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Supplementary Table 2 from Protein Tyrosine Phosphatase Receptor Type γ Is a Functional Tumor Suppressor Gene Specifically Downregulated in Chronic Myeloid Leukemia

Chronic myelogenous leukemia (CML) is the most common myeloproliferative disease. Protein tyrosine phosphatase receptor type γ (PTPRG) is a tumor suppressor gene and a myeloid cell marker expressed by CD34+ cells. Downregulation of PTPRG increases colony formation in the PTPRG-positive megakaryocytic cell lines MEG-01 and LAMA-84 but has no effect in the PTPRG-negative cell lines K562 and KYO-1. Its overexpression has an oncosuppressive effect in all these cell lines and is associated with myeloid differentiation and inhibition of BCR/ABL-dependent signaling. The intracellular domain of PTPRG directly interacts with BCR/ABL and CRKL, but not with signal transducers and activators of transcription 5. PTPRG is downregulated at the mRNA and protein levels in leukocytes of CML patients in both peripheral blood and bone marrow, including CD34+ cells, and is reexpressed following molecular remission of disease. Reexpression was associated with a loss of methylation of a CpG island of PTPRG promoter occurring in 55% of the patients analyzed. In K562 cell line, the DNA hypomethylating agent 5-aza-2′-deoxycytidine induced PTPRG expression and caused an inhibition of colony formation, partially reverted by downregulation of PTPRG expression. These findings establish, for the first time, PTPRG as a tumor suppressor gene involved in the pathogenesis of CML, suggesting its use as a potential diagnostic and therapeutic target. Cancer Res; 70(21); 8896–906. ©2010 AACR.

Supplementary Table 1 from Protein Tyrosine Phosphatase Receptor Type γ Is a Functional Tumor Suppressor Gene Specifically Downregulated in Chronic Myeloid Leukemia

Protein tyrosine phosphatase receptor type &gamma, (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. &beta, catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between &beta, catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene &beta, Catenin. PTPRG was found to be capable of dephosphorylating &beta, catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of &beta, catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and &beta, catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and &beta, catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells.

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.

Mycoplasma contamination represents a significant problem to the culture of mammalian cells used for research as it can cause disastrous effects on eukaryotic cells by altering cellular parameters leading to unreliable experimental results. Mycoplasma cells are very small bacteria therefore they cannot be detected by visual inspection using a visible light microscope and, thus, can remain unnoticed in the cell cultures for long periods. The detection techniques used nowadays to reveal mycoplasma contamination are time consuming and expensive with each having significant drawbacks. The ideal detection should be simple to perform with minimal preparation time, rapid, inexpensive, and sensitive. To our knowledge, for the first time, we employed Fourier transform infrared (FTIR) microspectroscopy to investigate whether we can differentiate between control cells and the same cells which have been infected with mycoplasmas during the culturing process. Chemometric methods such as HCA and PCA were used for the data analysis in order to detect spectral differences between control and intentionally infected cells, and spectral markers were revealed even at low contamination level. The preliminary results showed that FTIR has the potential to be used in the future as a reliable complementary detection technique for mycoplasma-infected cells. Graphical abstract FTIR microspectroscopy is able to differentiate between mycoplasma infected cells (LC for low contamination and HC for high contamination) and control non-infected cells (CN).

In lung cancer, the survival of patients with the same clinical stage varies widely for unknown reasons. In this two-phase study, we examined the hypothesis that germline variations influence the survival of patients with lung adenocarcinoma. First, we analyzed existing genotype and clinical data from 289 UK-resident patients with lung adenocarcinoma, identifying 86 single nucleotide polymorphisms (SNPs) that associated with survival (p

Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 μL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR-positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR-defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels. © 2014 International Society for Advancement of Cytometry.

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.

Item does not contain fulltext BACKGROUND: Cystic fibrosis (CF) is a common genetic disease caused by mutations of the CF transmembrane conductance regulator (CFTR) gene. Persistent lung inflammation, characterized by increasing polymorphonuclear leukocyte recruitment, is a major cause of the decline in respiratory function in CF patients, and is a leading cause of morbidity and mortality. CFTR is expressed in various cell types, including leukocytes, but its involvement in the regulation of leukocyte recruitment is unknown. METHODS: Ex vivo adhesion and chemotaxis, flow cytometry, immunofluorescence, GTPases activity assays. RESULTS: We found that chemoattractant-induced activation of beta1 and beta2 integrins and of chemotaxis is defective in mononuclear cells isolated from CF patients. In contrast, polymorphonuclear leukocyte adhesion and chemotaxis were normal. The functionality of beta1 and beta2 integrins was restored by treatment of CF monocytes with the CFTR correcting drugs VX325 and VX809. Moreover, treatment of healthy monocytes with the CFTR inhibitor CFTR inh-172 blocked integrin activation by chemoattractants. In a murine model of lung inflammation, we found that integrin-independent migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, integrin-dependent transmigration into the alveolar space was impaired. Finally, signal transduction analysis showed that in CF monocytes chemoattractant-triggered activation of RhoA and CDC42 rho small GTPases, controlling integrin activation and chemotaxis respectively, was strongly deficient. CONCLUSION: Altogether, these data highlight the critical regulatory role of CFTR in integrin activation by chemoattractants in monocytes and identify CF as a new, cell-type selective, leukocyte adhesion deficiency disease providing new insights on CF pathogenesis.

Vezzalini M, Aletta J M, Beghelli S, Moratti E, Della Peruta M, Mafficini A, Mojica W D, Mombello A, Scarpa A & Sorio C (2010) Histopathology57, 725–733 Immunohistochemical detection of arginine methylated proteins (MeRP) in archival tissues Aims: To (i) determine whether methylarginine-specific antibodies can be employed for standard immunohistochemical analysis of paraffin-embedded tissues, (ii) analyse methylarginine expression in normal and neoplastic tissues and (iii) correlate methylarginine expression with that of protein arginine methyltransferase (PRMT1), the predominant cellular arginine methyltransferase. Methods and results: Immunohistochemistry of normal and cancer tissues was performed utilizing three commercial polyclonal antibodies: anti-methylarginine-specific antibody (anti-mRG) raised against a methylarginine peptide, Control antibody (anti-RG), a control antiserum raised against a corresponding arginine peptide without any methylated residues and anti-PRMT1. Nuclear and/or cytoplasmic methylarginine expression was detected in all keratinized and non-keratinized epithelia. A preliminary survey of a series of thyroid, pancreatic, colonic and gastric cancers identified a different pattern of methylarginine expression in comparison with normal tissue. A correlation between methylarginine staining and PRMT1 expression was found in all normal and cancer tissues analysed. Conclusion: Methylarginine-specific antibodies are capable of recognizing methylarginine proteins (MeRP) in paraffin-embedded tissues. Methylarginine proteins are expressed widely and show differences in subcellular localization in various organs and neoplastic conditions. The efficient detection of methylproteins by standard immunohistochemistry provides a new tool to investigate the role of methylarginine proteins (MeRP) in biological processes including carcinogenesis.

Contains fulltext : 51777.pdf (Publisher’s version ) (Closed access) AIMS: To establish the conditions for protein tyrosine phosphatase gamma (PTPgamma) detection in paraffin tissues using two antibodies raised against its NH(2)- (anti-P4) and COOH-termini (gammaTL1); to analyse its expression in normal tissues and to perform an initial screening of neoplastic tissues. METHODS AND RESULTS: Membranous and/or cytoplasmic PTPgamma expression was detected in the majority of epithelial cell types and in endocrine cells, with the highest expression in adrenal medulla, endocrine cells of the gastrointestinal tract and pancreatic islets. Both antibodies stained the thyroid follicular epithelium, but only anti-P4 antibody stained the colloid matrix, suggesting shedding/secretion of the PTPgamma extracellular domain. Marked loss of PTPgamma immunoreactivity was detected in subsets of ovarian (21%), breast (56%) and lung (80%) neoplasms. Conversely, cytoplasmic positivity was found in 37% of lymphomas, mainly of high-grade histotypes, while normal lymphocytes were negative. Brain tissue showed PTPgamma expression in a few neuronal and glial elements and PTPgamma was overexpressed in the majority of high-grade astrocytomas. CONCLUSIONS: We have analysed PTPgamma expression in archival paraffin-embedded tissues for the first time, demonstrating particularly high expression in endocrine cells and both down- and up-regulation in neoplasia, the latter possibly reflecting the undifferentiated state of the neoplastic cells, suggesting a complex role for this phosphatase.

Item does not contain fulltext We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived parental Ba/F3 cells (Ba/F3#PAR), Ba/F3 cells transfected with p210(BCR/ABL) (Ba/F3#WT) and expressing high levels of protein tyrosine kinase (PTK), and human-derived BCR/ABL positive K562 leukemic cell sub-clones engineered to differently express receptor-type tyrosine-protein phosphatase gamma (PTPRG). Synchrotron radiation (SR) and conventional (globar) IR sources were used to perform microFTIR respectively, on single cells and over several cells within the same sample. Ex vivo time-course experiments were run, inducing maximal protein phosphorylation in PMNs by 100 nM N-formylated tripeptide fMLP. Within the specific IR fingerprint 1800-850 cm(-1) frequency domain, PCA identified two regions with maximal signal variance. These were used to model and test the robustness of PCA in representing the dynamics of protein phosphorylation/de-phosphorylation processes. An IR signal ratio marker reflecting the homeostatic control by protein kinases and phosphatases was identified in normal leukocytes. The models identified by microFTIR and PCA in normal leukocytes also distinguished BCR/ABL positive Ba/F3#WT from BCR/ABL negative Ba/F3#PAR cells as well as K562 cells exposed to functionally active protein tyrosine phosphatase recombinant protein ICD-Tat transduced in cells by HIV-1 Tat technology or cells treated with the PTK inhibitor imatinib mesylate (IMA) from cells exposed to phosphatase inactive (D1028A)ICD-Tat recombinant protein and untreated control cells, respectively. The IR signal marker correctly reflected the degrees of protein phosphorylation associated with abnormal PTK activity in BCR/ABL positive leukemic cells and in general was inversely related to the expression/activity of PTPRG in leukemic sub-clones. In conclusion, we have described a new, reliable and simple spectroscopic method to study the ex vivo protein phosphorylation/de-phosphorylation balance in cell models: it is suitable for biomedical and pharmacological research labs but it also needs further optimization and its evaluation on large cohorts of patients to be proposed in the clinical setting of leukemia.

In lung cancer, the survival of patients with the same clinical stage varies widely for unknown reasons. In this two-phase study, we examined the hypothesis that germline variations influence the survival of patients with lung adenocarcinoma. First, we analyzed existing genotype and clinical data from 289 UK-resident patients with lung adenocarcinoma, identifying 86 single nucleotide polymorphisms (SNPs) that associated with survival (p0.01). We then genotyped these candidate SNPs in a validation series of 748 patients from Italy that resulted genetically compatible with the UK series based on principal component analysis. In a Cox proportional hazard model adjusted for age, sex and clinical stage, four SNPs were confirmed on the basis of their having a hazard ratio (HR) indicating the same direction of effect in the two series and p0.05. The strongest association was provided by rs2107561, an intronic SNP of PTPRG, protein tyrosine phosphatase, receptor type, G; the C allele was associated with poorer survival in both patient series (pooled analysis loge HR = 0.31; 95% CI: 0.15-0.46, p = 8.5 × 10(-5) ). PTPRG mRNA levels in 43 samples of lung adenocarcinoma were 40% of those observed in noninvolved lung tissue from the same patients. PTPRG overexpression significantly inhibited the clonogenicity of A549 lung carcinoma cells and the anchorage-independent growth of the NCI-H460 large cell lung cancer line. These four germline variants represent promising candidates that, with further study, may help predict clinical outcome. In addition, the PTPRG locus may have a role in tumor progression.

Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 μL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR-positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR-defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels. © 2014 International Society for Advancement of Cytometry.

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.

We tested the ability of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) in combination with unsupervised Hierarchical Cluster Analysis (HCA) in identifying drug-resistance/sensitivity in leukemic cells exposed to tyrosine kinase inhibitors (TKIs). Experiments were carried out in a well-established mouse model of human Chronic Myelogenous Leukemia (CML). Mouse-derived pro-B Ba/F3 cells transfected with and stably expressing the human p210(BCR-ABL) drug-sensitive wild-type BCR-ABL or the V299L or T315I p210(BCR-ABL) drug-resistant BCR-ABL mutants were exposed to imatinib-mesylate (IMA) or dasatinib (DAS). MicroFTIR was carried out at the Diamond IR beamline MIRIAM where the mid-IR absorbance spectra of individual Ba/F3 cells were acquired using the high brilliance IR synchrotron radiation (SR) via aperture of 15 × 15 μm(2) in sizes. A conventional IR source (globar) was used to compare average spectra over 15 cells or more. IR signatures of drug actions were identified by supervised analyses in the spectra of TKI-sensitive cells. Unsupervised HCA applied to selected intervals of wavenumber allowed us to classify the IR patterns of viable (drug-resistant) and apoptotic (drug-sensitive) cells with an accuracy of95%. The results from microFTIR + HCA analysis were cross-validated with those obtained via immunochemical methods, i.e. immunoblotting and flow cytometry (FC) that resulted directly and significantly correlated. We conclude that this combined microFTIR + HCA method potentially represents a rapid, convenient and robust screening approach to study the impact of drugs in leukemic cells as well as in peripheral blasts from patients in clinical trials with new anti-leukemic drugs.

The receptor protein tyrosine phosphatase γ (Ptprg-RPTPγ) is a receptor protein widely expressed in many tissues, including the central nervous system (CNS). Several RPTPγ isoforms are expressed in the brain during development and in adulthood, but their distribution and role are unknown. In this study, we investigated the distribution of some RPTPγ isoforms in the adult brain using antibodies against the epitopes localized in the C- and in the N-terminal domains of the full length isoform of RPTPγ. We found a predominant and widespread neuronal positivity throughout the neocortex, hippocampus, striatum and in many nuclei of the brainstem and cerebellum. At least 2 distinct isoforms that can co-exist in various compartments in the same cell are detectable in different neuron types. Immunopositivity for epitopes located in both the N- and C-terminus domains were found in the neuropil of cortical and hippocampal neurons, whereas the N-terminal domain positivity was found in the soma, often without colocalization with its C-terminal counterpart. Among glial cells, some protoplasmic and perivascular astrocytes and the cerebellar Bergmann glia, express RPTPγ. The astrocytic expression of RPTPγ and putative processing isoforms of 120 and 80 kDa increases during neuroinflammation, in particular 24 h after LPS treatment. Activated astrocytes were found to be strongly positive for RPTPγ also in a mice model of Alzheimer’s disease. Our results confirm previous findings and enrich the current knowledge of RPTPγ distribution in the CNS, highlighting a role of RPTPγ during neuroinflammation processes.

BackgroundEvaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes.DescriptionWestern blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated, cell membrane-localized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ: 15 subjects), but it failed in monocytes from CF patients (44 subjects). We propose an index, which values in CF patients are significantly (pResults and significanceCFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials.

Aimed at developing accurate, reliable and cost-saving analytical techniques for drugs screening we evaluated the potential of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) as a quantitative pre-diagnostic approach for the rapid identification of IR signatures of drugs targeting specific molecular pathways causing Chronic Myeloid Leukemia (CML). To obtain reproducible FTIR absorbance spectra at the necessary spatial resolution we optimized sample preparation and acquisition parameters on a single channel Mercury-Cadmium-Telluride (MCT) detector in the spectral interval of frequencies from 4000 to 800 cm(-1). Single K562 cells were illuminated by Synchrotron Radiation (SR) and a number of ~15 K562 cells spread in monolayer were illuminated by a conventional IR source (Globar), respectively. Combining IR spectral data with the results of complementary biochemical investigations carried out in samples by different analytical methods we identified and cross-validated IR signatures of drugs targeting the oncogenic protein BCR/ABL and its associated abnormal tyrosine kinase activity in K562 cell line. Unsupervised pattern recognition performed by Hierarchical Cluster Analysis (HCA) clustered the spectra of single K562 cells in two distinct groups roughly corresponding to living and to apoptotic cells, respectively. The corresponding IR spectral profiles were assumed to represent drug-resistant and drug-sensitive cells. Significant variations with increasing percentages of apoptotic cells were observed after the treatment of K562 cells with drugs that directly or indirectly target BCR/ABL. In conclusion, we suggest that microFTIR associated with multivariate data analysis may be useful to assess drug compounds in ex vivo cancer cell models and possibly peripheral blast cells from CML patients.

85 Expression of Unfolded Protein Response (UPR) protein markers are increased in primary and cystic fibrosis (CF) nasal polyposis (NP) L. Jeanson1,2, C. Guerrera1,4, M. Baudouin-Legros1,5, S. Amselem2, A. Coste3, E. Escudier2, A. Edelman1,5. 1U845, Inserm, Paris, France; 2U933, Inserm, Paris, France; 3U955, Inserm, Creteil, France; 4Plateau Proteome Necker, IFR 94, Paris, France; 5Faculte de Medecine, Universite Paris Descartes, Paris, France

Background Chronic Myeloid Leukemia (CML), is a biologically and clinically heterogeneous disease that originates in a pluripotent bone marrow stem cell. The hallmark of CML is the presence of an aberrant gene, BCR-ABL1, which encodes for a constitutively activated tyrosine kinase. Protein tyrosine phosphatases can negatively regulate ABL1 mediated signaling by dephosphorylating the kinase and/or its substrates and impaired phosphatase activity has been linked to several solid cancers and leukemias. We have previously reported the role of the receptor-type phosphatase PTPRG as tumor suppressor gene and demonstrated a significant decrease in the PTPRGexpression level in CML. Here we have investigated the expression of PTPRG gene in 32 CML patients aiming at the evaluation of the clinical relevance of the PTPRGdown regulation. Methods All 32 CML patients included in this study were diagnosed in chronic phase. Thirteen untreated patients diagnosed with philadelphia-negative myelod disorders were included in the study as control group. The study was approved by the Local Ethics Committee, and informed consent in accordance with declaration of Helsinki was obtained from each patient. The expression level of PTPRG gene was evaluated by a sybr green real-time RT-PCR assay in 2 samples from each patient, taken at diagnosis and following TKI treatment. The beta-Actin (ACTB) housekeeping gene was used for normalization. Results were validated using predesigned TaqMan quantitative RT-PCR assays for PTPRG (intracellular domain) and ABL1 genes. Quantitative RT-PCR was used to quantify BCR-ABL1 gene fusion transcripts according to the European Leukemia Net guidelines. Statistical analysis and comparisons were performed by Mann-Whitney's and Student paired T tests using the SPSS software. Results The mean levels of PTPRG transcript, expressed as PTPRG/ACTB ratio, were significantly lower in CML samples at diagnosis compared to the non-CML control group (0,44%, range 0-0,37 vs 6,29%, range 0,09-52; p=0.020). Recovery of PTPRG mRNA expression at variable levels, ranging from 0.17 to 30%, was detected in 29/32 follow up samples, taken at different time points of treatment (from 3 months to 10 years). Differences in PTPRG gene expression levels between CML samples at diagnosis and after treatment were statistically significant (p=0,027). No statistically significant correlation was observed between PTPRG/ACTB and BCR-ABL/ABL1 ratios nor with deepness of molecular response or SOKAL and EUTOS scores, even though 2 of the 3 patients showing higher level of PTPRGmRNA at diagnosis than in the follow up sample showed resistance to TKI treatment. Conclusions In this study we confirm a down regulation of PTPRG in a high percentage of CML patients and show that its expression is restored upon treatment with TKIs. Deregulated expression of PTPRG phosphatase underline its role as a tumor suppressor gene in CML and highlights its potential use as a new bio-marker of disease potentially usable in association with BCR/ABL1, allowing for a better understanding of the molecular mechanism underlying the development of CML, and propose possible new avenues for therapeutic treatment. Disclosures: Yassin: qatar national research fund: Patents & Royalties, Research Funding.

Background: CML is the most common myeloproliferative disease observed among adults, its 1st line of treatment is IM with a response rate ranging between 55 – 90%. In Qatar the resistance rate is higher than 45%. Our collaborators in Italy recently reported on the relation between CML and PTPRG. Methods: One cohort of patients (n=25, period=3years) receiving Imatinib was studied for haematological, cytogenetic molecular and biochemical abnormalities. Our collaborators in Italy examined different CML cell lines and an independent cohort of patients for the level of expression of PTPRG using QPCR, clonogenic assays, methylation-specific PCR, flow cytometry and western blotting. Results: Our team reported previously on the high rate of resistance of CML to IM (45%). During this forum the team is further reporting on the possible underlying mechanisms behind this resistance (see Al-Dewik et al at this forum) Despite a few positive findings, no pattern could be identified to delineate a significant underlying mechanism. Our collaborators in Italy, identified that down-regulation of PTPRG increased colony formation in the PTPRG+ve megakaryocytic MEG-01 and LAMA-84, but had no effect in the PTPRG-ve K562 and KYO-1. Its over-expression had an oncosuppressive effect in all four cell lines and is associated with inhibition of BCR/ABL-dependent signalling. PTPRG was down-regulated at the mRNA and protein levels in CML patients in both PB and BM, including CD34+ cells, and is re-expressed following molecular remission of disease. This re-expression was associated with loss of methylation of a CpG island of PTPRG promoter in 55% patients. In K562 cells, the hypomethylating agent 5-aza-2’-deoxycytidine induced PTPRG expression and caused inhibition of colony formation that was partially reverted by antisense-mediated down-regulation of PTPRG expression. Conclusions: Although this study was done on 2 independent patient populations, it suggests that in CML populations with high resistance rate it might be worth examining the PTPRG expression level and correlate it with the pattern of resistance. Our group has secured 3 years funding from QNRF (NPRP 4-157-3-052) to investigate PTPRG signalling in CML, including the study of a possible link among the high CML resistance and the PTPRG expression levels.

Protein tyrosine phosphatase (PTPγ) is a receptor-like molecule with a known role in murine hematopoiesis. We analyzed the regulation of PTPγ expression in the human hematopoietic system, where it was detected in human peripheral blood monocytes and dendritic cells (DCs) of myeloid and plasmacytoid phenotypes. Its expression was maintained during in vitro monocyte differentiation to dendritic cells (moDC) and was further increased after maturation with bacterial lipopolysaccharide (LPS), CD40L, and TNFα. But PTPγ was absent when monocytes from the same donor were induced to differentiate in macrophages. B and T lymphocytes did not express PTPγ. Rather, PTPγ mRNA was expressed in primary and secondary lymphoid tissues, and the highest expression was in the spleen. PTPγ was detected by immunohistochemistry in subsets of myeloid-derived DCs and specialized macrophages (tingible bodies, sinus and alveolar macrophages). Classic macrophages in infective or reactive granulomatous reactions did not express PTPγ. Increased PTPγ expression was associated with a decreased ability to induce proliferation and interferon-γ secretion in T cells by moDCs from patients with advanced pancreatic cancer. Taken together, these results indicate that PTPγ is a finely regulated protein in DC and macrophage subsets in vitro and in vivo.