Leukemia (2014) 28, 787–793 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu

ORIGINAL ARTICLE DNA methylation of membrane-bound tyrosine phosphatase genes in acute lymphoblastic leukaemia

WS Stevenson1,2, OG Best2, A Przybylla2, Q Chen1,2, N Singh1, M Koleth1, S Pierce3, T Kennedy1, W Tong3, S-Q Kuang3 and G Garcia-Manero3

Aberrant DNA promoter methylation with associated gene silencing is a common epigenetic abnormality in acute lymphoblastic leukaemia (ALL) and is associated with poor survival. We have identified a family of transmembrane tyrosine phosphatase proteins as targets of hypermethylation in ALL and high-grade B cell lymphoma and demonstrated that this abnormal methylation correlates with transcript expression. PTPRG was methylated in 63% of ALL samples, PTPRK in 47%, PTPRM in 64% and PTPRO in 54% of cases, with most ALL samples containing methylation at multiple phosphatase loci. PTPRK promoter methylation was associated with a decreased overall survival in the cohort. Restoration of PTPRK transcript levels in leukaemia cells, where phosphatase transcript was silenced, reduced cell proliferation, inhibited colony formation and increased sensitivity to cytotoxic chemotherapy. These biological changes were associated with a reduction in levels of phosphorylated Erk1/2, Akt, STAT3 and STAT5 suggesting functional phosphatase activity after transcript re-expression. Methylation of the phosphatase promoters was reversible with decitabine and a histone deacetylase inhibitor, suggesting that PTPRK-mediated cell signalling pathways may be targeted with epigenetic therapies in lymphoid malignancy.

Leukemia (2014) 28, 787–793; doi:10.1038/leu.2013.270 Keywords: PTPRG; PTPRK; PTPRM; PTPRO; non-Hodgkin lymphoma

INTRODUCTION proliferation may be dysregulated with an upregulation of a Transcriptional repression due to CpG island methylation is positive signal, as with the increased protein phosphorylation frequently observed in adult acute lymphoblastic leukaemia associated with the BCR-ABL1 protein in ALL, or may be (ALL) cells and this abnormality may be associated with a worse dysregulated by the inhibition of a negative regulator. Transmem- overall survival after standard chemotherapy treatment.1–3 In ALL, brane tyrosine phosphatase proteins represent a large family of 6 some of the genes that are methylated are tumour suppressor 21 related enzymes that inhibit kinase-mediated signalling genes, whereas other genes may be silenced as a consequence of and represent potential candidates as tumour suppressor genes. dysregulated methylation within the cancer cell and may not have Membrane-bound tyrosine phosphatases are transmembrane any functional significance. Identification of functional tumour proteins with extracellular domains that are implicated in cell to suppressor genes silenced by methylation is important as this may cell interactions7–9 and intracellular domains with phosphatase identify pathways important for leukaemogenesis and may function.6 They have been implicated in oncogenesis in a identify genetic targets for pharmacological intervention. variety of cellular contexts including cancer of the breast,10 Silencing of groups of genes that modulate a single biological skin,11–13 lung14 and Epstein-Barr virus (EBV)-associated Hodgkin pathway may be more important than silencing of individual lymphoma.15 This family of proteins appear to modulate cell genes alone. The cyclin-dependent kinase inhibitor, CDKN1C (p57), biology by interacting with a range of different signalling path- is methylated in half of adult ALL patients but has no prognostic ways including the epidermal growth factor receptor signalling significance if examined in isolation. If two other members of this pathway,12,13 b-catenin localisation and signalling11,14 and cell cycle pathway, TP73 (p73) and CDKN2B (p15), are also signalling via transforming growth factor b.13,15 In T-cell ALL, examined with CDKN1C then methylation of this triad of genes chromosomal deletion of PTPN2 and inactivating mutations of imparts a poor prognosis.3 This observation has been validated at PTPRC have both been implicated in aberrant Janus kinase/signal a functional level with the protein expression of p57, p15 and p73 transducer and activator of transcription (JAK-STAT) signalling in adult ALL predicting prognosis that is independent of the through JAK1, promoting leukaemia cell proliferation and methylation status of the individual genes alone.4 Methylation of survival.16,17 these three cell cycle genes is infrequent during childhood ALL Adult patients with chemotherapy refractory ALL represent a suggesting that some epigenetic lesions may have specificity for particularly high-risk group for whom novel drug targets and the adult disease.5 therapies are required. A previous genome-wide study of DNA Kinase signalling pathways that regulate cell growth by methylation changes in patients with ALL refractory to the hyper- phosphorylation of cytoplasmic proteins may represent functional CVAD (cyclophosphamide, vincristine, adriamycin and dexametha- targets of gene silencing. Normal signals promoting blood cell sone alternating with methotrexate and cytosine arabinoside)

1Department of Haematology, Royal North Shore Hospital, Pathology North, Sydney, Australia; 2Northern Blood Research Centre, Kolling Institute of Medical Research, University of Sydney, Sydney, Australia and 3Department of Leukemia, University of Texas, MD Anderson Cancer Center, Houston, TX, USA. Correspondence: Dr WS Stevenson, Department of Haematology, Royal North Shore Hospital, Pathology North, Pacific Highway, St Leonards, Sydney, New South Wales 2065, Australia. E-mail: [email protected] Received 3 April 2013; revised 20 August 2013; accepted 3 September 2013; accepted article preview online 18 September 2013; advance online publication, 11 October 2013 Phosphatase genes in ALL WS Stevenson et al 788 chemotherapy protocol identified 404 methylated genes from (Becton Dickinson, Franklin Lakes, NJ, USA) and green fluorescent protein- 17 000 promoter-specific transcripts.18 These methylated promoters positive cells with 498% purity obtained. were spread across all chromosomes and were clustered in networks involved with cell growth and differentiation, metabolism, Cell proliferation assays and colony formation cell death, DNA replication, gene regulation and signal transduc- The MTT (3-(Deimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay tion. Membrane-bound tyrosine phosphatases were identified as 4 was performed with 1  10 of either PTPRK-transduced cells or empty hypermethylated during this screen, prompting the current study vector control cells incubated in a 96-well plate for 24–96 h. Blue crystals and the finding that this family of genes is frequently methylated produced by the viable cells present were solubilised by the addition in ALL. Further, studies concerning the function of one member of 100 ml dimethyl sulphoxide and readings were taken at 570 nM using of this family, PTPRK, indicate that this gene does modulate the a Biotek Powerwave XS plate reader (BioTech, Winooski, VT, USA). For phosphorylation status of intracellular signalling proteins and colony formation, 500 cells were plated separately in dishes containing demonstrates tumour suppressor function in ALL. 1.5 ml of Methocult H4330 semisolid medium without cytokines (Stem Cell Technologies, Vancouver, Canada). Dishes were incubated at 37 1Cin 5% CO2 for 7 days. Colonies were then enumerated by two independent observers under phase contrast microscopy at 4 magnification. Repre- MATERIALS AND METHODS  sentative images were acquired using an Olympus CKX41 microscope with Cell lines and patient samples an Olympus DP21 camera attachment at  40 magnification. Leukaemia cell lines were obtained from the American Type Culture Collection and were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Gemini Bio-Products, Flow cytometry West Sacromento, CA, USA) and penicillin-streptomycin (Invitrogen). Apoptosis was measured by incubation of cells with 60 nM DilC1(5) Cell suspensions from bone marrow aspiration specimens from patients (Invitrogen) for 15 min, and 5 mg/ml propidium iodide was added with leukaemia were obtained before any therapy and were stored immediately before analysis. Data acquisition was performed using a at established tissue banks at MD Anderson Cancer Center (MDACC) fluorescence-activated cell sorting Calibur (Becton Dickinson) instrument. following institutional guidelines. Cells that were positive for DilC1 (5) and negative for propidium iodide were considered viable. Phosphorylated STAT3 (p-STAT3) and phosphory- lated STAT5 (p-STAT5) were measured using intracellular flow cytometry. Pyrosequencing of CpG islands Cells were fixed with 1% paraformaldehyde for 30 min at 4 1C then washed DNA was modified by bisulfite treatment and pyrosequencing performed in a phosphate-buffered saline buffer containing 0.5% bovine serum 19,20 using two PCR reactions as previously described. In the first PCR albumin and 0.1% saponin and permeabilised by incubation in 100% reaction, bisulfite-treated DNA was amplified by forward and reverse methanol for at least 2 h at À 20 1C. Cells were then stained for 2 h in the primers (listed in Supplementary Table 1) designed to analyse three to four dark at room temperature using antibodies against p-STAT3 (S727) and CpG sites in the promoter region using Pyrosequencing Assay Design p-STAT5 (Y694) conjugated to fluorescein isothiocyanate and Alexa Fluor- Software from Biotage (Uppsala, Sweden). In the second reaction, a reverse 647 respectively (Becton Dickinson). primer with a 20-bp universal sequence was included in the reaction with a biotinylated universal primer. The biotin-labelled PCR product was then captured by Streptavidin Sepharose HP (Amersham Biosciences, Uppsala, Western blotting Sweden) and purified in a Pyrosequencing Vacuum Prep Tool (Pyro- Cells (1  106) were lysed in sample buffer and run on 4–12% gradient SDS- sequencing Inc, Westborough, MA, USA). A sequencing primer was then PAGE (Invitrogen). Proteins were transferred to polyvinylidene difluoride annealed to the single-stranded product and pyrosequencing performed membrane using the iblot rapid transfer system (Invitrogen) and blocked using the PSQ HS96 Pyrosequencing system. A gene was considered as in 0.5% milk/Tris-buffered saline with Tween-20 (TBST). Primary antibodies methylated if the methylation density of the promoter was X20%. to phospho-Erk1/2 (T202/Y204), Erk1/2, phospho-Akt (S473), Akt and b-Actin (all from Cell Signaling Technology) were applied to the blot Real-time reverse transcription-PCR overnight. Following washing in TBST a secondary anti-rabbit HRP antibody (BioLegend, San Diego, CA, USA) was applied to the blot for 1 h at Total RNA was isolated from cells with Trizol reagent (Invitrogen) and room temperature. Proteins were then visualised using WesternBright reverse transcribed with Superscript II reverse transcriptase (Invitrogen). chemiluminescent substrates (Advansta, Menlo Park, CA, USA) and a Taqman probes (PTPRG Hs00892788_m1, PTPRK Hs00267788_m1, PTPRM ChemiDoc XRS þ molecular biology imager (Bio-Rad, Hercules, CA, USA). Hs00267809_m1, PTPRO Hs00243097_m1 and GAPDH) were used in real- time PCR reactions in triplicate on the ABI Prism 7000 Detection System (Applied Biosystems, Foster City, CA, USA) to determine relative gene Statistical analysis expression according to the manufacturer’s instructions. Comparison between two groups were made by Student’s t-test. Multi- variate Cox proportional hazard modelling was performed with Stata12.1. Treatment of leukaemia cell lines Cell lines were cultured in media supplemented with 3 mmol/l of 5-aza- 20-deoxycytidine (DAC; Eisai, Woodcliff, NJ, USA) for 4 days, or 3 mmol/l of RESULTS 5-aza-20-deoxycytidine for 3 days and 500 nmol/l suberolyanilide hydroxamic Leukaemia cell lines demonstrate CpG island methylation in the acid (SAHA; Merck, Whitehouse station, NJ, USA) for 1 day. Raji and Jurkat promoter regions of membrane-bound tyrosine phosphatase genes cells were treated with 0.001–50 mmol/l of cytosine arabinoside (AraC; Sigma, Genome-wide analysis of promoter CpG island methylation St Louis, MO, USA) or dimethyl sulphoxide alone as a vehicle control for in bone marrow samples from patients with ALL refractory 2days. to standard chemotherapy identified a number of aberrantly methylated membrane-bound tyrosine phosphatase genes.18 Lentivirus constructs and gene transduction Seven genes in this family (PTPRA, PTPRG, PTPRJ, PTPRK, PTPRM, Human full-length PTPRK (a kind gift from GJ Fisher, Ann Arbor, MI, USA) PTPRO and PTPRU) were initially selected for further study based was inserted into a bicistronic lentiviral expression vector (pLVX-IRES- on the presence of promoter methylation on CpG array18 and ZsGreen, Clonetech, Mountain View, CA, USA). Lentivirus production was evidence of predicted gene expression in haemopoietic cells and conducted by co-transfection of 293T cells with the lentiviral expression leukaemia.21–24 The presence of promoter methylation in these vector carrying PTPRK or an empty vector as a control with the envelope (pMD2.G, Addgene, Cambridge, MA, USA) and packaging plasmids seven genes was then examined in 22 leukaemia cell lines by (psPAX2, Addgene). Raji and Jurkat cells were transduced by inoculation pyrosequencing after a bisulfite reaction had been performed with suspension from 293T cells in Dulbecco’s Modified Eagle medium (Figure 1). In this panel of genes screened, PTPRG, PTPRK, PTPRM with polybrene (10 mg/ml) and protamine (10 mg/ml). Transduced cells were and PTPRO all demonstrated significant levels of methylation in sorted on a fluorescence-activated cell sorting vantage SE cell sorter the majority of leukaemia cells tested. PTPRO was methylated in all

Leukemia (2014) 787 – 793 & 2014 Macmillan Publishers Limited Phosphatase genes in ALL WS Stevenson et al 789

Figure 1. The CpG islands present within the promoter of seven members of the membrane-bound tyrosine phosphatase gene family show variable amounts of methylation in different leukaemia cell lines. Quantitation of methylation was performed by pyrosequencing and is presented as the mean percentage across the total number of CpG dinucleotides within the region (0–19% normal to low methylation in white, 20–40% intermediate methylation in grey and 440% is highly methylated in black).

Figure 2. Methylation of the CpG islands present within the promoter of PTPRG, PTPRK, PTPRM and PTPRO shows that hypermethylation is frequently observed in ALL and high-grade B-cell lymphoma samples (0–19% normal to low methylation in white, 20–40% intermediate methylation in grey and 440% is highly methylated in black).

cell lines examined, whereas PTPRG, PTPRK and PTPRM had The 57 ALL samples studied were obtained from adults aged variable methylation patterns in both myeloid and lymphoid cells. 17–79 years (median 38 years) that had predominantly B-lineage disease (91%). Methylation changes in phosphatase genes were observed in both male and female patients and throughout the Methylation of PTPRG, PTPRK, PTPRM and PTPRO is frequent in range of ages studied (Supplementary Table 2). Methylation primary ALL samples and PTPRK methylation correlates with changes were observed in patients with high- and low risk disease. overall survival Hypermethylation was observed in individuals with high and The presence of promoter methylation in PTPRG, PTPRK, PTPRM low presenting white cell counts and in normal and abnormal and PTPRO was then examined in a cohort of 57 primary ALL karyotypes with no correlation to specific karyotypic abnormalities samples using the same pyrosequencing methodology (Figure 2). (Supplementary Table 2). Significant levels of methylation were observed in the promoter All patients with ALL received hyper-CVAD-based chem- of PTPRG in 63% of ALL samples, PTPRK in 47% of samples, PTPRM otherapy at MDACC. To examine whether gene methylation had in 64% of samples and PTPRO in 54% of samples. Many of the an impact on survival, Cox proportional hazard modelling samples demonstrated promoter methylation of multiple phos- was performed on the four phosphatase genes with methylation phatase genes with at least two of the four gene promoters studied as a continuous variable with age in ALL. In this methylated in 72% of individuals with ALL. Methylation was also multivariate model, methylation of PTPRK (hazard ratio 1.015 per frequently observed in high-grade B-cell lymphoma in leukaemic 1% methylation, 95% confidence interval 1.000–1.028) and age phase (Burkitt and Burkitt-like lymphoma) across the four gene (hazard ratio 1.052, 95% confidence interval 1.027–1.28) had a loci with 73% of phosphatase promoters demonstrating methyla- significant impact on overall survival (Figure 3). tion greater than 20%. In contrast, methylation of these genes was rare in samples from patients with chronic lymphocytic leukaemia (7%, n ¼ 14) and acute myeloid leukaemia or myelodysplastic The PTPRK, PTPRM, PTPRG and PTPRO loci are epigenetically syndromes (4%, n ¼ 28) and normal samples without haematolo- regulated in ALL gical disease (2%, n ¼ 11). This pattern of methylation of the To determine whether the expression of selected phosphatase selected phosphatase genes suggests specificity for high-grade genes was modified by alterations in CpG island methylation, the lymphoid malignancy. expression of phosphatase transcripts was examined in lymphoid

& 2014 Macmillan Publishers Limited Leukemia (2014) 787 – 793 Phosphatase genes in ALL WS Stevenson et al 790

Figure 3. Cox proportional hazards regression function for PTPRK promoter methylation modelled at 10, 40 and 70% in ALL.

cell lines by real-time PCR. In Molt4 cells, the PTPRO promoter is methylated but the PTPRG, PTPRK and PTPRM loci show no significant evidence of methylation (Figure 1). This was consistent with baseline measurements of gene expression that demonstrate relatively high levels of PTPRK and PTPRM expression and inter- mediate expression of PTPRG compared with PTPRO expression (Figure 4a). These same four phosphatase genes in Raji cells are highly methylated (Figure 1) and demonstrate very low or unmeasurable transcript expression consistent with methylation- associated gene silencing (Figure 4b). To determine whether these Figure 4. Expression of phosphatase genes in ALL cell lines before loci are modified by epigenetic drug exposure, gene transcript and after epigenetic drug exposure. (a) The Molt4 cell line showed expression was measured in these two cell lines after DAC or DAC relatively high expression of PTPRK and PTPRM, intermediate expres- plus SAHA treatment. In Molt4 cells, DAC or DAC plus SAHA sion of PTPRG and low PTPRO expression before drug treatment treatment did not cause consistent changes in gene expression (white). There is no consistent change in phosphatase gene across the four gene loci examined (Figure 4a). In contrast, expression after DAC (grey) or DAC plus SAHA (black) treatment. treatment with DAC and DAC in combination with SAHA produced (b) Raji cells had low or unmeasureable expression of PTPRG, PTPRK, PTPRM and PTPRO gene transcript before drug exposure (white). consistent increases in phosphatase gene expression in Raji cells Expression of all four genes increased after exposure to DAC (grey) (Figure 4b). After the treatment of Raji cells with DAC, promoter and then further increased after exposure to DAC plus SAHA (black). methylation as measured by pyrosequencing, was reduced by an Gene expression was measured by real-time PCR with phosphatase average of 46% across the PTPRG, PTPRK, PTPRM and PTPRO loci gene transcript expressed relative to the GAPDH gene. All results and this was associated with a marked increase in gene expression represent mean±s.d. from three experiments. of all four phosphatase gene transcripts (Figure 4b). This increase in expression was further enhanced upon the addition of SAHA with all gene loci experiencing a minimum of a sixfold increase in 2 days (Figure 5a). After day 2, control cells proliferated faster that phosphatase gene expression over baseline. These data suggest PTPRK-expresssing cells and this difference was significant by the that gene transcription at these four gene loci in ALL cells is fourth day of culture with 31% less cells present in the cultures related to the relative methylation of the gene promoter and containing PTPRK transcripts (P 0.05). Similar results were observed when these loci are silenced by promoter methylation, gene o for Jurkat cells in which control cells proliferated faster than PTPRK- expression is inducible with epigenetic drug exposure. transduced cells after 24 h (Po0.05). To further investigate growth characteristics of these cells, PTPRK and control Raji cells were seeded Expression of PTPRK in leukaemia cells inhibits cell proliferation into semisolid medium and the number of colonies enumerated after PTPRK was selected for further study as this gene has been 7days.PTPRK-transduced cells formed fewer colonies in vitro implicated in the pathogenesis of EBV-related Hodgkin lymphoma,15 compared with control cells (Po0.05, Figure 5b) and the colonies and methylation of this gene locus appears to be associated with expressing PTPRK typically displayed a more dispersed colony survival in multivariate modelling of this ALL cohort. To investigate morphology compared with the predominantly tightly packed the biological significance of silencing PTPRK in lymphoid leukaemia colonies observed in control cells (Figure 5b). cells, full-length PTPRK was expressed via lentiviral transduction in PTPRK has an intracellular phosphatase domain. To determine Raji and Jurkat cell lines. Cells containing PTPRK or an empty vector whether these observed changes in growth were associated were isolated after transduction by fluorescence-activated cell sorting with changes in the phosphorylation status of signalling proteins, on green fluorescent protein expression and expression of PTPRK the levels of p-STAT3 and p-STAT5 were measured by intracellular measured by real-time PCR. In Raji cells, the PTPRK locus is heavily flow cytometry (Figure 6a) and p-Erk1/2 and p-Akt measured methylated (94%, Figure 1) and transcriptionally silent (Figure 4b, by western blotting (Figure 6b). Mean fluorescence intensity for PTPRK relative gene expression 2.4 Â 10 À 7±2 Â 10 À 7). After trans- p-STAT3 and p-STAT5 were lower in PTPRK-transduced cells duction, green fluorescent protein-positive Raji cells expressed PTPRK (mean fluorescence intensity p-STAT3 36.0±24.8 and p-STAT5 at similar levels to those observed in Molt4 cells in which the locus is 31.0±20.9) compared with control cells (mean fluorescence not methylated (Raji PTPRK expression 0.017±0.001 compared with intensity p-STAT3 63.4±45.6, Po0.05; p-STAT5 89.8±26.2, Molt4 0.013±0.008). Jurkat cells have a moderately methylated Po0.05). PTPRK expression in Raji cells also resulted in a significant PTPRK locus (27%, Figure 1) and increased PTPRK expression by downregulation of Erk1/2 activity and complete abrogation of Akt approximately fourfold after transduction (PTPRK expressing cells activity without any significant change in the expression of either 0.015±0.0006 compared with empty vector 0.004±0.0003). total protein (Figure 6b). Taken together, these observations Raji cell proliferation measured by the MTT assay was similar for suggest that PTPRK may act as a functional phosphatase in PTPRK-transduced cells and the empty vector control cells for B-lymphoid cells.

Leukemia (2014) 787 – 793 & 2014 Macmillan Publishers Limited Phosphatase genes in ALL WS Stevenson et al 791

Figure 5. Expression of PTPRK inhibits cell proliferation. (a) Prolifera- tion of Raji and Jurkat cells transduced with PTPRK (black) or an empty vector control (white) were measured by the MTT assay over 4 days in culture. (b) Control Raji cells generated 73% more cell Figure 7. Apoptosis of Raji (a) and Jurkat (b) cells measured by flow colonies than Raji cells transduced with PTPRK after 7 days culture in cytometry after 48 h exposure to increasing concentration of AraC in semisolid media (Po0.05). Representative images illustrate the culture. The proportion of cells undergoing apoptosis was greater in differences in the colonies formed; the control Raji cells (top) the cells transduced with PTPRK (black) compared with cells generated dense colonies, whereas Raji cells transduced with PTPRK transduced with an empty vector (white). Control cells were not showed a more dispersed colony morphology. exposed to drug treatment. All results represent mean±s.d. from three experiments.

Expression of PTPRK restores sensitivity to cytotoxic drug exposure in ALL cells Initial promoter methylation screening was performed on samples from patients with ALL that displayed resistance to standard chemotherapy.18 To determine whether restoration of phosphatase function alters sensitivity of ALL cells to cytotoxic drug treatment, control and PTPRK-transduced Raji and Jurkat cells were cultured in increasing concentrations of AraC and apoptosis of cells measured by flow cytometry at 48 h (Figure 7). PTPRK-transduced Raji and Jurkat cells were significantly more sensitive to AraC than untreated control cells. In the measured dose range, PTPRK expressing Raji cells demonstrated increased apoptosis compared with empty vector control cells at each AraC concentration examined (Po0.01 for all comparisons). PTPRK-transduced Jurkat cells also demonstrated increased apoptosis after exposure to AraC in the concentration range of 50–500 nM (Po0.05). Data demonstrating the antiproliferative and pro-apoptotic changes in Raji and Jurkat cells transduced with PTPRK suggest that this membrane-bound phosphatase exhibits tumour suppressor functions in both B and T-lineage lymphoid cells.

DISCUSSION In normal cell homoeostasis, the phosphorylation status of intra- cellular signalling pathways is regulated by the balanced action Figure 6. Expression of PTPRK in Raji cells alters phosphatase of kinases promoting phosphorylation and protein phosphatases signalling. (a) The presence of PTPRK transcript significantly inhibiting phosphorylation. Aberrant tyrosine kinase activity downregulated levels of phosphorylated forms of STAT3 and causing excessive activation of signalling pathways is frequently STAT5 (Po0.05). Results are presented as fold change in mean fluorescence intensity with control cells (white) normalised to a observed in many different types of leukaemia with the best value of 1. (b) Western blotting demonstrates decreased levels of characterised being the aberrant BCR-ABL1 kinase in chronic phosphorylated forms of Erk1/2 and Akt after expression of PTPRK myeloid leukaemia and ALL causing excess activation of down- in Raji cells. stream pathways including JAK-STAT and the mitogen-activated

& 2014 Macmillan Publishers Limited Leukemia (2014) 787 – 793 Phosphatase genes in ALL WS Stevenson et al 792 protein kinase pathway. In contrast to the tyrosine kinases, much but it is possible they also promote leukaemogenesis and less is understood about phosphatase function in leukaemia, chemotherapy refractoriness through similar pathways. although it is likely they represent important genes in onco- Methylation defects associated with functional silencing of the genesis. At present, SHP2 is the only well-characterised phospha- PTPRO locus have been identified in childhood ALL.28,29 These tase enzyme with oncogenic potential with mutations described abnormalities are particularly observed at relapse perhaps in specific myeloid leukaemia syndromes.25 There are a large suggesting a role for this gene in the development of resistance numbers of phosphatase enzymes6 and there is an increasing to standard chemotherapy agents. In the K562 myeloid cell line, evidence that functional silencing of phosphatase genes may the PTPRO protein interacts with the activated kinase BCR-ABL1 cooperate with oncogenic kinase signalling in leukaemogenesis causing decreased phosphorylation of the downstream target and promote dysregulated cell growth. In T-cell ALL, loss of proteins Crkl and STAT5.30 Expression of active PTPRO decreased function of PTPN2 via chromosomal deletion has been implicated the proliferation of these leukaemia cells and inhibited the ability in aberrant signalling through JAK1 and ABL1 promoting of the cells to form colonies in semisolid media suggesting that leukaemia cell proliferation and survival.16 This appears to be a this phosphatase gene may modulate anchorage-dependent cell co-operative mutation as it occurs in close association with growth. This change in colony formation in myeloid cells is similar mutant kinase function in the NUP214-ABL1 translocation and with to the altered colony morphology observed in this report with activating mutations in JAK1. In vitro experiments demonstrate PTPRK expression in Raji cells. The typical tight clusters seen in that reduction of functional Ptpn2 in Ba/F3 cells facilitates the cells where PTPRK was silenced were more dispersed after the transformation of cells to cytokine-independent growth when phosphatase gene was introduced into the cell. This observation is co-transfected with various JAK1 mutations. Transformed cells consistent with structural modelling of the PTPRM protein that exhibit increased JAK/STAT signalling and this cell growth was suggests this family of proteins may act as a distance gauge reversed by pharmacological JAK1 inhibition suggesting specificity between cells that regulate growth when cells are in proximity.7 for this signalling pathway. Similarly, loss of function mutations in To our knowledge, there have been no functional studies of PTPRC occur in association with activating mutations of IL-7R, JAK1 PTPRM in cancer cells to date; however, there is a suggestion and LCK in rare cases of T-cell ALL.17 This functional silencing of of functional significance for PTPRM in clinical data from gene PTPRC function is associated with increased signalling through the expression profiles in high-risk cases of childhood ALL. In a poor JAK/STAT pathway and cell proliferation. Thus, functional silencing of risk cohort of older children with high white cell counts, increased PTPN2 and PTPRC appear to be relatively rare events in T-cell ALL but expression of PTPRM along with four other non-phosphatase both appear to have the similar functional outcomes with increased genes in an expression signature was associated with a better JAK/STAT signalling and associated increased cell proliferation. overall outcome following chemotherapy.31 This suggests that loss In contrast to the relatively rare occurrence of chromosomal of function of the PTPRM gene, perhaps via methylation-induced deletion of PTPN2 and mutation of PTPRC in T-lineage ALL, silencing, may be associated with poorer outcome in this group of methylation of PTPRG, PTPRK, PTPRM and PTPRO are common paediatric ALL patients. events in adult ALL. In this cohort, at least two of these genes Chemotherapy refractory ALL is a high-risk disease that requires were methylated in 73% of cases suggesting a relatively high novel therapies. Methylation profiling has identified membrane- prevalence of methylation in this gene family compared with bound phosphatases as a family of genes that may promote other methylated genes studied in ALL.3,26,27 This promoter leukaemia progression and drug resistance in this setting. Methyla- methylation was associated with gene silencing in ALL cell lines tion defects in PTPRG, PTPRK, PTPRM and PTPRO are common and as the degree of methylation correlated well with phosphatase potentially reversible with epigenetic drug therapy. In this study, one transcript expression. Importantly, this functional gene silencing of these genes, PTPRK, does have antiproliferative and pro-apoptotic was reversible with epigenetic drug therapy. function in ALL cells and may represent a novel target for epigenetic Introduction of PTPRK into lymphoid leukaemia cells where it modulation. In addition, PTPRK interacts with various signalling had been silenced by methylation produced biological changes proteins and downstream effects of PTPRK silencing may identify consistent with its proposed function as a tumour suppressor pathways for pharmacological intervention. gene. After PTPRK transduction into Raji and Jurkat cells, the proliferation rate in culture slowed and cytotoxic chemotherapy sensitivity was increased. These results were consistent with the CONFLICT OF INTEREST role of PTPRK in other cancer models. In breast cancer cells The authors declare no conflict of interest. overexpressing HER2, PTPRK protein is suppressed compared with normal mammary cells, and this loss of phosphatase function is associated with accelerated cell cycle progression and enhanced ACKNOWLEDGEMENTS responses to epidermal growth factor with increased epidermal WSS was supported by a Cancer Institute NSW International Clinical and Research 10 growth factor receptor phosphorylation. Similar changes are Fellowship. noted in keratinocytes, where PTPRK dephosphorylates the epidermal growth factor receptor.12,13 PTPRK is also implicated 11 14 in cell to cell adhesion in both melanoma and lung cancer cells REFERENCES with evidence suggesting that PTPRK binds b-catenin and alters its 1 Garcia-Manero G, Daniel J, Smith TL, Kornblau SM, Lee MS, Kantarjian HM et al. distribution within the cell. 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