Nageswara Rao Dunna

Association of GSTP1 gene (I105V) polymorphism with acute leukaemia

Glutathione S-transferase P1 (GSTP1) enzyme plays a key role in biotransformation and bioactivation of certain environmental pollutants such as benzo[a]pyrene-7, 8-diol-9, 10-epoxide (BPDE) and other diol epoxides of polycyclic aromatic hydrocarbons (Hengstler et al. 1998) and catalyses detoxification of base propanols that arise from DNA oxidation thus offering cellular protection against oxidative stress. GSTP1 gene belongs to the pi class gene family, located on chromosome 11q13 (Autrup 2000). It comprises of seven exons (Morrow et al. 1989; Bora et al. 1997) and codes for cytosolic GST enzyme (Fryer et al. 1986). The first polymorphism identified is an A–G polymorphism at nucleotide 313 in exon 5 of GSTP1 gene which leads to an amino acid substitution of isoleucine (IE) by valine (val) at 105 amino acid position (Ile105Val). This substitution results in three GSTP1 genotypes: they are isoleucine/isoleucine (Ile/Ile) homozygous wildtype, isoleucine/valine (Ile/Val) heterozygote and valine/valine (Val/Val) homozygous variant.GSTP1 codon 105 polymorphism might play an important role in leukaemiogenesis, as it potentially alters protein function, diminishing its detoxification ability for certain mutagens and carcinogens, which could result in increased DNA damage and mutation, and a greater risk of developing cancer. Biochemical studies indicated that GSTP1 Val105 allele has a lower thermal stability than GSTP1 Ile105 allele (Zimniak et al. 1994; Johanson et al. 1998), and Val homozygotes had a lower conjugating activity than Ile homozygotes, with heterozygotes displaying intermediate activity (Watson et al. 1998). Individuals with at least one Val allele at codon 105 of GSTP1 enzyme might have an underlying predisposition to cancer when exposed to environmentally derived

TP53 Codon 72 Polymorphism and Risk of Acute Leukemia

TP53 is the mostly commonly mutated gene in many cancers and the P53 tumor suppressor protein is involved in multiple cellular processes, including transcription, DNA repair, genomic stability, senescence, cell cycle control and apoptosis. A common single nucleotide polymorphism located within the proline rich region of TP53 gene at codon 72 in exon 4 encodes either proline or arginine. TP53 Arg 72 is more active than TP53 Pro 72 in inducing apoptosis. The aim of this study was to understand the association of the 72 codon polymorphism with acute leukemia development and prognosis. A total of 288 acute leukemia cases comprising 147 acute lymphocytic leukemia (ALL) and 141 acute myeloid leukemia (AML), as well as 245 controls were recruited for analysis of the TP53 72 polymorphism using PCR-RFLP method. Significant association of homozygous arginine genotype with AML was observed (χ2- 133.53; df-2, p < 0.001. When data were analyzed with respect to clinical variables, elevation in mean WBC, blast %, LDH levels and slight reduction in DFS in ALL cases with the arginine genotype was observed. In contrast, AML patients with Pro/Pro had elevated WBC, Blast%, LDH levels with slightly reduced DFS. Our study indicates that Arg/Arg genotype might confer increased risk to development of acute myeloid leukemia.

Deletion of GSTM1 and T1 Genes as a Risk Factor for Development of Acute Leukemia

The glutathione S-transferases (GSTs) are a family of enzymes involved in the detoxification of a wide range of chemicals, including important environmental carcinogens, as well as chemotherapeutic agents. In the present study 294 acute leukemia cases, comprising 152 of acute lymphocytic leukemia (ALL) and 142 of acute myeloid leukemia, and 251 control samples were analyzed for GSTM1 and GSTT1 polymorphisms through multiplex PCR methods. Significantly increased frequencies of GSTM1 null genotype (M0), GSTT1 null genotype (T0) and GST double null genotype (T0M0) were observed in the both ALL and AML cases as compared to controls. When data were analyzed with respect to clinical variables, increased mean levels of WBC, Blast %, LDH and significant reduction in DFS were observed in both ALL and AML cases with T0 genotype. In conclusion, absence of both GST M and GST T might confer increased risk of developing ALL or AML. The absence of GST enzyme might lead to oxidative stress and subsequent DNA damage resulting in genomic instability, a hallmark of acute leukemia. The GST enzyme deficiency might also exert impact on clinical prognosis leading to poorer DFS. Hence GST genotyping can be made mandatory in management of acute leukemia so that more aggressive therapy such as allogenic stem cell transplantation may be planned in the case of patients with a null genotype.

High Affinity Pharmacological Profiling of Dual Inhibitors Targeting RET and VEGFR2 in Inhibition of Kinase and Angiogeneis Events in Medullary Thyroid Carcinoma

Clinical evidence shows that dual inhibition of kinases as well angiogenesis provides ideal therapeutic option in the treatment of medullary thyroid carcinoma (MTC) than inhibiting either of these with the events separately. Although treatment with dual inhibitors has shown good clinical responses in patients with MTC, it has been associated with serious side effects. Some inhibitors are active agents for both angiogenesis or kinase activity. Owing to narrow therapeutic window of established inhibitors, the present study aims to identify high affinity dual inhibitors targeting RET and VEGFR2 respectively for kinase and angiogenesis activity. Established inhibitors like Vandetanib, Cabozantinib, Motesanib, PP121, RAF265 and Sunitinib served as query parent compounds for identification of structurally similar compounds by Tanimoto-based similarity searching with a threshold of 95% against the PubChem database. All the parent inhibitors and respective similar compounds were docked against RET and VEGFR2 in order to retrieve high affinity compounds with these two proteins. AGN-PC-0CUK9P PubCID: 59320403 a compound related to PPI21 showed almost equal affinity for RET and VEGFR2 and unlike other screened compounds with no apparent bias for either of the receptors. Further, AGN- PC-0CUK9P demonstrated appreciable interaction with both RET and VEGFR2 and superior kinase activity in addition to showed optimal ADMET properties and pharmacophore features. From our in silico investigation we suggest AGN-PC-0CUK9P as a superior dual inhibitor targeting RET and VEGFR2 with high efficacy which should be proposed for pharmacodynamic and pharmacokinetic studies for improved treatment of MTC.

Multiclass Comparative Virtual Screening to Identify Novel Hsp90 Inhibitors: A Therapeutic Breast Cancer Drug Target

Since the discovery of Hsp90, a decade ago, it has surfaced as a potential target in breast cancer therapy along with other cancers. In present study, we have selected seven established Hsp inhibitors viz., PU3, CCT-018159, CNF-2024, SNX-5422, NVP (AUY-922), EGCG and IPI-504 used in the treatment of cancer. Considering these seven inhibitors as a parent compound, ligand based search was carried out with 90% similarity in Pubchem database (31 million compounds). All the similar molecules belonging to respective parent compound along with similar compound were subjected to virtual screening using MolDock and PLP algorithm aided molecular docking. Compounds with highest docking rerank scores were selected and filtered through Lipinskis drug-likeness filters and toxicity parameters. New candidate (Pubchem CID: 11363378) qualified to demonstrate considerable affinity towards Hsp90. The selected compound was further pharmcophorically incited for receptor- ligand interactions like H-bond, electrostatic, hydrophobic interactions etc.

Significance of ATM Gene Polymorphisms in Chronic Myeloid Leukemia - a Case Control Study from India

BACKGROUND Development of chronic myeloid leukemia (CML) involves formation of double strand breaks (DSBs) which are initially sensed by the ataxia telangiectasia mutated (ATM) signal kinase to induce a DNA damage response (DDR). Mutations or single nucleotide polymorphisms in ATM gene are known to influence the signaling capacity resulting in susceptibility to certain genetic diseases such as cancers. MATERIALS AND METHODS In the present study, we have analyzed -5144A>T (rs228589) and C4138T (rs3092856) polymorphisms of theATM gene through polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) in 925 subjects (476 CML cases and 449 controls). RESULTS The A allele of -5144A>T polymorphism and T allele of C4138T polymorphism which were known to be influencing ATM signaling capacity are significantly associated with enhanced risk for CML independently and also in combination (evident from the haplotype and diplotype analyses). Significant elevation in the frequencies of both the risk alleles among high risk groups under European Treatment and Outcome Study (EUTOS) score suggests the possible role of these polymorphisms in predicting the prognosis of CML patients. CONCLUSIONS This study provides the first evidence of association of functional ATM gene polymorphisms with the increased risk of CML development as well as progression.

LCN2 Promoter Methylation Status as Novel Predictive Marker for Microvessel Density and Aggressive Tumor Phenotype in Breast Cancer Patients.

LCN2 (Lipocalin 2) is a 25 KD secreted acute phase protein, reported to be a novel regulator of angiogenesis in breast cancer. Up regulation of LCN2 had been observed in multiple cancers including breast cancer, pancreatic cancer and ovarian cancer. However, the role of LCN2 promoter methylation in the formation of microvessels is poorly understood. The aim of this study was to analyze the association of LCN 2 promoter methylation with microvessel formation and tumor cell proliferation in breast cancer patients. The LCN2 promoter methylation status was studied in 64 breast cancer tumors by methylation specific PCR (MSP). Evaluation of microvessel density (MVD) and Ki67 cell proliferation index was achieved by immunohistochemical staining using CD34 and MIB-1 antibodies, respectively. LCN2 promoter unmethylation status was observed in 43 (67.2%) of breast cancer patients whereas LCN2 methylation status was seen in 21 (32.8%). Further, LCN2 promoter unmethylation status was associated with aggressive tumor phenotype and elevated mean MVD in breast cancer patients.

Role of the MDM2 Promoter Polymorphism (-309T>G) in Acute Myeloid Leukemia Development

BACKGROUND The human homologue of the mouse double minute 2 (MDM2) gene is a negative regulator of Tp53. MDM2-309T>G a functional promoter polymorphism was found to be associated with overexpression thereby attenuation of Tp53 stress response and increased cancer susceptibility. We have planned to evaluate the possible role of MDM2-309T>G polymorphism with risk and response to chemotherapy in AML. MATERIALS AND METHODS A total of 223 de novo AML cases and 304 age and sex matched healthy controls were genotyped for the MDM2-309T>G polymorphism through the tetra-primer amplification refractory mutation system (ARMS)-PCR method. In order to assess the functional relationship of -309T>G SNP with MDM2 expression level, we quantified MDM2 mRNA in 30 primary AML blood samples through quantitative RT-PCR. Both the (-309T>G) genotypes and the MDM2 expression were correlated with disease free survival (DFS) rates among patients who have achieved complete remission (CR) after first induction chemotherapy. RESULTS MDM2-309T>G polymorphism was significantly associated with AML development (p<0.0001). The presence of either GG genotype or G allele at MDM2-309 confered 1.79 (95% CI: 1.12-2.86; p<0.001) and 1.46 fold (95%CI: 1.14-1.86; p=0.003) increased AML risk. Survival analysis revealed that CR+ve cases with GG genotype had significantly increased DFS rates (16months, p=0.05) compared to CR+ve TT (11 months) and TG (9 months) genotype groups. Further, MDM2 expression was also found to be significantly elevated in GG genotype patients (p=0.0039) and among CR+ve cases (p=0.0036). CONCLUSIONS The MDM2-309T>G polymorphism might be involved in AML development and also serve as a good prognostic indicator.

Abstract 4625: Influence of intrinsic apoptotic pathway gene polymorphisms on the development and progression of acute myeloid leukemia: Case-control study

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Apoptosis plays a pivotal role in maintaining cellular homeostasis mainly in rapidly proliferating tissue like hematopoietic tissue. Defective apoptosis is one of the hallmarks of cancer as it plays a crucial role in malignant transformation through disruption of homeostasis mechanism resulting in acquired chemo-resistance. Aim: The functional single nucleotide polymorphisms (SNPs) in apoptotic genes can influence the gene expression leading to altered apoptosis that may promote malignancy. Moreover, these SNPs might also influence the treatment outcome, since most of the chemotherapeutic drugs being used are aimed to induce apoptosis of malignant cells. The present study mainly aims to evaluate the role of SNPs in BCL2 (-938C>A (rs2279115); BAX (-248G>A (rs4645878), CASP9 [-1263A>G (rs4645978); -712C>T (rs4645981); -293del (rs4645982); Ex5+32G>A (rs1052576)] with development of acute myeloid leukemia (AML) and treatment outcome in terms of disease free survival (DFS) among patients who have achieved complete remission(CR) rates after first induction chemotherapy. Methods: The study includes 225 AML cases and 307 age-gender matched controls for case-control comparison. The study was approved by ethical committee of Osmania University. Before collecting 5ml of blood sample, each participant had provided written informed consent. Baseline line clinical characteristics and follow-up data were collected from the tumor registries with the help of oncologist. Genomic DNA was extracted and SNPs were genotyped using PCR, RFLP, Tetra primer techniques. The genotype data was subjected to various statistical analyses to correlate with disease occurrence and treatment outcome in terms of CR and DFS rates using SPSS Software. We have also performed non parametric analysis to know gene-gene interaction through Multi Dimensionality Reduction (MDR). Results: The single SNP analysis had revealed that BCL2 (rs2279115), BAX (rs4645878), CASP9 (rs4645978) and CASP9 (rs4645982) were significantly associated with the origin of AML. Survival analysis had shown that minor alleles of both CASP9 (rs4645978 and rs4645982) SNPs were associated with reduced DFS rates (log rank p = 0.02). Further, cox regression analysis had also confirmed these observations where CASP9 rs4645978 (HR = 1.91; 95%CI = 1.19-3.07; p = 0.008) and rs4645982 (HR = 1.88; 95%CI = 1.06-3.35; p = 0.03) had elevated risk for relapse and shorter DFS rates. The MDR analysis had revealed that CASP9 (rs4645981) is the highest predicting variable. The best interaction model was found to be CASP9 (-1263A>G (rs4645978); -712C>T (rs4645981); and BCL2 (rs2279115) with CVC = 8/10 and TAB = 0.604. Conclusion: Our results suggest that CASP9 (rs4645978 and rs4645982) SNPs might influence the AML development and DFS rates after first induction chemotherapy. Citation Format: Anuradha Cingeetham, Sugunakar Vuree, Nageswara Rao Dunna, Manjula Gorre, Raghunadharao Digumarti, Sudha Sinha, Vishnupriya Satti. Influence of intrinsic apoptotic pathway gene polymorphisms on the development and progression of acute myeloid leukemia: Case-control study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4625. doi:10.1158/1538-7445.AM2015-4625