Protein Tyrosine Phosphatase Receptor Type Γ Is a Functional Tumor Suppressor Gene Specifically Downregulated in Chronic Myeloid Leukemia

Published OnlineFirst October 19, 2010; DOI: 10.1158/0008-5472.CAN-10-0258

Tumor and Stem Cell Biology Cancer Research Protein Tyrosine Phosphatase Receptor Type γ Is a Functional Tumor Suppressor Gene Specifically Downregulated in Chronic Myeloid Leukemia

Marco Della Peruta1, Giovanni Martinelli4, Elisabetta Moratti1, Davide Pintani1, Marzia Vezzalini1, Andrea Mafficini1,3, Tiziana Grafone4, Ilaria Iacobucci4, Simona Soverini4, Marco Murineddu5, Fabrizio Vinante2, Cristina Tecchio2, Giovanna Piras5, Attilio Gabbas5, Maria Monne5, and Claudio Sorio1,3

Abstract 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.

Introduction For this reason, much attention has been focused on naturally occurring negative regulators of tyrosine kinase Chronic myelogenous leukemia (CML), also known as signaling: the protein tyrosine phosphatase (PTP) family chronic myeloid or chronic myelocytic leukemia, is a malig- of enzymes. nant cancer of the bone marrow myeloid lineage. It ac- The human PTP family contains 107 members, 38 of which counts for ∼15% to 20% of all cases of leukemia (1–1.5 belong to the phosphotyrosine-specific (“classic”)PTPsub- cases per 100,000 population per year; ref. 1) and originates family (subdivided in receptor- and nonreceptor-like types) from a pluripotential stem cell in which a 9:22 translocation and 61 belong to the so-called “dual-specific phosphatases.” results in the production of BCR/ABL fusion protein. This To date, only two tyrosine phosphatases, PTP1B and SHP-1, has a constitutive tyrosine kinase activity and deregulates are known to dephosphorylate and partially inhibit the trans- signal transduction pathways, leading to leukemia (2). Phos- formation potential of BCR/ABL (4, 5). Serine-threonine phorylation of key residues is required for the full transform- phosphatase PP2A is inhibited in blast crisis CML (6). These ing activity of BCR/ABL (3). PTPs belong to the nonreceptor class of enzymes. Recently,

Authors' Affiliations: 1Department of Pathology and Diagnostics and performed flow cytometry. I. Iacobucci: BCR/ABL analysis. S. Soverini: 2Department of Clinical and Experimental Medicine, University of Verona; management of clinical data (Bologna). M. Murineddu: flow cytometry 3ARC-Net Research Center, University of Verona, Policlinico G.B. Rossi, and patient selection. F. Vinante: patient selection and clinical information Verona, Italy; 4Institute of Hematology and Medical Oncology, «Lorenzo (Verona). C. Tecchio: performed research. A. Mafficini: performed flow e Ariosto Seragnoli», University of Bologna, Bologna, Italy; and 5Centro cytometry. G. Piras: statistical analysis. A. Gabbas: provided clinical data. di Diagnostica Biomolecolare e Citogenetica Emato-Oncologica, “San M. Monne: patient selection and clinical information, methylation studies Francesco” Hospital, ASL3, Nuoro, Italy in patients (Nuoro). C. Sorio: designed research, wrote manuscript. Note: Supplementary data for this article are available at Cancer Research Corresponding Author: Claudio Sorio, Department of Pathology and Online (http://cancerres.aacrjournals.org/). Diagnostics, General Pathology Section, University of Verona, Strada Author contribution statement: M. Della Peruta: designed and performed Le Grazie 8, 37134 Verona, Italy. Phone: 39-45-8027688; Fax: 39-45- experiments, analyzed data. G. Martinelli: patient selection and clinical 8027127; E-mail: [email protected]. information (Bologna). E. Moratti: performed experiments, coimmunopre- doi: 10.1158/0008-5472.CAN-10-0258 cipitation studies. D. Pintani: performed clonogenic assays. M. Vezzalini: QPCR analysis, clonogenic and proliferation assays. T. Grafone: ©2010 American Association for Cancer Research.

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PTPROt, a receptor-like PTP, has been found to interfere be in complete cytogenetic remission (CCR) based on the with BCR/ABL signaling in K562 cells (7). BIOMED 2 standardized protocols. PTP receptor type γ (PTPRG) is a member of the receptor- like PTPs (8), is expressed in myeloid cells, including CD34+ Cells precursors, and can affect hematopoietic differentiation K562 (22) and MEG-01 (23) were from American Type (9–11). It is a candidate tumor suppressor gene in solid Culture Collection; KYO-1 (24) and LAMA-84 (25) were from tumors (12–14) owing to its reduced expression in ovarian, DSMZ. HEK293 was purchased from Invitrogen. K562 transfec- breast, and lung tumors (15, 16). Somatic mutations and epi- tants were selected, adding 0.50 mg/mL G418 (Invitrogen) to genetic silencing were reported (17–21). the culture medium. All were characterized by cytogenetic anal- Here, we show that PTPRG acts as a functional tumor ysis and antigen expression. Human samples were derived from suppressor gene in CML, interacting with BCR/ABL and in- Ficoll-purified cells (mRNA analysis) or whole blood samples hibiting downstream signaling events. PTPRG is specifically (flow cytometry analysis). downregulated in peripheral blood and bone marrow leukocytes of CML patients, at least in part by a mechanism Cell transfection and selection of CML cell lines involving hypermethylation of a PTPRG CpG island located Full-length (FL) human PTPRG (26) and antisense (AS) in the 5′ untranslated region. These findings imply that cDNA and cell lines were described (10, 27). The D1028A PTPRG might represent a potential diagnostic and thera- PTPRG cDNA was obtained by site-directed mutagenesis, peutic target in CML. wherein the Asp 1028 codon was replaced with an Ala codon (NP_002832.3) and verified by sequencing. Materials and Methods To avoid the source of error associated with clonal variation within cell lines, we selected a K562 clone (named B4) Tissue samples that maintains the capability to differentiate when treated Patients were recruited at the hematology departments of with the well-known inducers sodium butyrate and hemin Bologna, Nuoro, and Verona, Italy among newly diagnosed (28, 29) and performed all the transfection and selection CML patients during the first chronic phase. Presence of processes starting from this well-characterized clone. the Philadelphia chromosome and p210BCR-ABL rearrange- ment was a prerequisite for enrollment. CML samples were Transfection of HEK293 cells taken at diagnosis and after the initiation of therapy with HEK293 cells were transfected with FL cDNA coding for imatinib mesylate (IM). Age- and sex-matched samples p210BCR/ABL cDNA in pLNL 5LX cytomegalovirus, derived from from individuals diagnosed as not affected by malignant pLNL-XHC (30), kindly provided by Dr. Paolo Vigneri (Univer- disease were used as a reference group. Analysis of the sity of Catania, Italy). FL human PTPRG cDNA and D1028A CD34+ population for the group of patients in molecular PTPRG cDNA were previously described. Transfection was remission was not possible, as these samples were not performed with Lipofectamine 2000 reagent (Invitrogen) ac- available at the time of evaluation. Table 1 reports clinical cording to the manufacturer's instructions using 1.5 μgplasmid data. Written informed consent was obtained in accordance DNA (for single transfection) or 1 + 1 μgplasmidDNA(fordouble with the Declaration of Helsinki. Cytogenetic response was transfection) following the manufacturer's instructions. Cultures classified as complete, and the patients were considered to were cultured for 48 hours, washed with cold TBS, and lysed.

Table 1. Clinical data of patients

Diagnosis (n = 22) Remission (n =8) Median (range) Median (range)

Age 54.12 (47–63) 60.42 (33–84) Female (%) 12.5 33.4 Bcr-Abl/Abl (%) 69.13 (30.84–100) 0.10 (0–1) Hemoglobin level (g/dL) 10.99 (7–13.8) 11.3 (5.90–15.5) Platelets (×109/L) 346.25 (6–589) 193.7 (88–291) WBC (×109/L) 218.75 (137–296) 4.77 (0.13–7.73) Peripheral blood blasts (%) 4.4 (2–6) 0 Peripheral blood basophils (%) 7.6 (0–15) 0.006 (0.002–0.01) BM blasts (%) 3.2 (1–4) 0 Palpable splenomegaly (%) 57.14% 0.00

Abbreviations: WBC, white blood cells; BM, bone marrow.

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Immunoblotting and antisera Aldrich) resuspended in sterile PBS were added, and cells Cells (20 × 106/1 mL) were solubilized in lysis buffer (LB) as were lysed according to the manufacturer's instructions. described (10) or directly lysed in sample buffer [40 mmol/L Absorbance at 570 nm was read in an ELX808iu Ultra Micro- Tris-HCl (pH 6.8), 183 mmol/L β-mercaptoethanol, 1% SDS, plate Reader (Bio-Tek Instruments, Inc.). 5% glycerol], heated at 95°C for 5 minutes, passed through a 23-gauge needle to fragment DNA, resolved on SDS-PAGE, Xenografting in nude mice and electroblotted onto polyvinylidene difluoride membranes In vivo studies using 4-week-old nu/nu Swiss mice weigh- (Millipore Corp.). The antibodies used were antiphospho- ing 18 to 22 g (Charles River) for each experimental condition tyrosine [clone 4G10 (Upstate Biotechnology) and clone were performed exactly as described (31). PY99 (Santa Cruz Biotechnology)]; anti-PTPRG P4 (26), anti-Crk-L (H-62), and anti-c-Abl (Santa Cruz Biotechnology); Reverse transcription-PCR and real-time and anti-phospho-Crk-L (Tyr207), signal transducers and acti- quantitative PCR vators of transcription 5 (STAT5), and rabbit anti-phospho- Total RNA was isolated using TRIZOL (Invitrogen) accord- STAT5 (Tyr694; Cell Signaling, Boston). Bound antibodies ing to the manufacturer's indications. PTPRG cDNA expres- were visualized using the enhanced chemiluminescence sion was analyzed by PCR and quantitative PCR (QPCR) detection system (Amersham). Membranes were treated for as described (11). The primers used for amplification of 30 minutes at 65°C in 0.5 mmol/L Tris (pH 6.7), 2% SDS, and PTPRC-CD45, PTPRJ-CD148, PTPRE-PTPε,PTPRU-PTPμ, 100 mmol/L β-mercaptoethanol and washed before probing PTPN1-PTP1B, PTPN6-SHP-1, and PPP2R4-PP2A are shown with additional antibodies. in Supplementary Table S1. To evaluate the change of PTPRG and BCR/ABL mRNA Immunoprecipitation with Protein G-Sepharose 4 levels in CML patients during follow-up, we applied the Fast Flow formula (T − U)/(T + U) adapted from Mauri and collea- Total protein content was assessed using Bradford assay gues (32). For each individual, the difference (T − U) be- (Sigma). Cell lysates (400 µg of total protein for each sam- tween the mRNA levels of both PTPRG and BCR/ABL on ple) were incubated with 3 µg of specific antibodies for 3 (T) and before treatment (U) with IM was divided by the hours at 4°C. Twenty microliters of protein G-Sepharose sum of the same values (T + U). When T equals U,the (Sigma) for each sample were added, and the mixture was ratio is zero, which corresponds to no change in the ex- incubated for 1 hour at 4°C with gentle rocking, washed, pression level between the two conditions. This ratio and subjected to SDS-PAGE. equals −1.0 when no expression is detectable in the treated patient (T = 0). When U = 0, the ratio will be +1.0. Inter- Expression and purification of PTPRG intracellular mediate ratio values between −1.0 and +1.0 correspond to domain and pull-down assay different expression levels. PTPRG intracellular domain (ICD) and D1028A mutant (amino acid residues 797–1145 of NP_002832 sequence) were Methylation-specific PCR cloned in the T7-based HisG-tagged vector expression pRSET Genomic DNA (1 μg) was subjected to bisulfite modifica- A (Invitrogen) using the unique BamHI and EcoRI sites, se- tion using the CpGenome DNA modification kit (Chemicon) quenced, and expressed in BL21 (DE3) pLysS Escherichia coli, according to the manufacturer's protocol. PCR conditions and the recombinant proteins were purified. Approximately were 95°C for 8 minutes followed by 45 cycles at 95°C 2 μg of PTPRG and 10 μg of enhanced green fluorescent pro- for 30 seconds and an annealing temperature of 60°C tein (EGFP) affinity purified protein were added to 500 μgof (methylation-specific PCR) or 61°C (unmethylation-specific K562 lysed in LB for 3 hours at 4°C. The beads were collected, PCR) for 1 minute followed by a final extension at 72°C for washed three times with LB, and then subjected to SDS-PAGE 7 minutes. PCR products (168 bp for unmethylated PTPRG and Western blotting with specific antibodies as described. and 166 bp for methylated PTPRG)amplifyingtheregion from −576 to −410 relative to the transcription start were Clonogenic and proliferation assays resolved in a 2.5% agarose gel. K562 (3 × 103 cells/mL), KYO-1 (2.4 × 103 cells/mL), LAMA-84, and MEG-01 (2 × 104 cells/mL) in MethoCult Flow cytometry of purified cells and whole blood H4230 (StemCell Technologies) were transfected and trans- Cell lines and whole blood samples were stained with the ferred in 24-well plates with 0.5 mg/mL G418. After 8 days, anti-PTPRG antibody chPTPRG IgY or preimmune IgY and with each well was scored for the presence and number of colonies. monoclonal anti-CD34 PE (clone AC136, Miltenyi Biotech) as When indicated, K562 cells were exposed to 2 μmol/L described (10). Flow cytometry was performed on a Becton 5-aza-2′-deoxycytidine (DAC) for 24 hours, washed, and then Dickinson FACScan flow cytometer. Data analysis was plated in a drug-free medium. The same experiment was re- performed with FCS Express V3 software (De Novo Software). produced in mock-transfected K562, and two independent clones were stably transfected with PTPRG-AS cDNA that Statistical analysis was harvested and plated in a 96-well plate (5 × 103 cell/ Data analysis was performed using unpaired and one- 100 μL/well) or in MethoCult as described previously. At sample t test (GraphPad Instat software). A P <0.05was the indicated time points, 10 μL of 5 mg/mL MTT (Sigma- considered statistically significant.

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Results PTPRE-PTPε, and PTPRU-PTPμ)ortotheirroleinCML [PTPN1-PTP1B (4), PTPN6-SHP-1 (5), and PPP2R4-PP2A (6)]. PTPRG expression specifically correlates with decreased PTPRG affects the clonogenic capability of both PTPRG- clonogenic capability and growth in CML cell lines positive and -negative cell lines. As shown in Fig. 1C, PTPRG K562, KYO-1, LAMA-84, and MEG-01 were plated in Metho- expression reduced the clonogenicity in all cell lines from 31% Cult. Each well was scored for the presence and number of to 40% relative to controls. Down-modulation of PTPRG expres- colonies after 8 days. Fig. 1A shows the number of colonies sion by transfection of an AS PTPRG construct (27) resulted in grown (top) and their volumes (bottom). Only PTPRG expres- an increase of colony number only in PTPRG-positive LAMA-84 sion correlates with a reduced clonogenic capability and vol- and MEG-01 cell lines. Interestingly, these cell lines are of mega- ume of the colonies (Fig. 1B). The choice of the PTP panel was karyocytic origin, and it is known that PTPRG is expressed in linked either to a receptor-like structure and known expres- human megakaryocytes (16). The construct is capable to down- sion in hematopoietic lineages (PTPRC-CD45, PTPRJ-CD148, regulate PTPRG as shown in Supplementary Table S2.

Figure 1. PTPRG expression and clonogenic capability in CML cell lines. A, colony formation assay. Cells were plated in 24-well plates in MethoCult as described. Top, clonogenic capability was evaluated as the ratio between the number of cells plated and the number of colonies developed. Bottom, volume of colonies (n = 3). B, reverse transcription-PCR analysis of PTPs. Expression of the indicated phosphatases. ACTB expression was used as an invariant control in all experiments (one representative of a minimum of two experiments). C, effect of PTPRG modulation on clonogenic capability. CML cell lines were transfected with empty vector (mock), PTPRG FL, and AS cDNA and, the day after, plated in MethoCult in the presence of 0.5 mg/mL G418. Results are percentages (%) of colonies grown compared with the control group (cell transfected with empty plasmid, 100%). n =3.

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Figure 2. PTPRG expression in K562 cells inhibits clonogenic capability and p210BCR-ABL signaling. A, stable transfectants (mock, FL, and D1028A cells) were characterized by immunoblotting analysis with anti-P4 antibody (arrow indicates the band corresponding to FL PTPRG) and by flow cytometric analysis with chPTPRG antibody (n = 4). B, level of PTPRG expression detected in blood monocytes compared with the level of expression in K562 mock and FL cell lines (left). Glycophorin (top) and CD13 (bottom) expression in mock, PTPRG cDNA (FL), and PTP inactive (D1028A) transfected K562 cells (right). C, top, clonogenic assay in MethoCult. Cells were plated at 2 × 103/mL, and after 8 d, each well was scored for the number, expressed as a percentage relative to the mock-transfected cells. K562 cells mock (left), PTPRG FL (middle), and PTPRG D1028A (right) are shown (n = 7). Bottom, xenograft assay in nu/nu mice: 10 million cells per mouse (mock-, FL-, and D1028A-transfected cells, five mice each) were inoculated for each experimental condition. Tumor volumes were recorded at the indicated time points. D, immunoblotting analysis of K562 cell lines. Same amounts of whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with a mixture of antiphosphotyrosine antibodies (PY99 and 4G10) and after stripping with anti-ABL, anti-CRKL, anti-phospho-CRKL (p-CRKL), anti-STAT5, and phospho-STAT5 (pSTAT5) antibodies as described. Bottom, actin and Ponceau staining, which showed equal protein loading (n = 3).

PTPRG expression has an oncosuppressive effect in the BCR/ABL is a substrate of PTPRG K562 cell line both in vitro and in vivo As BCR/ABL dephosphorylation occurred in PTPRG- Clones expressing comparable protein levels of PTPRG transfected K562 (Fig. 2D), we hypothesized that BCR/ABL and D1028A mutant cDNA were selected (Fig. 2A; Supple- might represent a direct substrate for PTPRG. Histidine- mentary Table S2). Protein expression was comparable but tagged EGFP protein (mock), WT, and D1028A ICD bound slightly lower than those recorded in human monocytes to affinity resin were then incubated with K562 cell lysates. and was associated with the increase of the myeloid differ- WT and D1028A ICD precipitated a complex containing entiation marker CD13 (Fig. 2B). Clonogenic capability of BCR/ABL, ABL, and its direct substrate CRKL (Fig. 3A). The the cells was decreased in FL compared with mock- and specificity of the interaction was supported by the lack of co- D1028A-transfected cells. In in vivo proliferation assay per- precipitation of STAT5 with the complex as well as by the lack formed in xenografted nude mice, K562 FL clone formed of any signal in the lane containing larger amounts of EGFP very small tumors compared with mock and D1028A protein (mock; Fig. 3A and C). BCR/ABL precipitated from clones (Fig. 2C). the WT PTPRG ICD is specifically dephosphorylated as the Wild-type (WT) PTPRG induces a reduction of total and same protein bound to the catalytic inactive enzyme main- p210BCR-ABL-specific tyrosine phosphorylation of its direct tains its tyrosine phosphorylated status, thus ruling out the substrate CRKL (33–35) and of STAT5, a transcription factor presence of other tyrosine phosphatase activities within the that represents a downstream target of BCR/ABL (36, 37), complex (Fig. 3B). The interaction also occurs when both pro- and is not occurring in mock-transfected and phosphatase- teins are coexpressed within HEK293 (Fig. 3D). dead (D1028A)–transfected clones (Fig. 2D). These results indicate that the tumor suppressor effect of PTPRG PTPRG expression in CML patients in K562 cells is mediated by interference with BCR/ABL- PTPRG downregulation also occurred in CML patients: dependent signaling. transcript level was 12.2-fold in Ficoll-purified bone

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marrow, whereas it was 4.68-fold lower in peripheral blood Fig. 4B, center). PTPRG expression in a group of CML mononuclear cells (PBMC) than in samples derived from patients in CCR regained levels to that seen in healthy do- healthy donors (Fig. 4A, left and middle). Thirteen CML nors (MFI ratio, 2.25; range, 0.72–3.67; Fig. 4B, center). CD34+ patients were quantitatively analyzed for the expression hemopoietic progenitors in both bone marrow and peripher- of PTPRG and BCR/ABL at diagnosis and during molecular al blood are among the highest PTPRG-expressing cells (10): remission, showing an inverse correlation between the two PTPRG was highly expressed on CD34+ cells of healthy genes (Fig. 4A, right). donors (MFI ratio, 3.91; range, 1.42–9.84) and decreased The correspondence with protein expression was evaluat- (MFI ratio, 1.24; range, 1.09–2) by 90% in CML patients ed by flow cytometry (Fig. 4B, left). MFI ratio between im- (Fig. 4B, right). mune and preimmune PTPRG IgY was 2.43 (range, 1.6–5) in Taken together, these results show that the lack of PTPRG age- and sex-matched healthy donors (control group), expression was associated with the occurrence of the disease whereas the value in CML cells was almost undetectable and rule out intrinsic defects of the healthy hemopoietic cells (93% reduction), with a MFI ratio of 1.10 (range, 0.44–1.83; of the subjects to express PTPRG.

Figure 3. PTPRG pull-down assay and PTPRG-specific BCR/ABL dephosphorylation. Purified recombinant His-tagged PTPRG ICDs of native or phosphatase-inactive (D1028A) PTPRG were bound to nichel beads and reacted with K562 cell line lysates. A, Western blotting with anti-ABL, CRKL, and STAT5 antibodies. B, the blot was reacted with antiphosphotyrosine antibodies PY20 and 4G10 (pY). Dephosphorylation of BCR/ABL specifically occurred only when BCR/ABL was incubated with ICD. B, the presence of the protein in the D1028A-coated beads was confirmed by the reaction of the stripped blot with anti-ABL antibody. C, Ponceau staining of the same membrane showed the extent of protein purification achieved by affinity purification of the baits and show that the amount of control protein (EGFP) present in the control beads is higher that the specific baits. One representative of two independent experiments. D, the results of single and combined cDNA transfection with the indicated cDNA (top). HEK293 cells were transfected with p210BCR-ABL (p210), PTPRG (FL), PTPRG D1028A (D1028A), and a combination of p210 + FL and p210 + D1028A cDNA. Equal amounts of HEK293 lysate were probed with anti-ABL and anti-PTPRG-P4 antibodies. Bottom, the result of coprecipitation of HEK293-transfected cells. The lysates were immunoprecipitated with anti-PTPRG-P4 antibody and immunoblotted with anti-ABL antibody and vice versa, showing that PTPRG-p210 interaction occurs between endogenously expressed proteins.

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Figure 4. PTPRG mRNA and protein expression in patients. A, expression of PTPRG mRNA in samples from healthy donors or CML patients was evaluated by QPCR: bone marrow (BM; left) and PBMC (center). Right, opposite variation of PTPRG and BCR/ABL mRNA levels in 13 CML patients on treatment with IM. The difference of mRNA levels during (T) and before (U) treatment for each patient has been divided by the sum of the same values, yielding normalized fold change values within the range (−1, +1). B, left, expression of PTPRG antigen in samples from healthy donors (control) and CML patients was evaluated by flow cytometry of whole blood (gate on the left) with chPTPRG antibody; an example of the analysis is shown. Center, geometric median fluorescent intensity (MFI) ratio values of the samples analyzed, including controls and CML at diagnosis and at CCR. Right, geometric MFI ratio values of PTPRG antigen in CD34+ cells from peripheral blood of healthy donors (control) and CML patients. Data are average geometric MFI ratios between the chPTPRG-stained sample and the preimmune chicken IgY. In the absence of PTPRG antigen expression, the MFI ratio equals one.

Demethylating agents induce PTPRG expression 6.5% and 1.1% at 3 and 6 days, respectively, on DAC treat- PTPRG downregulation by epigenetic modification has ment of AS-transfected clones. been reported in various cancer types (18–21). The hypomethylating agent (DAC) induced reexpression of PTPRG Reduced promoter methylation and recovery of PTPRG (Fig. 5A) followed by a marked inhibition of colony formation expression in a subset of patients and cell proliferation/survival in semisolid and liquid media CpG methylation analysis in the same patients at diagnosis partially reversed by the concomitant inhibition of PTPRG ex- and after successful treatment (all characterized by upregu- pression through transfection with a PTPRG AS carrying plas- lation of PTPRG expression) indicated that the recovery of mid in both transiently (not shown) as well as stably PTPRG expression is associated with reduced methylation transfected K562 cell line clones (Fig. 5B and C). The pres- of a region of its promoter in a substantial fraction of ence of AS construct was effective in inhibiting DAC-induced patients (Fig. 5D). PTPRG expression as evaluated after 3 and 6 days from the removal of the drug (Fig. 5C). Flow cytometry confirmed the Discussion efficacy of the construct: the percentage of PTPRG-positive cells at 3 and 6 days after DAC treatment in control K562 In this study, we first observed a specific association bet- clone was 13.2% and 16.2%, respectively, which dropped to ween loss of PTPRG expression and increased clonogenic

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capability in CML cell lines that did not occur with seven reduced extent, prompted us to speculate that the level other phosphatases analyzed. The observation that the of PTPRG expression/activity could determine the two CML cell lines that express PTPRG were still capable growth capability and differentiation of these cells. This of growing in methylcellulose, although at a much hypothesis was confirmed by the results of the PTPRG

Figure 5. PTPRG promoter hypermethylation in K562 cell lines and primary cells. Involvement of PTPRG in the oncosuppressive effect of DAC. A, expression of PTPRG in K562 after 3 and 6 days of treatment with DAC; cells were exposed to 2 μmol/L DAC for 24 h and then replated in drug-free medium. RNA was extracted on the third and sixth days after treatment to verify PTPRG gene expression by PCR (ATCB, 22 cycles; PTPRG, 40 cycles). One representative experiment of three experiments. B, cells were transfected with mock and PTPRG AS cDNA vector and selected for G418 resistance. The cells were then exposed to 2 μmol/L DAC. At 24 h after drug addition, cells were washed with drug-free medium and then replated at 3 × 103/mL; after 8 d, each well was scored for colony number and expressed as a percentage relative to untreated transfected cells. Top, a detail of the colonies grown (magnification, 10×); bottom, MTT-stained wells (n = 3). The same experiment was reproduced in transiently transfected K562 clones (mock and AS). C, same cells of B cultured in liquid medium. The percentage of live cells after DAC treatment was calculated, setting the value of the mock and AS K562 cell lines treated with vehicle (DMSO) to 100%. Top, MTT staining was performed at 3 and 6 d. Bottom, at the same time points, an aliquot of cells for each experimental condition was analyzed for PTPRG expression by flow cytometry (reported in the text) and mRNA levels by QPCR. n =2. D, PTPRG methylation-specific PCR in CML patients. All patient (1–11) samples at diagnosis were hypermethylated for PTPRG. Examples of PCR amplification data are shown: M, methylated amplicon; UM, unmethylated amplicon. Patients 1 to 5 were hypermethylated at the follow-up after treatment, whereas patients 6 to 11 were unmethylated. No alterations in the methylation pattern of PTPRG were observed in non-CML disease (n = 5, data not shown) under the same experimental conditions.

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modulation experiments, wherein overexpression deter- of this application, such as ease of use, enhanced repro- mined a reduction of clonogenic capability whereas ducibility, and cost-effectiveness. downregulation was associated with an increase in clo- Members of the tyrosine phosphatase family are in- nogenic capability only in the two PTPRG-positive cells volved in the pathogenesis of CML (4–6, 41, 42), and a lines MEG-01 and LAMA-84. therapeutic strategy based on PTP activation has been pro- K562 cell line showed activity-dependent increase of posed (43). Having identified a molecular target, ligand- the myeloid differentiation marker CD13 and decrease stimulated activation of the residual PTPRG molecules in clonogenic capability both in vitro and in vivo follow- expressed on the surface of Ph+ myeloid blasts and the de- ing PTPRG cDNA transfection, associated with a reduced velopment of specific chemical activators can be readily total, BCR/ABL-, CRKL-, and STAT5-specific tyrosine proposed. phosphorylation. No detectable PTPRG degradation Downregulation of PTPRG expression associated with was found at variance with other studies reporting that methylation of specific promoter regions has been recently the association of BCR/ABL with the serine-threonine described in various cancers (18–21). This result, along phosphatase PP2A leads to SHP-1 activation followed by with the protective effect of the AS construct on DAC BCR/ABL dephosphorylation and proteasome-dependent treatment, is in accordance with these findings and adds proteolysis (6). PTPRG likely acts through a different pathway CML to the list of neoplastic diseases where methylation- activating myeloid differentiation and a different pattern of dependent PTPRG downregulation occurs. This observa- dephosphorylation and/or protein association in comparison tion might explain the molecular mechanism of epigenet- with PP2A/SHP-1 whose mRNA, at variance with PTPRG, is ic drugs found to be active in CML (44). Even if we expressed in K562 (Fig. 1B). observed this association in 55% of the patients analyzed, CRKL dephosphorylation directly links PTPRG to the in- still not all of them show the effect. However, an associ- hibition of BCR/ABL (35). Pull-down assay confirms a di- ation between PTPRG downregulation and promoter rect interaction between the ICD domain of PTPRG and hypermethylation within this region was reported, despite BCR/ABL along with its direct substrate CRKL. BCR/ABL this phenomenon occurring in 27% of the patients affect- acts as a bona fide substrate for PTPRG, as it is depho- ed by cutaneus T-cell lymphoma (20). It is likely that CpG sphorylated only by precipitation with the catalytic active islands within other PTPRG promoter regions might enzyme. This interaction occurs most likely in the ABL be involved, and this possibility needs to be thoroughly portion of the aberrant kinase, as suggested by the ability analyzed in future studies using high-throughput of the bait to coprecipitate ABL, and also occurs when approaches. both targets are coexpressed in HEK293 cells. The absence The loss of PTPRG expression in CD34+ cells indicates that of signal using higher amounts of an unrelated protein this is an early event in the pathogenesis of CML, although it (EGFP) and the absence of STAT5 within the complex tes- is still unclear if the maintenance of critical levels of PTPRG tify to the specificity of the interaction. This last observa- expression can act as a “gatekeeper” in the molecular events tion is in agreement with the fact that, although known to leading to the clinical manifestation of CML. be dependent on phosphorylation and activation by BCR/ Overall, our findings provide the first compelling evi- ABL, STAT5 was never reported to coprecipitate with it dence of the tumor-suppressive effect of PTPRG in CML, (36, 38, 39). indicating that downregulation, but not necessarily a com- A near-complete lack of expression was associated with plete loss of PTPRG expression, is associated with the de- the occurrence of CML in primary patient samples, velopment of CML and that restoration of expression whereas we observed a recovery to values close to those levels similar to, but even lower than those present in nor- of healthy donors for both mRNA and protein levels fol- mal myeloid cells seems to exert a strong oncosuppressive lowing molecular remission. This shows that the lack of effect in Ph+ cells. expression was not due to other factors independent of These results point for the first time to PTPRG as a relevant the disease status. The inverse correlation with BCR/ new player in the pathogenesis of CML and suggest it as a ABL mRNA levels before and after treatment suggests potential target for diagnostic and therapeutic applications. that the loss of the oncogenic clone is followed by the recovery of a nonneoplastic hemopoiesis characterized by PTPRG expression. Along these lines, we show that, Disclosure of Potential Conflicts of Interest similar to the well-established molecular analysis for identification of the t(9;22)(q34.1;q11.21) chromosomal No potential conflicts of interest were disclosed. translocation (40), the measurement of the PTPRG transcript might offer a new diagnostic tool in association with the former. This potential application needs, of Acknowledgments course, a more extensive validation by multicenter studies. Moreover, we provide for the first time the proof of We thank Dr. Emanuela Parmagnani for the subcloning and characterization principle that flow cytometric analysis of PTPRG expres- of the K562-B4 cell line and Dr. Maria Antonietta Fancello for her skillful technical assistance. KYO-1 and MEG-01 cell lines were kindly provided sion might represent a potentially useful and unique ad- by Drs. Livia Manzella and Paolo Vigneri (University of Catania). We ditional biomarker for CML, with the advantages typical acknowledge the role of “Consorzio per gli Studi Universitari di Verona” for

8904 Cancer Res; 70(21) November 1, 2010 Cancer Research

PTPRG in Chronic Myeloid Leukemia

its critical support during the earliest phase of the study and throughout the The costs of publication of this article were defrayed in part by the following developments. payment of page charges. This article must therefore be hereby marked Grant Support advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Support for this study was provided by the Consorzio Studi Universitari in Verona and by the Associazione Italiana per la Ricerca sul Cancro (grant Received 01/21/2010; revised 07/28/2010; accepted 07/29/2010; published IG 4667). OnlineFirst 10/19/2010.

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8906 Cancer Res; 70(21) November 1, 2010 Cancer Research

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

Marco Della Peruta, Giovanni Martinelli, Elisabetta Moratti, et al.

Cancer Res 2010;70:8896-8906. Published OnlineFirst October 19, 2010.

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