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1.  Quantitative characterization of T-cell repertoire and biomarkers in kidney transplant rejection 
BMC Nephrology  2016;17:181.
Background
T-cell-mediated rejection (TCMR) remains a major cause of kidney allograft failure. The characterization of T-cell repertoire in different immunological disorders has emerged recently as a novel tool with significant implications. We herein sought to characterize T-cell repertoire using next generation sequencing to diagnose TCMR.
Methods
In this prospective study, we analyzed samples from 50 kidney transplant recipients. We collected blood and kidney transplant biopsy samples at sequential time points before and post transplant. We used next generation sequencing to characterize T-cell receptor (TCR) repertoire by using illumina miSeq on cDNA synthesized from RNA extracted from six patients’ samples. We also measured RNA expression levels of FOXP3, CD8, CD4, granzyme and perforin in blood samples from all 50 patients.
Results
Seven patients developed TCMR during the first three months of the study. Out of six patients who had complete sets of blood and biopsy samples two had TCMR. We found an expansion of the TCR repertoire in blood at time of rejection when compared to that at pre-transplant or one-month post transplant. Patients with TCMR (n = 7) had significantly higher RNA expression levels of FOXP3, Perforin, Granzyme, CD4 and CD8 in blood samples than those with no TCMR (n = 43) (P = 0.02, P = 0.003, P = 0.002, P = 0.017, and P = 0.01, respectively).
Conclusions
Our study provides a potential utilization of TCR clone kinetics analysis in the diagnosis of TCMR. This approach may allow for the identification of the expanded T-cell clones associated with the rejection and lead to potential noninvasive diagnosis and targeted therapies of TCMR.
doi:10.1186/s12882-016-0395-3
PMCID: PMC5117555  PMID: 27871261
T-cell; Kidney transplant; T cell mediated rejection; T cell sequencing
2.  Expression and polymorphism (rs4880) of mitochondrial superoxide dismutase (SOD2) and asparaginase induced hepatotoxicity in adult patients with acute lymphoblastic leukemia 
The pharmacogenomics journal  2016;10.1038/tpj.2016.7.
Asparaginase, which depletes asparagine and glutamine, activates amino acid stress response. Oxidative stress mediated by excessive reactive oxygen species (ROS) causes enhanced mitochondrial permeabilization and subsequent cell apoptosis and is considered a plausible mechanism for drug-induced hepatotoxicity, a common toxicity of asparaginase in adults with acute lymphoblastic leukemia (ALL). Studies investigating the pharmacogenetics of asparaginase in ALL are limited and focused on asparaginase-induced allergic reaction common in pediatric patients. Here, we sought to determine a potential association between the variant rs4880 in SOD2 gene, a key mitochondrial enzyme that protects cells against ROS, and hepatotoxicity during asparaginase-based therapy in 224 patients enrolled on CALGB-10102, a treatment trial for adults with ALL. We report that the CC genotype of rs4880 is associated with increased hepatotoxicity following asparaginase-based treatment. Thus, rs4880 likely contributes to asparaginase-induced hepatotoxicity, and functional studies investigating this SNP are needed to develop therapeutic approaches that mitigate this toxicity.
doi:10.1038/tpj.2016.7
PMCID: PMC5089920  PMID: 27019981
Acute Lymphoblastic Leukemia; Asparaginase; SOD2; rs4880; polymorphism; hepatotoxicity
3.  SUV420H1 enhances the phosphorylation and transcription of ERK1 in cancer cells 
Oncotarget  2015;6(41):43162-43171.
The oncogenic protein ERK, a member of the extracellular signal-regulated kinase (ERK) cascade, is a well characterized signaling molecule involved in tumorigenesis. The ERK signaling pathway is activated in a large proportion of cancers and plays a critical role in tumor development. Functional regulation by phosphorylation of kinases in the ERK pathway has been extensively studied, however methylation of the ERK protein has not been reported to date. Here, we demonstrated that the protein lysine methyltransferase SUV420H1 tri-methylated ERK1 at lysines 302 and 361, and that substitution of methylation sites diminished phosphorylation levels of ERK1. Concordantly, knockdown of SUV420H1 reduced phosphorylated ERK1 and total ERK1 proteins, and interestingly suppressed ERK1 at the transcriptional level. Our results indicate that overexpression of SUV420H1 may result in activation of the ERK signaling pathway through enhancement of ERK phosphorylation and transcription, thereby providing new insights in the regulation of the ERK cascade in human cancer.
PMCID: PMC4791223  PMID: 26586479
ERK1; SUV420H1; protein lysine methyltransferase; non-histone protein methylation
4.  Targeting Suppressor of Variegation 3-9 Homologue 2 (SUV39H2) in Acute Lymphoblastic Leukemia (ALL)1 
Translational Oncology  2015;8(5):368-375.
Although recent progress in understanding the biology and optimizing the treatment of acute lymphoblastic leukemia (ALL) has improved cure rates of childhood ALL to nearly 90%, the cure rate in adult ALL remains less than 50%. The poor prognosis in adult ALL has in part been attributed to larger proportion of high-risk leukemia showing drug resistance. Thus, identifying novel therapeutic targets in ALL is needed for further improvements in treatment outcomes of adult ALL. Genetic aberration of chromatin-modifying molecules has been recently reported in subtypes of ALL, and targeting components of chromatin complexes has shown promising efficacy in preclinical studies. Suppressor of variegation 3-9 homologue 2 (SUV39H2), also known as KMT1B, is a SET-domain–containing histone methyltransferase that is upregulated in solid cancers, but its expression is hardly detectable in normal tissues. Here, we show that SUV39H2 is highly expressed in ALL cells but not in blood cells from healthy donors and also that SUV39H2 mRNA is expressed at significantly higher levels in bone marrow or blood cells from patients with ALL obtained at diagnosis compared with those obtained at remission (P = .007). In four ALL cell lines (Jurkat and CEM derived from T-ALL and RS4;11 and REH derived from B-ALL), SUV39H2 knockdown resulted in a significant decrease in cell viability (~ 77%, P < .001), likely through induction of apoptosis. On the other hand, SUV39H2 overexpression made cells more resistant to chemotherapy. We conclude that SUV39H2 is a promising therapeutic target and further investigation of this therapeutic approach in ALL is warranted.
doi:10.1016/j.tranon.2015.07.003
PMCID: PMC4631083  PMID: 26500027
5.  T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3-ITD mutated acute myeloid leukemia 
Oncotarget  2015;6(32):33410-33425.
Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.
PMCID: PMC4741775  PMID: 26450903
AML; FLT3-ITD; TOPK; CEBPA; kinase inhibitor
6.  Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia 
Oncotarget  2014;5(23):12371-12382.
Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients.
PMCID: PMC4323011  PMID: 25365263
MELK; AML; OTS167
7.  Determination of cellular uptake and intracellular levels of Cenersen (Aezea®, EL625), a p53 Antisense Oligonucleotide in Acute Myeloid Leukemia Cells 
TP53 encodes for tumor protein p53. The suppression of p53 protein results in interruption of DNA repair mechanisms in dividing malignant cells thereby increasing the DNA damage and activating p53-independent mechanisms of apoptosis. This ultimately may translate into enhanced cytotoxic effects of standard chemotherapy. Based on this rationale, Cenersen a phosphorothioate oligonucleotide antisense to p53-mRNA was synthesized and tested in clinical trials for patients with acute myeloid leukemia (AML). An important component of Cenersen clinical development is to develop a sensitive and specific method to quantify plasma and intracellular levels of Cenersen in different biologic matrices in order to determine tissue and intracellular distribution of the parent compound and its metabolites. Ultimately, this will allow us to determine pharmacokinetic and pharmacodynamic relationship for dose-effect correlation and design effective regimen to be rapidly translate into the clinic. An ELISA-based assay was adapted for assay development and validation of Cenersen in mouse plasma and cell lysate. Cellular uptake of Cenersen was studied in MV4-11 and KASUMI-1 AML cell lines. Real-time RT-PCR was used to measure P53-mRNA expression changes in treated cells. The assay had a limit of quantification of 35pmol/L in mouse plasma. Within-day and between-day precision of <15% and accuracy nearly 100% were observed in a linear range of 10-2000pmol/L (R2=0.99) in AML cell lysate. The selectivity of this assay examined as cross-reactivity with its 3`N-1, 3`N-2-metabolites, was 16.8% and 0.4%, respectively, and with its mismatch and the scramble oligonucleotides was 0.06% and 0.4% respectively. Cenersen was stable in mouse plasma up to 8 hrs at 37C°. When exposed to 0.1-1μmol/L Cenersen, MV4-11 and KASUMI-1 cells showed intracellular concentration in the range of 9.97-45.34nmol//mg protein and 0.1-2.1nmol/mg protein, respectively. Successful downregulation of p53-mRNA expression was observed in Cenersen treated cells. This ELISA-based assay was applicable to plasma and intracellular concentration measurement of Cenersen. Assessment of achievable concentration of Cenersen in different biologic matrices will be useful to elucidate the biological and clinical activity of this promising drug and define its recommended dose in future clinical trials.
doi:10.1016/j.jpba.2012.08.011
PMCID: PMC4201859  PMID: 22944355
Cenersen; p53 Antisense; Acute Myeloid Leukemia (AML); enzyme-linked immunosorbent assay (ELISA); Intracellular uptake
8.  SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome 
The Journal of Clinical Investigation  2014;124(4):1512-1524.
Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.
doi:10.1172/JCI70921
PMCID: PMC3973087  PMID: 24590286
9.  Silvestrol exhibits significant in vivo and in vitro antileukemic activities and inhibits FLT3 and miR-155 expressions in acute myeloid leukemia 
Background
Activating mutations [internal tandem duplication (ITD)] or overexpression of the FMS-like tyrosine kinase receptor-3 (FLT3) gene are associated with poor outcome in acute myeloid leukemia (AML) patients, underscoring the need for novel therapeutic approaches. The natural product silvestrol has potent antitumor activity in several malignancies, but its therapeutic impact on distinct molecular high-risk AML subsets remains to be fully investigated. We examined here the preclinical activity of silvestrol in FLT3-ITD and FLT3 wild-type (wt) AML.
Methods
Silvestrol in vitro anti-leukemic activity was examined by colorimetric cell viability assay, colony-forming and flow cytometry assays assessing growth inhibition and apoptosis, respectively. Pharmacological activity of silvestrol on FLT3 mRNA translation, mRNA and protein expression was determined by RNA-immunoprecipitation, qRT-PCR and immunoblot analyses, respectively. Silvestrol in vivo efficacy was investigated using MV4-11 leukemia-engrafted mice.
Results
Silvestrol shows antileukemia activity at nanomolar concentrations both in FLT3-wt overexpressing (THP-1) and FLT3-ITD (MV4-11) expressing AML cell lines (IC50 = 3.8 and 2.7 nM, respectively) and patients’ primary blasts [IC50 = ~12 nM (FLT3-wt) and ~5 nM (FLT3-ITD)]. Silvestrol increased apoptosis (~4fold, P = 0.0001), and inhibited colony-formation (100%, P < 0.0001) in primary blasts. Silvestrol efficiently inhibited FLT3 translation reducing FLT3 protein expression by 80–90% and decreased miR-155 levels (~60%), a frequently co-regulated onco-miR in FLT3-ITD-positive AML. The median survival of silvestrol-treated vs vehicle-treated mice was 63 vs 29 days post-engraftment, respectively (P < 0.0001).
Conclusions
Silvestrol exhibits significant in vivo and in vitro antileukemic activities in AML through a novel mechanism resulting in inhibition of FLT3 and miR-155 expression. These encouraging results warrant a rapid translation of silvestrol for clinical testing in AML.
doi:10.1186/1756-8722-6-21
PMCID: PMC3623627  PMID: 23497456
10.  Nicotinic α5 receptor subunit mRNA expression is associated with distant 5′ upstream polymorphisms 
CHRNA5, encoding the nicotinic α5 subunit, is implicated in multiple disorders, including nicotine addiction and lung cancer. Previous studies demonstrate significant associations between promoter polymorphisms and CHRNA5 mRNA expression, but the responsible sequence variants remain uncertain. To search for cis-regulatory variants, we measured allele-specific mRNA expression of CHRNA5 in human prefrontal cortex autopsy tissues and scanned the CHRNA5 locus for regulatory variants. A cluster of six frequent single nucleotide polymorphisms (rs1979905, rs1979906, rs1979907, rs880395, rs905740, and rs7164030), in complete linkage disequilibrium, fully account for a >2.5-fold allelic expression difference and a fourfold increase in overall CHRNA5 mRNA expression. This proposed enhancer region resides more than 13 kilobases upstream of the CHRNA5 transcription start site. The same upstream variants failed to affect CHRNA5 mRNA expression in peripheral blood lymphocytes, indicating tissue-specific gene regulation. Other promoter polymorphisms were also correlated with overall CHRNA5 mRNA expression in the brain, but were inconsistent with allelic mRNA expression ratios, a robust and proximate measure of cis-regulatory variants. The enhancer region and the nonsynonymous polymorphism rs16969968 generate three main haplotypes that alter the risk of developing nicotine dependence. Ethnic differences in linkage disequilibrium across the CHRNA5 locus require consideration of the upstream enhancer variants when testing clinical associations.
doi:10.1038/ejhg.2010.120
PMCID: PMC2995013  PMID: 20700147
Nicotinic receptor; alpha5 subunit; gene expression; nicotine dependence; lung cancer; enhancer
11.  Nicotinic α5 receptor subunit mRNA expression is associated with distant 5′ upstream polymorphisms 
CHRNA5, encoding the nicotinic α5 subunit, is implicated in multiple disorders, including nicotine addiction and lung cancer. Previous studies demonstrate significant associations between promoter polymorphisms and CHRNA5 mRNA expression, but the responsible sequence variants remain uncertain. To search for cis-regulatory variants, we measured allele-specific mRNA expression of CHRNA5 in human prefrontal cortex autopsy tissues and scanned the CHRNA5 locus for regulatory variants. A cluster of six frequent single-nucleotide polymorphisms (rs1979905, rs1979906, rs1979907, rs880395, rs905740, and rs7164030), in complete linkage disequilibrium (LD), fully account for a >2.5-fold allelic expression difference and a fourfold increase in overall CHRNA5 mRNA expression. This proposed enhancer region resides more than 13 kilobases upstream of the CHRNA5 transcription start site. The same upstream variants failed to affect CHRNA5 mRNA expression in peripheral blood lymphocytes, indicating tissue-specific gene regulation. Other promoter polymorphisms were also correlated with overall CHRNA5 mRNA expression in the brain, but were inconsistent with allelic mRNA expression ratios, a robust and proximate measure of cis-regulatory variants. The enhancer region and the nonsynonymous polymorphism rs16969968 generate three main haplotypes that alter the risk of developing nicotine dependence. Ethnic differences in LD across the CHRNA5 locus require consideration of upstream enhancer variants when testing clinical associations.
doi:10.1038/ejhg.2010.120
PMCID: PMC2995013  PMID: 20700147
nicotinic receptor; α5 subunit; gene expression; nicotine dependence; lung cancer; enhancer
12.  Allelic mRNA expression of sortilin-1 (SORL1) mRNA in Alzheimer’s autopsy brain tissues 
Neuroscience letters  2008;448(1):120-124.
Polymorphisms in the gene encoding SORL1, involved in cellular trafficking of APP, have been implicated in late-onset Alzheimer’s disease, by a mechanism thought to affect mRNA expression. To search for regulatory polymorphisms, we have measured allele-specific mRNA expression of SORL1 in human autopsy tissues from the prefrontal cortex of 26 Alzheimer’s patients, and 51 controls, using two synonymous marker SNPs (rs3824968 in exon 34 (11 heterozygous AD subjects and 16 controls), and rs12364988 in exon 6 (8 heterozygous AD subjects)). Significant allelic expression imbalance (AEI), indicative of the presence of cis-acting regulatory factors, was detected in a single control subject, while allelic ratios were near unity for all other subjects. We genotyped 7 SNPs in two haplotype blocks that had previously been implicated in Alzheimer’s disease. Since each of these SNPs was heterozygous in several subjects lacking AEI, this study fails to support a regulatory role for SORL1 polymorphisms in mRNA expression.
doi:10.1016/j.neulet.2008.10.034
PMCID: PMC2612539  PMID: 18938222
Alzheimer’s disease; SORL1; Allelic expression imbalance

Results 1-12 (12)