A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients

Abstract

BackgroundProtein 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.MethodsTPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry.ResultsCo-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.ConclusionsThe 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 materialThe online version of this article (doi:10.1186/s13045-017-0494-z) contains supplementary material, which is available to authorized users.

Regulative Loop between β-catenin and Protein Tyrosine Receptor Type γ in Chronic Myeloid Leukemia

“…Cells from the LAMA-84 cell line (1 × 10 5 ) treated with PTPRG siRNA or scramble sequence, were subject to cytospin (Thermo Fisher Scientific, Milan, Italy) and immediately fixed in 4% paraformaldehyde for 10 min at 4 • C. After three washing steps, slides were incubated overnight at 4 • C with primary antibodies: 5 µg/mL phospho-β-catenin (Tyr 654) (ECM Biosciences, Versailles, KY, USA), 5 µg/mL total-β-catenin (Santa Cruz Biotechnology Inc, Heidelberg Germany) and 5 µg/mL anti-PTPRG [44], in a buffer containing PBS/Tween20 0.05%/BSA 1%/NaN 3 4mM, followed by the proper secondary Alexa Fluor-conjugated antibodies (1:2000, Thermo Fisher Scientific, Milan, Italy), for 1 h at room temperature. After mounting with an anti-fading solution, samples were analyzed by fluorescence microscopy (DM6000B, Leica Microsystems, Wetzlar, Germay) and confocal microscopy (TCS-SP5, Leica Microsystem, Wetzlar, Germay).…”

BCR-AblSilencing by siRNA: A Potent Approach to Sensitize Chronic Myeloid Leukemia Cells to Tyrosine Kinase Inhibitor Therapy

“…It will be important to determine whether the combination approach will be functional in other BCR-Abl-positive leukemias, such as acute lymphoblastic leukemia that sometimes presents a BCR-Abl-positive phenotype. It remains to be seen whether other protein kinase inhibitors and antibodies can be explored as a synergistic pair with BCR-Abl siRNAs to develop a replacement for conventional therapies against drug-resistant CML phenotypes [49,50].…”

Chronic Myeloid Leukemia Preceded by Tuberculosis

“…Exposure to ionizing radiation is the only known risk factor for CML [4]. Tyrosine kinase inhibitors are a cornerstone in the management of CML around the world with good results [5][6][7][8][9]. This case is shedding light on a possible association between TB and the development of CML.…”