Deletion of chromosome 20q (del(20q)) is a common chromosomal abnormality associated with myeloid neoplasms including myeloproliferative neoplasms (MPNs), myelodysplastic syndrome, myelodysplastic syndrome/MPN overlap disorders and acute myeloid leukemia.1, 2 The del(20q) lesion is present in patients with myelofibrosis (MF) at a high frequency (23%) and is thus considered to be one of the most frequent cytogenetic abnormalities in MF.3 However, the identity of the target tumor suppressor gene(s) within 20q involved in the pathogenesis of MF and other myeloid neoplasms remains elusive.
The PTPN1 gene encoding protein tyrosine phosphatase non-receptor type 1 (PTPN1; also known as PTP1B) is located on human chromosome 20q13.1-q13.2. Both oncogenic and tumor suppressor functions for PTPN1 have been suggested. PTPN1 is overexpressed in breast cancer and deletion of Ptpn1 inhibits ErbB2-induced breast tumorigenesis in mice.4 Somatic loss-of-function mutations in PTPN1 have been associated with mediastinal B-cell lymphoma and Hodgkin lymphoma.5 Moreover, ablation of Ptpn1 accelerates B-cell lymphomagenesis and decreases survival in p53-null mice.6 PTPN1 can negatively regulate Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling,7 which is frequently found to be activated in MPNs.8 The common occurrence of del(20q) involving the PTPN1 locus in MPN and the negative regulatory role of the corresponding phosphatase in JAK/STAT signaling led us to hypothesize that PTPN1 is a potential tumor suppressor gene within 20q involved in MPN.
Using cytogenetic and single-nucleotide polymorphism array-based karyotyping, we detected the presence of 20q deletion and assessed the status of the PTPN1 gene locus in a cohort of 916 patients with myeloid malignancies (Supplementary Tables 1 and 2). The del(20q) involving PTPN1 deletion was identified in 11/135 cases (8%) of secondary acute myeloid leukemia, 1/120 cases (1%) of primary acute myeloid leukemia, 25/147 cases (17%) of MPN, 6/135 cases (4%) of myelodysplastic syndrome/MPN overlap disorders and 47/379 cases (12%) of myelodysplastic syndrome (Supplementary Figure 1). Thus, del(20q) involving PTPN1 deletion was more commonly found in MPN in comparison with other myeloid neoplasms. We also determined that 22% of MF patients (25/116 cases) in our cohort had del(20q) with PTPN1 deletion and 53% of MF patients (62/116 cases) were positive for the JAK2V617F mutation, 10% (12/116 cases) were positive for CALR mutation and 7% (8/116 cases) were positive for MPL mutation (Figure 1a). Among the 25 MF patients who had del(20q) with PTPN1 deletion, 17 were also positive for the JAK2V617F mutation, 2 patients were positive for CALR mutation, whereas 6 other del(20q) patients did not have any of the known MPN-associated mutations (Supplementary Figure 2). Interestingly, six cases with various myeloid neoplasms had somatic microdeletion of the PTPN1 locus (data not shown), but none of these cases had MF and coexisting MPN driver mutations. Fluorescence in situ hybridization using a PTPN1-specific probe confirmed the presence of monoallelic deletion of PTPN1 in MPN/MF bone marrow (BM) cells (Supplementary Figure 3). Overall, these data suggest that PTPN1 deletion occurs in myeloid malignancies and is more frequently present in MPN/MF.
To test the effects of Ptpn1 deletion on hematopoiesis, we crossed Ptpn1-floxed (Ptpn1fl/fl) mice9 with Mx1-Cre mice.10 At 4 weeks after birth, control (Ptpn1fl/fl; no Cre) and Mx1-Cre;Ptpn1fl/fl (referred as Ptpn1cKO) mice were injected with three doses of 2 μg polyinosine–polycytosine (pI–pC) to induce Ptpn1 deletion in the mice hematopoietic compartments. As shown in Figure 1b, Ptpn1 was efficiently deleted in Ptpn1cKO mice BM upon pI–pC induction. Compared with control animals, Ptpn1cKO mice exhibited significantly increased white blood cells and neutrophils in their peripheral blood (Figures 1c and d). However, no significant difference was observed in red blood cell and platelet counts between control and Ptpn1cKO mice (data not shown). Deletion of Ptpn1 also resulted in enlargement of spleen size/weight in Ptpn1cKO mice (Figure 1e), suggesting that loss of Ptpn1 induces extramedullary hematopoiesis. Mice with heterozygous Ptpn1 deletion, however, did not exhibit any significant phenotype (data not shown).
Flow cytometric analyses showed significant expansion of myeloid (Gr-1+/Mac-1+) precursors in the BM and spleens of Ptpn1cKO mice compared with control animals (Figure 1f and Supplementary Figure 4). Megakaryocytic (CD41+/CD61+) precursors were also significantly increased in the spleens of Ptpn1cKO mice (Figure 1f and Supplementary Figure 4). Flow cytometric analyses also revealed significant increases in the numbers of LSK cells (Lin−Sca1+c-kit+) and its subsets including long-term hematopoietic stem cell and short-term hematopoietic stem cell in Ptpn1cKO mice compared with control animals (Figure 1g and Supplementary Figure 5a). Total number of common myeloid progenitors, granulocyte–macrophage progenitors and megakaryocyte–erythroid progenitors were also significantly increased in Ptpn1cKO mice (Figure 1h and Supplementary Figure 5b). Hematopoietic progenitor colony assays showed significant increases in colony-forming unit-granulocyte and monocyte colonies in the BM and spleens of Ptpn1cKO mice compared with control animals (Figure 1i). We also observed significantly increased numbers of colony-forming unit-megakaryocytic colonies in the BM of Ptpn1cKO mice (Figure 1j). Taken together, these data suggest that loss of Ptpn1 promotes the expansion of HSC and increases myeloid/megakaryocytic differentiation.
Histopathologic analyses of the BM sections from Ptpn1cKO mice showed increased granulocytic precursors with decreased erythroid precursors and mildly increased megakaryocytes (Figure 1k). Spleen sections from Ptpn1cKO mice exhibited high myeloid to erythroid ratios and increased megakaryocytes. Reticulin staining demonstrated fibrosis (grade 2) in the BM and spleens of Ptpn1cKO mice at 52 weeks after pI–pC induction, whereas control animals did not exhibit fibrosis at that age (Figure 1k). Taken together, these results suggest that deletion of Ptpn1 induces an MPN-like phenotype, which progresses to MF over time.
To determine whether the MPN/MF-like phenotype observed in Ptpn1cKO mice was cell intrinsic, we transplanted BM cells from control and Ptpn1cKO mice into lethally irradiated C57BL/6 wild-type recipient mice as outlined in Figure 2a. Compared with recipients of control BM (bone marrow transplantation (BMT)-Control), transplanted animals receiving Ptpn1cKO mice BM (BMT-Ptpn1cKO) exhibited significantly increased white blood cell and neutrophil counts in their peripheral blood (Figures 2b and c). Histologic analysis confirmed the development of fibrosis in the BM and spleens of BMT-Ptpn1cKO mice at 18 weeks after transplantation (Figure 2d). These results suggest that the observed effect of Ptpn1 deletion in the development of MPN/MF was cell intrinsic.
To evaluate the effects of Ptpn1 deletion in hematopoietic stem cell reconstitution, we performed competitive repopulation assays as outlined in Figure 2e. The frequency and total number of donor-derived (CD45.2+) LSK cells was significantly increased in the BM and spleens of recipients of Ptpn1cKO BM compared with recipients of control BM (Supplementary Figure 6a and Figure 2f). We also observed significantly greater percentages and increased numbers of donor-derived Gr-1+ myeloid cells in the BM and spleens of recipients of Ptpn1cKO BM compared with control BM (Supplementary Figure 6b and Figure 2g). Taken together, these results suggest that loss of Ptpn1 increases the repopulating capacity of HSC and promotes expansion of myeloid lineage cells.
To investigate the effects of Ptpn1 deletion on hematopoietic signaling, we performed immunoblot analysis on total BM cell extracts from control and PTPN1cKO mice. We observed markedly increased phosphorylation of STAT5, AKT and ERK in PTPN1cKO BM compared with control BM cells (Figure 2h). We also observed enhanced phosphorylation of STAT5, AKT and ERK upon PTPN1 knockdown in human peripheral blood mononuclear cells as well as in murine hematopoietic Ba/F3 cells (Supplementary Figures 7 and 8a). In addition, PTPN1 knockdown significantly increased proliferation in Ba/F3 cells (Supplementary Figure 8b). Constitutive phosphorylation of JAK2/STAT5, AKT and ERK has been frequently observed in MPN/MF.8 Moreover, STAT5, AKT and/or ERK signaling pathways have been shown to have important roles in the pathogenesis of MPN/MF.11, 12, 13 Therefore, it is plausible that enhanced phosphorylation/activation of STAT5, AKT and ERK caused by Ptpn1 deletion is likely to contribute to the development of MPN/MF-like phenotype in Ptpn1cKO mice.
Consistent with previous reports suggesting that only heterozygous deletions of 20q are associated with myeloid malignancies,1, 2, 14 we observed monoallelic deletion of PTPN1 in MPN/MF. Monoallelic deletion does not always correspond to 50% expression of the gene, as has been shown for other 20q target genes, L3MBTL1, SGK2 and MYBL2, where heterozygous deletion results in almost complete loss or ~80% reduction in expression of these genes in patients.14, 15 We identified monoallelic PTPN1 deletion in MPN patient-derived UKE-1 cell line carrying del(20q) (Supplementary Figure 9a). We observed more than 90% reduction in PTPN1 protein expression in UKE-1 cell line compared with MPN patient-derived SET-2 cell line expressing wild-type PTPN1 (Supplementary Figures 9a and b). Mechanisms involving aberrant methylation and/or elevated expression of microRNAs have been suggested to cause further reduction in expression of tumor suppressor genes.14, 15 Future studies will determine if any such mechanisms exist in the regulation of PTPN1 expression in patients with MPN/MF.
In summary, our data suggest a tumor suppressor role for PTPN1 in MPN/MF. However, we cannot exclude the existence of additional target genes in 20q involved in the pathogenesis of MPN/MF. It remains possible that haploinsufficiency of other target genes within 20q or additional genetic lesions outside of 20q may cooperate with PTPN1 deletion in the pathogenesis of MPN/MF. We observed coexistence of PTPN1 deletion and JAK2V617F mutation in significant cases of MF. Our preliminary data show that heterozygous or homozygous deletion of PTPN1 increases the severity of MPN in Jak2V617F-knock-in mice. Future studies will determine whether loss of PTPN1 cooperates with the JAK2V617F mutation in the pathogenesis of MPN.
Asimakopoulos FA, White NJ, Nacheva E, Green AR . Molecular analysis of chromosome 20q deletions associated with myeloproliferative disorders and myelodysplastic syndromes. Blood 1994; 84: 3086–3094.
Huh J, Tiu R, Gondek LP, O'Keefe CL, Jasek M, Makishima H et al. Characterization of chromosome arm 20q abnormalities in myeloid malignancies using genome-wide single nucleotide polymorphism array analysis. Genes Chromosomes Cancer 2010; 49: 390–399.
Wassie E, Finke C, Gangat N, Lasho TL, Pardanani A, Hanson CA et al. A compendium of cytogenetic abnormalities in myelofibrosis: molecular and phenotypic correlates in 826 patients. Br J Haematol 2015; 169: 71–76.
Julien SG, Dubé N, Read M, Penney J, Paquet M, Han Y et al. Protein tyrosine phosphatase 1B deficiency or inhibition delays ErbB2-induced mammary tumorigenesis and protects from lung metastasis. Nat Genet 2007; 39: 338–346.
Gunawardana J, Chan FC, Telenius A, Woolcock B, Kridel R, Tan KL et al. Recurrent somatic mutations of PTPN1 in primary mediastinal B cell lymphoma and Hodgkin lymphoma. Nat Genet 2014; 46: 329–335.
Dubé N, Bourdeau A, Heinonen KM, Cheng A, Loy AL, Tremblay ML . Genetic ablation of protein tyrosine phosphatase 1B accelerates lymphomagenesis of p53-null mice through the regulation of B-cell development. Cancer Res 2005; 65: 10088–10095.
Myers MP, Anderson JN, Cheng A, Tremblay ML, Horvath CM, Parisien JP et al. TYK2 and JAK2 are substrates of protein-tyrosine phosphatase 1B. J Biol Chem 2001; 276: 47771–47774.
Levine RL, Gilliland DG . Myeloproliferative disorders. Blood 2008; 112: 2190–2198.
Bence KK, Delibegovic M, Xue B, Gorgun CZ, Hotamisligil GS, Neel BG et al. Neuronal PTP1B regulates body weight, adiposity and leptin action. Nat Med 2006; 12: 917–924.
Kühn R, Schwenk F, Aguet M, Rajewsky K . Inducible gene targeting in mice. Science 1995; 269: 1427–1429.
Yan D, Hutchison RE, Mohi G . Critical requirement for Stat5 in a mouse model of polycythemia vera. Blood 2012; 119: 3539–3549.
Khan I, Huang Z, Wen Q, Stankiewicz MJ, Gilles L, Goldenson B et al. AKT is a therapeutic target in myeloproliferative neoplasms. Leukemia 2013; 27: 1882–1890.
Kong G, Wunderlich M, Yang D, Ranheim EA, Young KH, Wang J et al. Combined MEK and JAK inhibition abrogates murine myeloproliferative neoplasm. J Clin Invest 2014; 124: 2762–2773.
Aziz A, Baxter EJ, Edwards C, Cheong CY, Ito M, Bench A et al. Cooperativity of imprinted genes inactivated by acquired chromosome 20q deletions. J Clin Invest 2013; 123: 2169–2182.
Heinrichs S, Conover LF, Bueso-Ramos CE, Kilpivaara O, Stevenson K, Neuberg D et al. MYBL2 is a sub-haploinsufficient tumor suppressor gene in myeloid malignancy. Elife 2013; 2: e00825.
We thank Dr Benjamin Neel (Laura and Isaac Perlmutter Cancer Center, New York University) for providing the Ptpn1-floxed mouse and Ptpn1 cDNA construct. We also thank Dr Constance Stein (SUNY Upstate Medical University) for help with fluorescence in situ hybridization analysis. This work was supported by US National Institute of Health (NIH) Grants R21 CA187128 (to GM), R01 HL095685 (to GM), R01 HL082983 (to JPM), U54 RR019391 (to JPM) and R01 CA113972 (to JPM) and by a grant from the Leukemia and Lymphoma Society TRP 624-13 (to JPM). GM is a Scholar of the Leukemia and Lymphoma Society.
FJ performed research, analyzed data and wrote the manuscript; BP, TK, HM and BP collected and analyzed data; YY analyzed data; REH conducted histopathologic analysis and revised the manuscript; KKB provided study material; JPM provided clinical specimens, analyzed data and revised the manuscript; GM designed the research, analyzed data and wrote the manuscript.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Leukemia website
About this article
Cite this article
Jobe, F., Patel, B., Kuzmanovic, T. et al. Deletion of Ptpn1 induces myeloproliferative neoplasm. Leukemia 31, 1229–1234 (2017). https://doi.org/10.1038/leu.2017.31
Hematology/Oncology Clinics of North America (2020)
Hippo kinase loss contributes to del(20q) hematologic malignancies through chronic innate immune activation
Deficiency in Protein Tyrosine Phosphatase PTP1B Shortens Lifespan and Leads to Development of Acute Leukemia
Cancer Research (2018)