Search

Background: Several studies showed that the aberrant DNA methylations are involved in leukemia and cancer pathogenesis. PTPRG is a natural inhibitory mechanism that is found to be down-regulated in Chronic Myeloid Leukemia (CML) disease. The mechanism behind its down-regulation has not been fully elucidated. Aim: The study investigates the role of methylation as a possible mechanism of PTPRG under-expression in CML patients. Method: Peripheral blood samples from CML patients treated with tyrosine kinase inhibitors (TKIs) and healthy controls were collected. DNA was extracted and treated with bisulfite treatment, followed by PCR and sequencing of 25 of CpG in Promoter and 26 of CpG in the Intronic regions of PTPRG. Bisulfite-sequencing technique as a high-resolution method was employed. Results : CML groups (New Diagnosed and Failed treatment) have significantly higher methylation levels in the Promoter and Intron regions compared to the healthy group. There were also significant differences in methylation levels of CpG sites in the Promoter and Intronic regions amongst the groups. Conclusion: Aberrant methylation of PTPRG is documented and potentially is one of the possible mechanisms of PTPRG down-regulation in CML.

Background Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients. Methods TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Results Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells. Conclusions The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically detect its target in various experimental conditions. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0494-z) contains supplementary material, which is available to authorized users.

Background: Chronic myeloid leukemia (CML) is a clonal myeloid stem cell disorder associated with increased myeloid cells spectrum in peripheral blood. The disease is due to the genetic translocation t(9;22)(q34;q11.2) encoding for the BCR-ABL1 fusion oncogene which is the hallmark of CML. The resultant chimeric protein P210 kD of BCR-ABL1 causes a constitutive activation of tyrosine kinase domain that leads to continuous activation of several cell signaling pathways. The CML is currently treated with several generations of tyrosine kinase inhibitors (TKIs). However, treatment failure, resistance and disease progression are still reported and the molecular mechanisms still remain poorly understood. On the other hand, protein tyrosine phosphatase receptor gamma (PTPRG) is the natural regulatory mechanism to tyrosine kinase family, acts as tumor suppressor gene, well documented to be downregulated in CML patients while its re-expression led to reduced tyrosine phosphorylation and induced apoptosis in CML cells. PTPRG is also an important protein in the JAK/STAT pathway, has a vital role in the downstream of BCR-ABL protein kinase. Several studies suggested that the aberrant DNA methylation is involved in TKIs resistance in CML leading to leukemic clone escape and disease propagation. In different types of tumors, several mechanisms have been suggested for the loss of function of PTPRG activities such as mutations, deletions, or loss of heterozygosity LOH and gene silencing by methylation. Aim: In this study, we aimed to evaluate and characterize the methylation status of PTPRG gene function in 15 CML Patients 1- Group A: CML patients at diagnosis and 2- Group B) CML patients failed treatments according to European Leukemia Net (ELN) 2013 criteria and 10 healthy individuals. (Labeled as Normal in the figure 1) Materials and Methods: Thirty subsequent peripheral blood samples have been collected from the 15 CML patients at diagnosis and at time of failure. Genomic DNA was isolated from 400 ml of blood on EDTA tubes using Maxwell DNA Purification Kits, the quality and quantity of DNA were measured on Nanodrop Spectrophotometer and gel electrophoresis. The methylation-specific PCR and bisulfite-sequencing were adapted, EZ DNA Methylation Kit was utilized for bisulfite-conversion. Methylation Primers were designed to target the promotor and intronic CpG islands of the PTPRG gene. Gradient PCR was performed to detect the two bands of 321bp and 218bp for CpG islands of promoter and intron of PTPRG respectively. Bisulfite-specific PCR technique was carried out to amplify a fragment of bisulfite-converted DNA that has been pre-defined by the designed bisulfite-specific primers. The Methylation PCR amplified product was sequenced via Sanger Sequencing (Applied Biosystems 3130 Genetic Analyzer). The methylation analysis was performed to study the methylation status of the CpG islands using the ESME (Epigenetic Sequencing Methylation) Analysis Software. Results: Results of the methylation in 25 CpG sites of Promoter region of PTPRG: There are significant differences in the mean of methylation status amongst CML patients at diagnosis and healthy individuals of CpG promoter of PTPRG (p value is ˂0.04). However, there are no significant differences in the mean of methylation status between patients failed treatments and other groups (Figure1) Results of the methylation in 26 CpG sites of Intronic region of PTPRG: There are also significant differences in the mean of methylation status amongst all groups (p value is ˂0.003). (Figure1) Discussion and Conclusions: This is the first prospective study to evaluate epigenetic changes of PTPRG gene from CML patients in Qatar. We previously documented that the expression level of PTPRG is inversely correlated with the expression of BCR-ABL1 in CML patients. In this study, the mechanism behind the downregulation of PTPRG in our cohorts of CML patients was explored and the aberrant methylation of PTPRG could be a mechanism associated with CML disease progression and resistance to TKIs. However, further studies are still needed including larger patient's cohort and CML patients responding to the TKIs. Finally, the methylation of PTPRG in the CML patients at diagnosis and failed treatment may allow the identification of new therapeutic targets and using methylation inhibitors could be also suggested to prevent or reverse TKIs resistance and restore TKIs sensitivity. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: Chronic Myelocytic Leukemia (CML) is a clonal myeloproliferative disorder characterized by constitutive phosphorylation of Protein Tyrosine kinases (PTKS) that continuously activates multiple proliferative and antiapoptotic signaling pathways. Protein Tyrosine Phosphatases (PTPs) on the other hand is potential natural inhibitory mechanism for regulating the tyrosine kinase activities in which phosphorylation is reciprocally controlled and maintained in equilibrium state by PTKs and PTPs. As a member of PTPs family, Protein Tyrosine Phosphatase Receptor Gamma (PTPRG) was found to act as a tumor suppressor gene. This negative regulatory mechanism of PTPRG was observed to be down-regulated and disabled in CML and one of the possible mechanisms that alter the negative regulatory effect of PTPs is mutations. Several mutations have been identified in PTPs in many different leukemias such as Acute Myeloid Leukemia (AML), Juvenile MyeloMonocytic Leukemia (JMML), Myelodysplasic Syndrome (MDS), B-cell Acute Lymphoblastic Leukemia (B-ALL) and these mutations are associated with hyper-cellular proliferation, disease progression and poor outcome. However, relatively little is known about PTPRG mutations and no studies on CML are available in the literature while mutations inBCR-ABL1tyrosine kinase have been extensively characterized. Thus, understanding the role of PTPRG in antagonizing the PTK phosphorylation of BCR-ABL1 will be important to determine its role in CML development and progression. Aim: 1) To identify potential genetic alterations causing inactivation of PTPRG and 2) correlate the PTPRG findings with patients' response to the Tyrosine kinase Inhibitors. Methods: 16 CML patients, 9 from Qatar and 7 from Italy respectively, were studied for PTPRG mutations by exome sequencing. Custom primers were designed for Human PTPRG gene (5 Kb of exonic region of interest) using Ion AmpliSeq Designer. Target regions were enriched and amplified for the 16 DNA samples using Ion AmpliSeq Library kit 2.0. The amplicons were partially digested with FuPa reagent and phosphorylated prior to ligation of Ion Xpress Barcode Adapters followed by cleanup using HighPrep reagent. The adapter ligated molecules were enriched with adapter specific primers using a limited cycle PCR followed by a cleanup using HighPrep reagent. The final libraries were quantified on Qubit Flurometer using Qubit dsDNA HS Assay Kit and Agilent Bioanalyzer using Agilent High Sensitivity DNA Kit. All samples were pooled according to the concentrations on the Bioanalyzer and loaded on Ion 318TM Chip kit V2 to be sequenced on Ion Personal Genome Machine (PGM) system. European Leukemia Net (ELN) 2013 criteria were employed to assess the response/resistance of patients to treatment. Responses are defined at the hematological, cytogenetic and molecular levels. Patients response was classified into optimal and failure Results: Four mutations/variants were identified in PTPRG genes, three were missense Y92H, G574S, S561Y and 1 was frameshift Y285fs in the 16 CML patients. PTPRG Y92H was identified in 5 (1 Homozygous and 4 heterozygous alleles) patients and the 5 patient failed the Imatinib Mesylate (IM) treatment. On the other hand, The PTPRG G574S was identified in 6 (2 homozygous and 4 heterozygous alleles) patients. Out of the 6 patients, 4 were classified as failure to the treatment and 2 responded optimally. In addition, the PTPRG S561Y and Y285fs were identified on 1 and 3 patients respectively and these patients responded optimally to IM treatment. Discussion and Conclusions: This is the first prospective pilot study to investigate PTPRG gene mutations as underlying mechanism to explain treatment failure. Our preliminary data showed that the identified variant PTPRG Y92H might be associated with IM failure although it has been reported as Single Nucleotide Polymorphisms (SNPs) (rs62620047) and this could be attributed that some polymorphisms might behave like a mutation. On the other hand, PTPRG G574S variant (rs2292245) showed various clinical outcomes regardless to its allele zygosity as 67% (4/6) of patients failed the TKIs treatment. From the results of our pilot study we recommend carrying out PTPRG sequencing in a significantly larger cohort of patients to further explore and pinpoint the crucial mutations that can be correlated with CML resistance/response to treatment. Disclosures No relevant conflicts of interest to declare.