatment of 72 h, ApoG2 induced a maximum of 32% of apoptotic cell death. Western blot experiments showed that treatment with ApoG2 led to cleavage of caspase-3, caspase-9 and PARP. Moreover, pretreatment of DLCL2 cells with caspase-3, -9 and broad spectrum caspase inhibitors significantly blocked growth inhibition induced by ApoG2. In conclusion, ApoG2 effectively inhibits growth of DLCL2 cells at least partly by inducing apoptosis. It is an attractive small molecule inhibitor of the Bcl-2 family proteins to be developed further for the treatment of diffuse large cell lymphoma.
PMCID: PMC4106024  PMID: 18769131
small molecule inhibitors; Bcl-2 family of protein; diffuse large cell lymphoma; apoptosis; chemotherapy; animal model; toxicity
6.  MDM2 inhibitor MI-319 in combination with cisplatin is an effective treatment for pancreatic cancer independent of p53 function 
Small molecule inhibitors (SMIs) of MDM2 are known to restore the apoptotic and cell cycle regulatory functions of p53 by disrupting the MDM2-p53 interaction. In principle, these SMIs are not effective against tumours with mut-p53, which is known to be present in approximately 50% of all cancers. In this study we are reporting, for the first time, that MI-319 in combination with cisplatin induced cell growth inhibition and apoptosis in pancreatic cancer (PC) cells irrespective of their p53 mutational status. MI-319-cisplatin combination synergistically suppressed cell growth (MTT CI<1) and colony formation (clonogenic assay) and induced apoptosis. Western blot analysis and siRNA silencing studies in mutant as well as p53 null cells highlighted a mechanism involving p73 which is also known to be under the regulation of MDM2, and unlike p53, it is rarely mutated in PC. Down regulating MDM2 using siRNA enhanced p73 reactivation and increased cell death. Further, the combination effectively reduced tumour growth in both wt-p53 and mut-p53 tumour xenograft models (50% Capan-2 animals were tumour free). Consistent with our in vitro results, remnant tumour tissue analysis showed up-regulation of p73 and the cell cycle regulator p21. In conclusion, this study highlights a new role of MDM2 inhibitors in combination with cisplatin, and thus warrants further clinical investigation in human pancreatic tumours containing both wt-p53 and mut-p53.
doi:10.1016/j.ejca.2010.01.015
PMCID: PMC4106027  PMID: 20156675
MDM2 and p53; Small molecule inhibitors; Cisplatin; Apoptosis; Pancreatic cancer
7.  A Potent Small-Molecule Inhibitor of the MDM2-p53 Interaction (MI-888) Achieved Complete and Durable Tumor Regression in Mice 
Journal of medicinal chemistry  2013;56(13):10.1021/jm4005708.
We previously reported the discovery of a class of spirooxindoles as potent and selective small-molecule inhibitors of the MDM2-p53 interaction (MDM2 inhibitors). We report herein our efforts to improve their pharmacokinetic properties and in vivo antitumor activity. Our efforts led to the identification of 9 (MI-888) as a potent MDM2 inhibitor (Ki = 0.44 nM) with a superior pharmacokinetic profile and enhanced in vivo efficacy. Compound 9 is capable of achieving rapid, complete, and durable tumor regression in two types of xenograft models of human cancer with oral administration and represents the most potent and efficacious MDM2 inhibitor reported to date.
doi:10.1021/jm4005708
PMCID: PMC3880646  PMID: 23786219
8.  BM-1197: A Novel and Specific Bcl-2/Bcl-xL Inhibitor Inducing Complete and Long-Lasting Tumor Regression In Vivo 
PLoS ONE  2014;9(6):e99404.
Bcl-2 and Bcl-xL are critical regulators of apoptosis that are overexpressed in a variety of human cancers and pharmacological inhibition of Bcl-2 and Bcl-xL represents a promising strategy for cancer treatment. Using a structure-based design approach, we have designed BM-1197 as a potent and efficacious dual inhibitor of Bcl-2 and Bcl-xL. BM-1197 binds to Bcl-2 and Bcl-xL proteins with Ki values less than 1 nM and shows >1,000-fold selectivity over Mcl-1. Mechanistic studies performed in the Mcl-1 knockout mouse embryonic fibroblast (MEF) cells revealed that BM-1197 potently disassociates the heterodimeric interactions between anti-apoptotic and pro-apoptotic Bcl-2 family proteins, concomitant with conformational changes in Bax protein, loss of mitochondrial membrane potential and subsequent cytochrome c release to the cytosol, leading to activation of the caspase cascade and apoptosis. BM-1197 exerts potent growth-inhibitory activity in 7 of 12 small cell lung cancer cell lines tested and induces mechanism-based apoptotic cell death. When intravenously administered at daily or weekly in H146 and H1963 small-cell lung cancer xenograft models, it achieves complete and long-term tumor regression. Consistent with its targeting of Bcl-xL, BM-1197 causes transit platelet reduction in mice. Collectively, our data indicate that BM-1197 is a promising dual Bcl-2/Bcl-xL inhibitor which warrants further investigation as a new anticancer drug.
doi:10.1371/journal.pone.0099404
PMCID: PMC4047118  PMID: 24901320
9.  A Potent Bivalent Smac Mimetic (SM-1200) Achieving Rapid, Complete and Durable Tumor Regression in Mice 
Journal of medicinal chemistry  2013;56(10):3969-3979.
We have designed, synthesized and evaluated a series of new compounds based upon our previously reported bivalent Smac mimetics. This led to the identification of compound 12 (SM-1200), which binds to XIAP, cIAP1 and cIAP2 with Ki values of 0.5 nM, 3.7 nM and 5.4 nM, respectively, inhibits cell growth in the MDA-MB-231 breast cancer and SK-OV-3 ovarian cancer cell lines with IC50 values of 11.0 nM and 28.2 nM, respectively. Compound 12 has a much improved pharmacokinetic profile over our previously reported bivalent Smac mimetics and is highly effective in induction of rapid and durable tumor regression in the MDA-MB-231 xenograft model. These data indicate that compound 12 is a promising Smac mimetic and warrants extensive evaluation as a potential candidate for clinical development.
doi:10.1021/jm400216d
PMCID: PMC3806058  PMID: 23651223
10.  Diastereomeric Spirooxindoles as Highly Potent and Efficacious MDM2 Inhibitors 
Small-molecule inhibitors that block the MDM2-p53 protein-protein interaction (MDM2 inhibitors) are being intensely pursued as a new therapeutic strategy for cancer treatment. We previously published a series of spirooxindole-containing compounds as a new class of MDM2 small-molecule inhibitors. We report herein a reversible ring opening-cyclization reaction for some of these spirooxindoles, which affords four diastereomers from a single compound. Our biochemical binding data showed that the stereo-chemistry in this class of compounds has a major effect on their binding affinities to MDM2; with >100-fold difference between the most potent and the least potent stereoisomers. Our study has led to the identification of a set of highly potent MDM2 inhibitors with a stereochemistry that is different from that of our previously reported compounds. The most potent compound (MI-888) binds to MDM2 with a Ki value of 0.44 nM and achieves complete and long-lasting tumor regression in an animal model of human cancer.
doi:10.1021/ja3125417
PMCID: PMC3806051  PMID: 23641733
11.  p53-mediated heterochromatin reorganization regulates its cell fate decisions 
p53 is a major sensor of cellular stresses, and its activation influences cell fate decisions. We identified SUV39H1, a histone code ‘writer’ responsible for the histone H3 Lys9 trimethylation (H3K9me3) mark for ‘closed’ chromatin conformation, as a target of p53 repression. SUV39H1 downregulation was mediated transcriptionally by p21 and post-translationally by MDM2. The H3K9me3 repression mark was found to be associated with promoters of representative p53 target genes and was decreased upon p53 activation. Overexpression of SUV39H1 maintained higher levels of the H3K9me3 mark on these promoters and was associated with decreased p53 promoter occupancy and decreased transcriptional induction in response to p53. Conversely, SUV39H1 pre-silencing decreased H3K9me3 levels on these promoters and enhanced the p53 apoptotic response. These findings uncover a new layer of p53-mediated chromatin regulation through modulation of histone methylation at p53 target promoters.
doi:10.1038/nsmb.2271
PMCID: PMC4009975  PMID: 22466965
12.  A Potent and Highly Efficacious Bcl-2/Bcl-xL Inhibitor 
Journal of medicinal chemistry  2013;56(7):3048-3067.
Our previously reported Bcl-2/Bcl-xL inhibitor, 4, effectively inhibited tumor growth but failed to achieve complete regression in vivo. We have now performed extensive modifications on its pyrrole core structure, which has culminated in the discovery of 32 (BM-1074). Compound 32 binds to Bcl-2 and Bcl-xL proteins with Ki values of < 1 nM and inhibits cancer cell growth with IC50 values of 1-2 nM in four small-cell lung cancer cell lines sensitive to potent and specific Bcl-2/Bcl-xL inhibitors. Compound 32 is capable of achieving rapid, complete and durable tumor regression in vivo at a well-tolerated dose-schedule. Compound 32 is the most potent and efficacious Bcl-2/Bcl-xL inhibitor reported to date.
doi:10.1021/jm4001105
PMCID: PMC3806060  PMID: 23448298
13.  RNF111-dependent neddylation activates DNA damage-induced ubiquitination 
Molecular cell  2013;49(5):897-907.
Summary
Ubiquitin-like proteins have been shown to be covalently conjugated to targets. However, the functions of these ubiquitin-like proteins are largely unknown. Here, we have screened most known ubiquitin-like proteins after DNA damage and found that NEDD8 is involved in the DNA damage response. Following various DNA damage stimuli, NEDD8 accumulated at DNA damage sites, and this accumulation was dependent on an E2 enzyme UBE2M and an E3 ubiquitin ligase RNF111. We further found that histone H4 was polyneddylated in response to DNA damage, and NEDD8 was conjugated to the N-terminal lysine residues of H4. Interestingly, the DNA damage-induced polyneddylation chain could be recognized by the MIU (Motif Interacting with Ubiquitin) domain of RNF168. Loss of DNA damage-induced neddylation negatively regulated DNA damage-induced foci formation of RNF168 and its downstream functional partners, such as 53BP1 and BRCA1, thus affecting the normal DNA damage repair process.
doi:10.1016/j.molcel.2013.01.006
PMCID: PMC3595365  PMID: 23394999
14.  Endocrine-Therapy-Resistant ESR1 Variants Revealed by Genomic Characterization of Breast-Cancer-Derived Xenografts 
Cell reports  2013;4(6):10.1016/j.celrep.2013.08.022.
SUMMARY
To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.
doi:10.1016/j.celrep.2013.08.022
PMCID: PMC3881975  PMID: 24055055
15.  Targeting Inhibitors of Apoptosis Proteins (IAPs) For New Breast Cancer Therapeutics 
Apoptosis resistance is a hallmark of human cancer. Research in the last two decades has identified key regulators of apoptosis, including inhibitor of apoptosis proteins (IAPs). These critical apoptosis regulators have been targeted for the development of new cancer therapeutics. In this article, we will discuss three members of IAP proteins, namely XIAP, cIAP1 and cIAP2, as cancer therapeutic targets and the progress made in developing new cancer therapeutic agents to target these IAP proteins.
doi:10.1007/s10911-012-9265-1
PMCID: PMC3534930  PMID: 23054134
16.  EFFECTS OF PRAMIPEXOLE ON THE REINFORCING EFFECTIVENESS OF STIMULI THAT WERE PREVIOUSLY PAIRED WITH COCAINE REINFORCEMENT IN RATS 
Psychopharmacology  2011;219(1):123-135.
Rationale
Dopamine D2-like agonists maintain responding when substituted for cocaine in laboratory animals. However, these effects appear to be mediated by an interaction with stimuli that were previously paired with cocaine reinforcement (CS).
Objectives
To evaluate the extent to which the pramipexole-maintained and -induced responding are influenced by cocaine-paired stimuli.
Methods
Rats were trained to nosepoke for cocaine under fixed ratio 1 (FR1) or progressive ratio (PR) schedules of reinforcement. In FR1-trained rats, pramipexole was substituted for cocaine with injections either paired with CSs, or delivered in their absence. The capacity of experimenter-administered pramipexole to induce FR1 and PR responding for CS presentation was evaluated. The effects of altering stimulus conditions, as well as pretreatments with D2- (L-741,626) and D3-preferring (PG01037) antagonists on pramipexole-induced PR responding were also evaluated.
Results
When substituted for cocaine, pramipexole maintained responding at high rates when injections were paired with CSs, but low rates when CSs were omitted. Similarly, experimenter-administered pramipexole induced dose-dependent increases in FR1 or PR responding, with high rates of responding observed when the CS was presented, and low rates of responding when CS presentation was omitted. D2 and D3 antagonists differentially affected pramipexole-induced PR responding, with L-741,626 and PG01037 producing rightward, and downward shifts in the dose-response curve for CS-maintained responding, respectively.
Conclusions
These data indicate that pramipexole is capable of enhancing the reinforcing effectiveness of conditioned stimuli, and raise the possibility that similar mechanisms are responsible for the increased occurrence of impulse control disorders in patients being treated with pramipexole.
doi:10.1007/s00213-011-2382-5
PMCID: PMC3800033  PMID: 21701814
17.  TW-37, a Small-Molecule Inhibitor of Bcl-2, Inhibits Cell Growth and Induces Apoptosis in Pancreatic Cancer: Involvement of Notch-1 Signaling Pathway 
Cancer research  2009;69(7):2757-2765.
Overexpression of Bcl-2 family proteins has been found in a variety of aggressive human carcinomas, including pancreatic cancer, suggesting that specific agents targeting Bcl-2 family proteins would be valuable for pancreatic cancer therapy. We have previously reported that TW-37, a small-molecule inhibitor of Bcl-2 family proteins, inhibited cell growth and induced apoptosis in pancreatic cancer. However, the precise role and the molecular mechanism of action of TW-37 have not been fully elucidated. In our current study, we found that TW-37 induces cell growth inhibition and S-phase cell cycle arrest, with regulation of several important cell cycle–related genes like p27, p57, E2F-1, cdc25A, CDK4, cyclin A, cyclin D1, and cyclin E. The cell growth inhibition was accompanied by increased apoptosis with concomitant attenuation of Notch-1, Jagged-1, and its downstream genes such as Hes-1 in vitro and in vivo. We also found that down-regulation of Notch-1 by small interfering RNA or γ-secretase inhibitors before TW-37 treatment resulted in enhanced cell growth inhibition and apoptosis. Our data suggest that the observed antitumor activity of TW-37 is mediated through a novel pathway involving inactivation of Notch-1 and Jagged-1.
doi:10.1158/0008-5472.CAN-08-3060
PMCID: PMC3798007  PMID: 19318573
18.  Structure-Based Discovery of BM-957 as a Potent Small-Molecule Inhibitor of Bcl-2 and Bcl-xL Capable of Achieving Complete Tumor Regression 
Journal of medicinal chemistry  2012;55(19):8502-8514.
Bcl-2 and Bcl-xL anti-apoptotic proteins are attractive cancer therapeutic targets. We have previously reported the design of 4,5-diphenyl-1H-pyrrole-3-carboxylic acids as a class of potent Bcl-2/Bcl-xL inhibitors. In the present study, we report our structure-based optimization for this class of compounds based upon the crystal structure of Bcl-xL complexed with a potent lead compound. Our efforts accumulated into the design of compound 30 (BM-957), which binds to Bcl-2 and Bcl-xL with Ki <1 nM and has low nanomolar IC50 values in cell growth inhibition in cancer cell lines. Significantly, compound 30 achieves rapid, complete and durable tumor regression in the H146 small-cell lung cancer xenograft model at a well-tolerated dose-schedule.
doi:10.1021/jm3010306
PMCID: PMC3495162  PMID: 23030453
19.  A Network of Substrates of the E3 Ubiquitin Ligases MDM2 and HUWE1 Control Apoptosis Independently of p53 
Science signaling  2013;6(274):ra32.
In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.
doi:10.1126/scisignal.2003741
PMCID: PMC3770270  PMID: 23652204
20.  TW-37, a small-molecule inhibitor of Bcl-2, inhibits cell growth and invasion in pancreatic cancer 
Bcl-2 family of proteins plays critical roles in human cancers, including pancreatic cancer, suggesting that the discovery of specific agents targeting Bcl-2 family proteins would be extremely valuable for pancreatic cancer therapy. We have previously reported the synthesis and characterization of TW-37, which seems to be a negative regulator of Bcl-2. In this investigation, we tested our hypothesis whether TW-37 could be an effective inhibitor of cell growth, invasion and angiogenesis in pancreatic cancer cells. Using multiple cellular and molecular approaches such as MTT assay, apoptosis enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, Western blotting, electrophoretic mobility shift assay for measuring DNA binding activity of NF-κB, migration, invasion and angiogenesis assays, we found that TW-37, in nanomolar concentrations, inhibited cell growth in a dose- and time-dependent manner. This was accompanied by increased apoptosis and concomitant attenuation of NF-κB, and downregulation of NF-κB downstream genes such as MMP-9 and VEGF, resulting in the inhibition of pancreatic cancer cell migration, invasion and angiogenesis in vitro and caused antitumor activity in vivo. From these results, we conclude that TW-37 is a potent inhibitor of progression of pancreatic cancer cells, which could be due to attenuation of Bcl-2 cellular signaling processes. Our findings provide evidence showing that TW-37 could act as a small-molecule Bcl-2 inhibitor on well-characterized pancreatic cancer cells in culture as well as when grown as tumor in a xenograft model. We also suggest that TW-37 could be further developed as a potential therapeutic agent for the treatment of pancreatic cancer.
doi:10.1002/ijc.23610
PMCID: PMC3766317  PMID: 18528859
Bcl-2; NF-κB; pancreatic cancer; invasion; angiogenesis
21.  Critical role of prostate apoptosis response-4 in determining the sensitivity of pancreatic cancer cells to small-molecule inhibitor-induced apoptosis 
Molecular cancer therapeutics  2008;7(9):2884-2893.
Role of prostate apoptosis response-4 (PAR-4) has been well described in prostate cancer. However, its significance in other cancers has not been fully elucidated. For the current study, we selected four pancreatic cancer cell lines (BxPC-3, Colo-357, L3.6pl, and HPAC) that showed differential endogenous expression of PAR-4. We found that nonpeptidic small-molecule inhibitors (SMI) of Bcl-2 family proteins (apogossypolone and TW-37; 250 nmol/L and 1 μmol/L, respectively) could induce PAR-4-dependent inhibition of cell growth and induction of apoptosis. Sensitivity to apoptosis was directly related to the expression levels of PAR-4 (R = 0.92 and R2 = 0.95). Conversely, small interfering RNA against PAR-4 blocked apoptosis, confirming that PAR-4 is a key player in the apoptotic process. PAR-4 nuclear localization is considered a prerequisite for cells to undergo apoptosis, and we found that the treatment of Colo-357 and L3.6pl cells with 250 nmol/L SMI leads to nuclear localization of PAR-4 as confirmed by 4′,6-diamidino-2-phenylindole staining. In combination studies with gemcitabine, pretreatment with SMI leads to sensitization of Colo-357 cells to the growth-inhibitory and apoptotic action of a therapeutic drug, gemcitabine. In an in vivo setting, the maximum tolerated dose of TW-37 in xenograft of severe combined immuno-deficient mice (40 mg/kg for three i.v. injections) led to significant tumor inhibition. Our results suggest that the observed antitumor activity of SMIs is mediated through a novel pathway involving induction of PAR-4. To our knowledge, this is the first study reporting SMI-mediated apoptosis involving PAR-4 in pancreatic cancer. [Mol Cancer Ther 2008;7(9):2884–93]
doi:10.1158/1535-7163.MCT-08-0438
PMCID: PMC3766350  PMID: 18790769
22.  High-affinity and selective dopamine D3 receptor full agonists 
We have designed, synthesized and evaluated a series of new compounds with the goal to identify potent and selective D3 ligands. The two most potent and selective new D3 ligands are compounds 38 and 52, which bind to the D3 receptors with a Ki value of <1 nM and display a selectivity of 450–494 times over the D2 receptors and >10,000 times over the D1 receptors. Both 38 and 52 are full agonists with high potency at the D3 receptor in a D3 functional assay.
doi:10.1016/j.bmcl.2012.07.003
PMCID: PMC3461280  PMID: 22871578
Dopamine 3 receptor; Full agonists; Structure-activity-relationships
23.  Anti-tumor and anti-angiogenic effects of metronomic dosing of BH3-mimetics 
Cancer research  2011;72(3):716-725.
Recent studies have shown that Bcl-2 functions as a pro-angiogenic signaling molecule in addition to its well-known effect as an inhibitor of apoptosis. The discovery of AT101, a BH3-mimetic drug that is effective and well tolerated when administered orally, suggested the possibility of using a molecularly targeted drug in a metronomic regimen. Here, we generated xenograft squamous cell carcinomas (SCC) with humanized vasculature in immunodeficient mice. Mice received taxotere in combination with either daily 10 mg/kg AT101 (metronomic regimen) or weekly 70 mg/kg AT101 (bolus regimen). The effect of single drug AT101 on angiogenesis, and combination AT101/taxotere on the survival of endothelial cells and SCC cells, were also evaluated in vitro. Metronomic AT101 increased mouse survival (p=0.02), decreased tumor mitotic index (p=0.0009), and decreased tumor microvessel density (p=0.0052), as compared to bolus delivery of AT101. Notably, the substantial potentiation of the anti-tumor effect observed in the metronomic AT101 group was achieved using the same amount of drug and without significant changes in systemic toxicities. In vitro, combination of AT101 and taxotere showed additive toxicity for endothelial cells and synergistic or additive toxicity for tumor cells (SCC). Interestingly, low-dose (sub-apoptotic) concentrations of AT101 potently inhibited the angiogenic potential of endothelial cells. Taken together, these data unveiled the benefit of metronomic delivery of a molecularly targeted drug, and suggested that patients with squamous cell carcinomas might benefit from continuous administration of low dose BH3-mimetic drugs.
doi:10.1158/0008-5472.CAN-10-2873
PMCID: PMC3748951  PMID: 22158856
Developmental therapeutics; targeted therapy; angiogenesis; Bcl-2; squamous cell carcinoma
24.  Structure-based Design of Potent Bcl-2/Bcl-xL Inhibitors with Strong in vivo Antitumor Activity 
Journal of medicinal chemistry  2012;55(13):6149-6161.
Bcl-2 and Bcl-xL are key apoptosis regulators and attractive cancer therapeutic targets. We have designed and optimized a class of small-molecule inhibitors of Bcl-2 and Bcl-xL containing a 4,5-diphenyl-1H-pyrrole-3-carboxylic acid core structure. A 1.4 Å resolution crystal structure of a lead compound, 12, complexed with Bcl-xL has provided a basis for our optimization. The most potent compounds, 14 and 15, bind to Bcl-2 and Bcl-xL with subnanomolar Ki values and are potent antagonists of Bcl-2 and Bcl-xL in functional assays. Compounds 14 and 15 inhibit cell growth with low nanomolar IC50 values in multiple small-cell lung cancer cell lines and induce robust apoptosis in cancer cells at concentrations as low as 10 nM. Compound 14 also achieves strong antitumor activity in an animal model of human cancer.
doi:10.1021/jm300608w
PMCID: PMC3417242  PMID: 22747598
25.  AT-406, an orally active antagonist of multiple inhibitor of apoptosis proteins, inhibits progression of human ovarian cancer 
Cancer Biology & Therapy  2012;13(9):804-811.
Ovarian carcinoma is the most deadly gynecological malignancy. Current chemotherapeutic drugs are only transiently effective and patients with advance disease often develop resistance despite significant initial responses. Mounting evidence suggests that anti-apoptotic proteins, including those of the inhibitor of apoptosis protein (IAP) family, play important roles in the chemoresistance. There has been a recent emergence of compounds that block the IAP functions. Here, we evaluated AT-406, a novel and orally active antagonist of multiple IAP proteins, in ovarian cancer cells as a single agent and in the combination with carboplatin for therapeutic efficacy and mechanism of action. We demonstrate that AT-406 has significant single agent activity in 60% of human ovarian cancer cell lines examined in vitro and inhibits ovarian cancer progression in vivo and that 3 out of 5 carboplatin-resistant cell lines are sensitive to AT-406, highlighting the therapeutic potential of AT-406 for patients with inherent or acquired platinum resistance. Additionally, our in vivo studies show that AT-406 enhances the carboplatin-induced ovarian cancer cell death and increases survival of the experimental mice, suggesting that AT-406 sensitizes the response of these cells to carboplatin. Mechanistically, we demonstrate that AT-406 induced apoptosis is correlated with its ability to down-regulate XIAP whereas AT-406 induces cIAP1 degradation in both AT-406 sensitive and resistance cell lines. Together, these results demonstrate, for the first time, the anti-ovarian cancer efficacy of AT-406 as a single agent and in the combination with carboplatin, suggesting that AT-406 has potential as a novel therapy for ovarian cancer patients, especially for patients exhibiting resistance to the platinum-based therapies.
doi:10.4161/cbt.20563
PMCID: PMC3679100  PMID: 22669575
Smac mimetic; carboplatin; chemosensitization; ovarian cancer; therapeutic agent

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