LETTERS TO THE EDITOR

ed to be shorter than the survival of patients with a high Loss of DEP-1 (Ptprj) promotes myeloproliferative PTPRJ expression level (P=0.07, Figure 1A). The data disease in FLT3-ITD acute myeloid leukemia from another study5 revealed a similar correlation (data not shown). In contrast, expression of PTPRJ did not corre- Acute myeloid leukemia (AML) is a heterogeneous late with overall survival in FLT3 WT AML (P=0.21) and group of diseases caused by oncogenic transformation of even showed improved survival for low PTPRJ expres- hematopoietic stem and progenitor cells. Mutations in sion (Figure 1B). the encoding the tyrosine kinase FLT3 leading to In order to directly study the role of PTPRJ in FLT3-ITD internal tandem duplications (ITDs) of sequence repre- induced disease, FLT3ITD/ITD mice6 were crossed with sent one of the most frequent genetic aberrations in Ptprj-/- mice.7 FLT3ITD/ITD Ptprj-/- mice showed an early onset human AML and are associated with a dismal prognosis.1 of disease and a shortened survival (Figure 2A). The sig- Herein, we show that genetic inactivation of the trans- nificantly reduced lifespan of FLT3-ITD mice was further membrane (receptor-like) protein-tyrosine phosphatase shortened in response to Ptprj inactivation. The (RPTP) PTPRJ/DEP-1 in FLT3-ITD knock-in mice ITD/ITD 6 ITD/ITD splenomegaly observed in FLT3 mice appeared less (FLT3 mice) promotes FLT3-ITD-mediated aberran- ITD/ITD -/- ITD/ITD -/- pronounced in FLT3 Ptprj mice (Figure 2B). While cies in hematopoiesis. FLT3 Ptprj mice were charac- ITD/ITD terized by enhanced extramedullary progenitor expan- the liver weight of FLT3 mice was not significantly elevated compared with WT mice, it was significantly sion, most notable in the spleen, increased colony form- ITD/ITD -/- ing unit-granulocyte monocyte (CFU-GM), and a more increased in the FLT3 Ptprj mice (Figure 2C). The severe myeloproliferative neoplasia (MPN). weight development during growth and the total weight Our previous work demonstrated that PTPRJ/DEP-1 of the adult animals did not differ among the respective functions as a negative regulator of physiological FLT3 genotypes (data not shown). signaling, but was partially inactivated in cells expressing Histological spleen sections demonstrated myeloid ITD/ITD -/- FLT3-ITD due to reversible oxidation by reactive oxygen infiltration in FLT3 mice, compared to WT or Ptprj ITD/ITD species (ROS) formation.2,3 Expression studies in human mice. This phenotype was more prominent in FLT3 -/- AML are also consistent with a possible contributing role Ptprj mice (Figure 2E). Concomitantly, lymphocyte num- of relative PTPRJ deficiency for FLT3-ITD driven disease. bers were decreased in the peripheral blood, bone mar- In general, according to transcriptomic data available row, and spleen (Figure 2D,G,H; Online Supplementary from bloodspot, PTPRJ appears to be highly expressed in Figure S1). While the histology of kidneys derived from AML patient samples compared to progenitor cells of FLT3ITD/ITD or Ptprj-/- mice was not different from that of healthy controls (data not shown). Of note, the expression the kidneys of WT animals, kidneys derived from of PTPRJ appeared to be lower in FLT3-ITD positive AML FLT3ITD/ITD Ptprj-/- mice showed massive infiltration of compared to patient samples without ITD mutations. leukemic cells in the renal medulla resulting in an obvi- One data set showed a significant downregulation in the ous reduction of the Bowman’s space around the FLT3-ITD positive AML patients compared to AML glomerular capsule (Figure 2E). Similarly, a massive infil- patients expressing FLT3 wild-type (WT; t-value=-2.48, tration of leukemic cells was observed in the livers of P=0.01).4 In addition, the overall survival of FLT3-ITD FLT3ITD/ITD Ptprj-/- mice. Infiltration occurred predominant- positive patients with a low PTPRJ expression level tend- ly around liver sinusoids, but was also widespread in the

AB

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Figure 1. PTPRJ expression is inversely correlated to survival of FLT3-ITD positive AML patients. (A, B). Overall survival of patients (Valk study,4,13 GEO accession GSE1159) with low (red, dotted) and high (blue) PTPRJ expression. Survival curves of AML FLT3-ITD positive (A: cutoff = 33.3, P=0.07) and FLT3 WT patients (B: cutoff = 34.9, P=0.21) are presented. The number of patient samples (n) is indicated.

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Figure 2. The inactivation of Ptprj results in enhanced myeloproliferation in FLT3ITD/ITD mice. (A) Kaplan-Meier survival curves of FLT3ITD/ITD Ptprj–/– and age- matched litter mates. The P values of the log test are indicated. The spleen (B) and liver (C) weight (normalized to total body weight) of 30 to 35-week-old WT, FLT3ITD/ITD, Ptprj–/– and FLT3ITD/ITD Ptprj–/– mice. *P<0.05, **P<0.01, ***P<0.001 compared to WT mice. (D) White blood cell count (WBC) differentials of peripheral blood. WBC differentials are shown in mean ± SEM for FLT3ITD/ITD Ptprj–/– mice and controls from 30 to 35-week-old mice and indicate a reduced lym- phocyte and increased neutrophil and monocyte population in Ptprj–/–FLT3ITD/ITD mice compared to FLT3ITD/ITD mice. (E) H&E histopathology showing spleen (top), liver (middle) and kidney (bottom) architecture from 30- to 35-week-old WT, FLT3ITD/ITD, Ptprj–/– and FLT3ITD/ITD Ptprj–/– mice (WP: white pulpa; RP: red pulpa; GC, glomerula capsule with arrows); bars indicate 100 mm. (F-H) Immunophenotype of the BM and spleen cells from FLT3ITD/ITD Ptprj–/– mice show an expansion of the granulocyte/monocyte population and lack of B cells. 30 to 35-week-old mice were analyzed. (F) Gating schema for the characterization of myeloid undif- ferentiated B and T cells (G) Graphical presentation of CD11b/Gr-1 expression in the BM (G) or spleen (H) and CD19/CD3e expression in the CD11b–/Gr-1– pop- ulation as the total cell number out of 106 analyzed cells. Values are given as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared to WT; #P<0.05com- pared to FLT3ITD/ITD. n.s: not significant; WT: wild-type.

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Figure 3. The inactivation of Ptprj in FLT3ITD/ITD mice affects the formation of progenitor cells. Lineage analysis of the BM and spleen cells from FLT3ITD/ITD Ptprj–/– mice shows an expansion of the Lin– c-+ Sca-1– and Lin– c-kit+ Sca-1+ population and amount of FLT3+ cells. Representative flow cytometric analysis of the BM or spleen cells derived from WT, FLT3ITD/ITD, Ptprj–/– and FLT3ITD/ITD Ptprj–/– mice are shown. (A) Dot plots illustrating the gating for Lin–, Lin– c-kit+ Sca-1– and LSK (Lin- c-kit+ Sca-1+) cell populations. An abundance of these cells in the BM (B) and spleen (C) is presented. Values are given as mean ± SEM; *P<0.05, **P<0.01, compared to WT. (D, E) The number of FLT3 positive cells of myeloid progenitors (Lin- c-kit+ Sca-1– , left) or LSK cell (right) derived from the BM (D) or spleen (E). (F) FLT3ITD/ITD Ptprj–/– mice show enhanced FLT3-ITD activity. Western blotting of the Lin- BM cells of 30-week-old FLT3ITD/ITD and FLT3ITD/ITD Ptprj–/– mice. BM cells were MACS-purified for Lin- immunophenotype. Cells were lysed, processed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and sub- sequently underwent immunoblotting using phospho site specific antibodies which recognized FLT3 pY591 and FLT3 pY589. Each blot was reprobed for panFLT3 antibodies and b-actin was used as the loading control. A representative blot is presented. Numbers under the phosphor-specific blots (mean +/- SEM) represent the quantification of the phosphor-specific signals of three independent experiments, normalized to the corresponding signals with pan-specific anti- bodies, and relative to the signals in FLT3 ITD mice, which was set to 1.0. *P<0.05. (G, H) The promotion of clonogenic growth of Lin- FLT3ITD/ITD Ptprj–/– spleen cells. Lin- BM (G) and spleen (H) cells from 30 to 35-week-old WT, FLT3ITD/ITD, Ptprj–/– and FLT3ITD/ITD Ptprj–/– mice were plated on M3434 methylcellulose medium (containing stem cell factor [SCF-1], interleukin-3 [IL-3], erythropoietin [EPO]) and scored for colony formation seven days later. ***P<0.001 compared to WT; #P<0.05; ###P<0.001 compared to FLT3ITD/ITD mice. WT: wild-type.

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tissue (Figure 2E). Myeloperoxidase (MPO) staining con- (HSC), long-term (LT) HSC and short-term (ST) HSC in firmed the infiltration of myeloid cells in the liver (Online Lin- Sca1+ c-kit+ cells using CD34 and FLT3 staining Supplementary Figure S2) and kidney (data not shown) of remained unchanged among the different mouse geno- FLT3ITD/ITD Ptprj-/- mice in contrast to age-matched litter- types (data not shown). mates of all other genotypes. Consistent with earlier The previously reported massive expansion of FLT3 findings in the FLT3ITD/ITD strain,6 the number of neu- positive cells8 was confirmed in our FLT3ITD/ITD mouse trophils and monocytes in the peripheral blood of 30- to model: in Lin– c-Kit+ progenitors derived from FLT3ITD/ITD 35-week-old mice compared to their WT littermates was mice, a 5- or 40-fold elevation of FLT3 positive cells in the 3- and 6-fold higher, respectively (Figure 2D, Online BM or spleen, respectively, was observed compared with Supplementary Table S1). This phenotype appeared even WT mice (Figure 3D,E). The inactivation of Ptprj resulted further pronounced in FLT3ITD/ITD Ptprj-/- animals. A white in a further increase of this cell population (Figure 3D,E) blood count (WBC) confirmed the expansion of leuko- confirming extramedullary hematopoiesis in the spleen. cytes in this mouse strain (Online Supplementary Figure Similarly, FLT3-ITD expressing LSK cells in FLT3ITD/ITD S3A). The red blood cell count (RBC) as well as the num- Ptprj–/– mice were enhanced in the spleen only. ber of platelets of FLT3ITD/ITD animals was significantly Given that PTPRJ can negatively regulate FLT3 signal- reduced compared to age-matched WT littermates ing, we expected that the transformation of the signaling (Online Supplementary Figure S3B,C). The loss of Ptprj in of FLT3-ITD in the absence of Ptprj would be enhanced, FLT3ITD/ITD mice did not further promote this effect. In thereby promoting the myeloproliferative phenotype. To FLT3ITD/ITD Ptprj-/- mice hemoglobin (HGB) was significant- assess FLT3-ITD signaling activity, Lin- cells from the BM ly enhanced compared to FLT3ITD/ITD mice (Online of 30-week-old mice were purified by magnetic-activated Supplementary Figure S3D). cell sorting (MACS) and subsequently probed for activity The previously reported increased number of mature specific phosphorylation of FLT3. While in WT or Ptprj-/- myeloid (Gr1+/CD11b+) cells in the bone marrow (BM), cells the level of FLT3 expression was too low to be and particularly in the spleen of FLT3ITD/ITD mice6 was fur- detected by western blotting, a significant increase of ther elevated in the spleen and in peripheral blood in FLT3-ITD pY591 autophosphorylation activity could be response to Ptprj inactivation (Figure 2G,H; Online detected in cells from FLT3ITD/ITD Ptprj-/-mice compared Supplementary Figure S1) demonstrating that the absence with cells from FLT3ITD/ITD mice (Figure 3F). In addition, an of Ptprj may lead to the acceleration of the myeloprolif- increased dominance of the immature FLT3 receptor of erative phenotype driven by FLT3-ITD. Comparative these cells further confirmed elevated activity.10 analysis of cytospins of age-matched littermates con- Unfortunately, altered FLT3-ITD mediated STAT5 activa- firmed an expanded monocyte population in the BM tion could not be demonstrated in vivo. Elevated kinase (Online Supplementary Figure S3E). In the spleen a massive activity of FLT3 is known to shift the dose-response infiltration of Gr1+ CD11b+ myeloid cells was observed in curve for tyrosine kinase inhibitors (TKI) towards some- FLT3ITD/ITD Ptprj-/- mice (Figure 2H). The number of CD19+ what lower sensitivity. Inversely, relative resistance to cells was drastically reduced in FLT3ITD/ITD mice. While in TKI11 may indicate higher FLT3 signaling activity. We the BM this reduction was irrespective of Ptprj activity, in employed 32D cells stably overexpressing FLT3-ITD12 the spleen Ptprj-/- resulted in a further abrogation of with inactivated Ptprj using CRISPR/Cas9 (Online + + CD19 cells (Figure 2G,H). The amount of CD3e T cells Supplementary Figure S5A). A slight elevation of pY589 of the Gr-1- CD11b– population did not change in any of site-specific phosphorylation was observed (Online the investigated genotypes (Figure 2G,H). These data Supplementary Figure S5B). Inactivation of Ptprj in these reveal that the inactivation of Ptprj did not promote an cells resulted in an elevated IC50 for the inflammatory phenotype in the FLT3ITD/ITD background FLT3-selective TKI quizartinib (AC220) and the more and aberrancies are due to altered hematopoiesis. broadly active TKI midostaurin (PKC412). These findings To address the question whether inactivation of Ptprj indicate that FLT3-ITD signaling activity appears to be in FLT3ITD/ITD mice affects the abundance of hematopoietic increased upon Ptprj inactivation. Moreover, data point progenitor cells, lineage negative (Lin-) cells from the BM to the possibility that patients with lower PTPRJ levels and spleen were characterized. While the single genetic may be relatively less sensitive to TKI treatment. lesions did not alter the number of Lin- cells in the BM, a To determine the potential of progenitor cells in the roughly 2.5-fold elevation in FLT3ITD/ITD Ptprj–/– mice was BM and spleen for CFU, cells were MACS-sorted for Lin- observed (Figure 3B). In the spleen of Ptprj–/– mice, a immunophenotype and subsequently evaluated in in vitro reduction of about 50% of this cell population compared clonogenic assays in M3434 methylcellulose. While the to WT or FLT3ITD/ITD littermates was detected. Previous number of CFU of BM granulocytes/ macrophages of reports have highlighted an increase of hematopoietic WT, FLT3ITD/ITD or Ptprj-/- mice showed no significant stem and progenitor cell numbers in FLT3ITD/ITD spleens.8,9 changes, the numbers of CFU-GM from FLT3ITD/ITD Ptprj–/– Importantly, we found a further increase in the number BM were significantly elevated (Figure 3G). The Lin- of Lin– Sca-1+ c-Kit+ (LSK) cells in the spleen of FLT3ITD/ITD spleen cells of FLT3ITD/ITD mice formed a similar number of Ptprj-/- mice (Figure 3C), whereas such differences were CFU-GM as cells from WT mice, but CFU-GMs were sig- not observed in the BM. Taken together, these data nificantly elevated in FLT3ITD/ITD Ptprj-/- mice (Figure 3G). demonstrate that the absence of Ptprj in FLT3ITD/ITD mice Cytospins of CFU-GM showed that cells derived from results in an increase of Lin- progenitor cells, which is FLT3ITD/ITD or FLT3ITD/ITD Ptprj-/- BM were characterized by predominantly apparent in the spleen. Further evaluation an accumulation of myelocytes, myeloblasts and mono- of the LSK compartment demonstrated reduced numbers cytes while the abundance of granulocytes and of megakaryocyte erythroid progenitors (MEP) and ele- macrophages was reduced compared to WT and Ptprj-/- lit- vated numbers of granulocyte-macrophage progenitor termates (data not shown). Almost no CFU of multipoten- (GMP) cells in FLT3ITD/ITD mice, but this did not alter in tial granulocyte, erythroid, macrophage, megakaryocyte response to the inactivation of Ptprj. (Online progenitor cells (CFU-GEMM) or erythroid progenitor Supplementary Figure S4). The number of common cells (BFU-E) were observed for all genotypes (data not myeloid progenitors (CMP) remained unchanged in all shown). We performed the re-plating of FLT3ITD/ITD Ptprj-/- genotypes. The abundance of hematopoietic stem cells BM cells in order to assess for a potential gain in self-

haematologica 2018; 103:e508 LETTERS TO THE EDITOR renewal capacity by combined FLT3-ITD expression and References Ptprj loss, however, FLT3ITD/ITD Ptprj-/- cells lacked re-plat- ITD/ITD -/- ing capacity, just like the FLT3 or Ptprj controls. In 1. Papaemmanuil E, Gerstung M, Bullinger L, et al. Genomic classifica- the absence of cytokines no colony formation of Lin- tion and prognosis in acute myeloid leukemia. N Engl J Med. 2016; cells in methylcellulose was observed (data not shown). 374(23):2209-2221. ITD/ITD Taken together, the inactivation of Ptprj in FLT3 2. Arora D, Stopp S, Böhmer SA, et al. Protein-tyrosine phosphatase mice resulted in a more pronounced infiltration of DEP-1 controls FLT3 signaling. J Biol Chem. + + myeloid (Gr-1 CD11b ) cells with an increased repres- 2011;286(13):10918-10929. sion of lymphocytes, which may indicate an enhanced 3. Godfrey R, Arora D, Bauer R, et al. Cell transformation by FLT3 ITD aggressiveness of a FLT3-ITD driven disease. The expan- ITD/ITD -/- in acute myeloid leukemia involves oxidative inactivation of the sion of the progenitor cells of FLT3 Ptprj mice, most tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ. notable in the spleen, indicated an increase of Blood. 2012;119(19):4499-4511. extramedullary hematopoiesis. Clonogenic assays 4. Valk PJ, Verhaak RG, Beijen MA, et al. Prognostically useful gene- showed an enhanced CFU-GM potential of Lin- spleen expression profiles in acute myeloid leukemia. N Engl J Med. 2004; cells. Moreover, the specific phosphorylation of FLT3 in Lin- BM cells derived from FLT3ITD/ITD Ptprj-/- mice was 350(16):1617-1628. enhanced. Thus, our data identify PTPRJ as a suppressor 5. Metzeler KH, Hummel M, Bloomfield CD, et al. An 86-probe-set of FLT3-ITD induced myeloproliferation. gene-expression signature predicts survival in cytogenetically normal acute myeloid leukemia. Blood. 2008;112(10):4193-4201. Anne Kresinsky,1 Reinhard Bauer,1 Tina M. Schnöder,2,3 6. Lee BH, Tothova Z, Levine RL, et al. FLT3 mutations confer enhanced Tobias Berg,4 Daria Meyer,5 Volker Ast,5 Rainer König,5 proliferation and survival properties to multipotent progenitors in a Hubert Serve,4 Florian H. Heidel,2,3 Frank- D. Böhmer1 and murine model of chronic myelomonocytic leukemia. Cancer Cell. Jörg P. Müller1 2007;12(4):367-380. 7. Trapasso F, Drusco A, Costinean S, et al. Genetic ablation of Ptprj, a 1Institute of Molecular Cell Biology, Center for Molecular mouse cancer susceptibility gene, results in normal growth and Biomedicine (CMB), Jena University Hospital; 2Innere Medizin II, Hämatologie und Onkologie, Jena University Hospital; 3Leibniz development and does not predispose to spontaneous tumorigene- Institute on Aging, Fritz-Lipmann-Institute (FLI), Jena; 4Department of sis. DNA Cell Biol. 2006;25(6):376-382. Medicine II, Hematology/Oncology, Goethe University, Frankfurt and 8. Li L, Bailey E, Greenblatt S, Huso D, Small D. Loss of the wild-type 5Network modelling, Leibniz Institute for Natural Product Research and allele contributes to myeloid expansion and disease aggressiveness in Infection Biology, Hans Knöll Institute, Jena, Germany FLT3/ITD knockin mice. Blood. 2011;118(18):4935-4945. 9. Li L, Piloto O, Nguyen HB, et al. Knock-in of an internal tandem Acknowledgments: we are thankful to Jörg Cammenga (Lund duplication mutation into murine FLT3 confers myeloproliferative University, Sweden) for kindly providing FLT3ITD/ITD mice and disease in a mouse model. Blood. 2008;111(7):3849-3858. Klaus Metzelder for providing additional array data. We thank Ilse 10. Schmidt-Arras DE, Bohmer A, Markova B, Choudhary C, Serve H, D. Jacobsen for kindly providing access to the Mindray Hematology system. Bohmer FD. Tyrosine phosphorylation regulates maturation of receptor tyrosine kinases. Mol Cell Biol. 2005;25(9):3690-3703. Funding: the work was supported by the Deutsche 11. Tse KF, Allebach J, Levis M, Smith BD, Bohmer FD, Small D. Forschungsgemeinschaft (grant Mu955/11-1) and by the Federal Inhibition of the transforming activity of FLT3 internal tandem dupli- Ministry of Education and Research (BMBF), Germany, FKZ cation mutants from AML patients by a tyrosine kinase inhibitor. 01ZX1302B, 01ZX1602B (CancerTel-Sys), FKZ: 01EO1002, Leukemia. 2002;16(10):2027-2036. 01EO1502 (CSCC). 12. Grundler R, Miething C, Thiede C, Peschel C, Duyster J. FLT3-ITD Correspondence: [email protected] and tyrosine kinase domain mutants induce 2 distinct phenotypes in doi:10.3324/haematol.2017.185306 a murine bone marrow transplantation model. Blood. 2005; Information on authorship, contributions, and financial & other disclo- 105(12):4792-4799. sures was provided by the authors and is available with the online version 13. Metzelder SK, Michel C, von Bonin M, et al. NFATc1 as a therapeutic of this article at www.haematologica.org. target in FLT3-ITD-positive AML. Leukemia. 2015;29(7):1470-1477.

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