Olga Ostrovsky

Genetic variations in the heparanase gene (HPSE) associate with increased risk of GVHD following allogeneic stem cell transplantation: effect of discrepancy between recipients and donors

Graft-versus-host disease (GVHD) is the most common cause of nonrelapse mortality and morbidity after hematopoietic stem cell transplantation (HSCT). The well-documented involvement of heparanase in the process of inflammation and autoimmunity led us to investigate an association between HPSE gene single-nucleotide polymorphisms (SNPs) and the risk of GVHD. The present study indicates a highly significant correlation of HPSE gene SNPs rs4693608 and rs4364254 and their combination with the risk of developing acute GVHD. Moreover, the study revealed that discrepancy between recipient and donor in these SNPs may elevate significantly the risk of acute GVHD. This association was statistically significant when the recipients possessed genotype combinations dictating higher levels of heparanase compared with their human leukocyte antigen (HLA)-matched donors. In addition, HPSE gene SNPs disclosed a correlation with extensive chronic GVHD, nonrelapse mortality, and overall survival. Our study indicates involvement of heparanase in the development of acute and extensive chronic GVHD. Moreover, it suggests a possible mechanism for the aggressive behavior of T lymphocytes leading to GVHD when the recipients possess genotype combinations that dictate high levels of heparanase mRNA compared with their HLA-matched donors expressing low levels of heparanase.

Association of heparanase gene (HPSE) single nucleotide polymorphisms with hematological malignancies

Heparanase, endo-β-D-glucuronidase, degrades heparan sulfate glycosaminoglycans – the principal polysaccharide of the basement membrane and extracellular matrix. Heparanase activity plays a decisive role in biological processes associated with remodeling of the extracellular matrix, such as cancer metastasis, angiogenesis and inflammation. In the hematopoietic system, heparanase is thought to be associated with normal differentiation and function of myeloid cells and platelets. We investigated heparanase polymorphisms in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), Hodgkins disease (HD) and multiple myeloma (MM). Significant correlation was found between rs11099592 and rs6535455 heparanase gene (HPSE) single nucleotide polymorphisms (SNPs) and ALL (χ21d.f.=4.96, P=0.026). Genotype frequency comparisons revealed a significant association with rs4693602 (χ22d.f.=7.276, P=0.026) in MM patients and rs4364254 (χ22d.f.=6.226, P=0.044) in AML patients. Examination of HPSE gene mRNA expression by real-time RT-PCR indicated a significant low HPSE gene expression level in ALL patients and a high expression level in MM and AML patients, compared to healthy controls. Moreover, statistically significant correlation was found between heparanase mRNA expression level and three HPSE gene SNPs (rs4693608, rs11099592 and rs4364254) among healthy individuals. These data suggest that certain HPSE gene SNPs may contribute to basal heparanase gene expression and that alterations in this gene are an important determinant in the pathogenesis of ALL, AML and MM.

Characterization of HPSE Gene Single Nucleotide Polymorphisms in Jewish Populations of Israel

Heparanase is a mammalian endoglucuronidase responsible for heparan sulfate (HS) degradation. HS is a major constituent of the extracellular matrix (ECM) and HS-degrading activity plays a decisive role in fundamental biological processes associated with remodeling of the ECM, such as cancer metastasis, angiogenesis and inflammation. There is great interest in the prospect of genome-wide association studies to identify genetic factors underlying complex diseases. It is important to establish a detailed description of the heparanase (HPSE) gene single nucleotide polymorphisms (SNPs). In this study, four Israeli Jewish populations (Ashkenazi, North African, Mediterranean and Near Eastern) were examined for 7 HPSE gene SNPs. Four out of 7 SNPs (rs4693608, db11099592, rs4364254, db6856901) were found to be polymorphic. Population comparisons revealed significant differences in SNPs allele frequency between Near Eastern and each of the other three populations. Genotype and allele frequencies in Jewish populations were different from non-Jewish populations, except for a certain similarity to Caucasians. Although the distance between SNPs is relatively small, the db11099592 SNP was in linkage disequilibrium (LD) only with the proximal SNP rs4693608. LD between distal SNPs rs4364254 and db6856901 was found only in Mediterraneans and North Africans. The current study provides a characterization of the normally occurring HPSE gene SNPs in different populations. This information is obligatory for further studies on the linkage between these SNPs and heparanase expression and function in various pathological processes, primarily cancer progression.

Genotype and allele frequencies of C3435T polymorphism of the MDR1 gene in various Jewish populations of Israel.

The human multidrug-resistant gene (MDR1) encodes for P-glycoprotein (P-gp), which is a membrane-bound effluxtransporter conferring resistance to a number of natural cytotoxic drugs and potentially toxic xenobiotics. The wobble C3435T polymorphism at exon 26 was associated with different expression levels of the MDR1 gene and substrate uptake. Differences in allele frequencies of the C3435T polymorphism have previously been demonstrated between racial groups. In this study, 500 individuals from 5 Jewish populations of Israel (Ashkenazi, Yemenite, North African, Mediterranean, Near-Eastern) were examined for C3435T polymorphism using a PCR-RFLP-based technique to calculate genotype and allele frequencies. Frequencies of the C allele were quite similar among the Ashkenazi (0.65), Yemenite (0.645), and North-African (0.615) Jewish populations. However, the frequency of this allele was slightly lower among Mediterranean Jews (0.58) and significantly lower among Near-Eastern Jews (0.445). The frequency of the C allele among Near-Eastern Jews is, therefore, significantly different from those of all other tested Jewish populations. In comparison to previously studied non-Jewish populations, the frequency of this allele among Near-Eastern Jews is different from that in West Africans (0.91) but is similar to that in whites (0.497). However, the C allele frequencies among the other 4 Jewish populations are significantly lower than that found among West Africans and significantly higher than among non-Jewish whites. These data may have important therapeutic and prognostic implication for P-gp-SYrelated drug dosage recommendation in Jewish populations.

Inverse correlation between HPSE gene single nucleotide polymorphisms and heparanase expression: possibility of multiple levels of heparanase regulation

Heparanase is an endo‐β‐glucuronidase that specifically cleaves the saccharide chains of heparan sulfate proteoglycans. Heparanase plays important roles in processes such as angiogenesis, tumor metastasis, tissue repair and remodeling, inflammation and autoimmunity. Genetic variations of the heparanase gene (HPSE) have been associated with heparanase transcription level. The present study was undertaken to identify haplotype or single nucleotide polymorphisms (SNPs) genotype combinations that correlate with heparanase expression both at the mRNA and protein levels. For this purpose, 11 HPSE gene SNPs were genotyped among 108 healthy individuals. Five out of the eleven polymorphisms revealed an association between the SNPs and heparanase expression. SNP rs4693608 exhibited a strong evidence of association. Analysis of haplotypes distribution revealed that the combination of two SNPs (rs4693608 and rs4364254) disclosed the most significant result. This approach allowed segregation of possible genotype combinations to three groups that correlate with low (LR: GG‐CC, GG‐CT, GG‐TT, GA‐CC), intermediate (MR: GA‐CT, GA‐TT) and high (HR: AA‐TT, AA‐CT) heparanase expression. Unexpectedly, LR genotype combinations were associated with low mRNA expressions level and high heparanase concentration in plasma, while HR genotype combinations were associated with high expression of mRNA and low plasma protein level. Because the main site of activity of secreted active heparanase is the extracellular matrix and cell surface, the origin and functional significance of plasma heparanase remain to be investigated. The current study indicates that rs4693608 and rs4364254 SNPs are involved in the regulation of heparanase expression and provides the basis for further studies on the association between HPSE gene SNPs and disease outcome.

Modification of heparanase gene expression in response to conditioning and LPS treatment: strong correlation to rs4693608 SNP

Heparanase is an endo‐β‐glucuronidase that specifically cleaves the saccharide chains of HSPGs, important structural and functional components of the ECM. Cleavage of HS leads to loss of the structural integrity of the ECM and release of HS‐bound cytokines, chemokines, and bioactive angiogenic‐ and growth‐promoting factors. Our previous study revealed a highly significant correlation of HPSE gene SNPs rs4693608 and rs4364254 and their combination with the risk of developing GVHD. We now demonstrate that HPSE is up‐regulated in response to pretransplantation conditioning, followed by a gradual decrease thereafter. Expression of heparanase correlated with the rs4693608 HPSE SNP before and after conditioning. Moreover, a positive correlation was found between recipient and donor rs4693608 SNP discrepancy and the time of neutrophil and platelet recovery. Similarly, the discrepancy in rs4693608 HPSE SNP between recipients and donors was found to be a more significant factor for the risk of aGVHD than patient genotype. The rs4693608 SNP also affected HPSE gene expression in LPS‐treated MNCs from PB and CB. Possessors of the AA genotype exhibited up‐regulation of heparanase with a high ratio in the LPS‐treated MNCs, whereas individuals with genotype GG showed down‐regulation or no effect on HPSE gene expression. HPSE up‐regulation was mediated by TLR4. The study emphasizes the importance of rs4693608 SNP for HPSE gene expression in activated MNCs, indicating a role in allogeneic stem cell transplantation, including postconditioning, engraftment, and GVHD.

Characterization of cyclin E expression in multiple myeloma and its functional role in seliciclib-induced apoptotic cell death.

Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator – cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator - cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion–mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy.

Identification of strong intron enhancer in the heparanase gene: effect of functional rs4693608 variant on HPSE enhancer activity in hematological and solid malignancies

Heparanase is an endo-β-glucuronidase that specifically cleaves the saccharide chains of heparan sulfate (HS) proteoglycans and releases HS-bound cytokines, chemokines, and bioactive growth-promoting factors. Heparanase plays an important role in the nucleus as part of an active chromatin complex. Our previous studies revealed that rs4693608 correlates with heparanase levels and increased risk of acute and extensive chronic graft vs. host disease (GVHD). Discrepancy between recipient and donor in this SNP significantly affected the risk of acute GVHD. In the present study, we analyzed the HPSE gene region, including rs4693608, and demonstrated that this region exhibits SNPs-dependent enhancer activity. Analysis of nuclear proteins from normal leukocytes revealed their binding to DNA probe of both alleles with higher affinity to allele G. All malignant cell lines and leukemia samples disclosed a shift of the main bands in comparison to normal leukocytes. At least five additional shifted bands were bound to allele A while allele G probe was bound to only one main DNA/protein complex. Additional SNPs rs4693083, rs4693084, and rs4693609 were found in strong linkage disequilibrium (LD) with rs11099592 (exon 7). Only rs4693084 affected protein binding to DNA in cell lines and leukemia samples. As a result of the short distance between rs4693608 and rs4693084, both SNPs may be included in a common DNA/protein complex. DNA pull-down assay revealed that heparanase is involved in self-regulation by negative feedback in rs4693608-dependent manner. During carcinogenesis, heparanase self-regulation is discontinued and the helicase-like transcription factor begins to regulate this enhancer region. Altogether, our study elucidates conceivable mechanism(s) by which rs4693608 SNP regulates HPSE gene expression and the associated disease outcome.

Reply to ‘Lack of association of the heparanase gene single-nucleotide polymorphism Arg307Lys with acute lymphoblastic leukemia in patients from Northern Ireland’ by Winter et al.

Reply to ‘Lack of association of the heparanase gene single-nucleotide polymorphism Arg307Lys with acute lymphoblastic leukemia in patients from Northern Ireland’ by Winter et al.