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Blood, Vol. 113, Issue 18, 4414-4424, April 30, 2009
Leukemogenic Ptpn11 causes fatal myeloproliferative disorder via cell-autonomous effects on multiple stages of hematopoiesis
Blood Chan et al.
113: 4414
Supplemental materials for: Chan et al
Generation of Lox-stop-Lox (LSL)-Ptpn11D61Y mice
A targeting vector was generated by ligating a long arm containing exon 3 (Apa I genomic fragment) into pBluescript SK containing PGK-dta. A loxP-bracketed transcriptional STOP cassette with a neo gene was placed 5′ of the long arm. A 1.3 kb short arm fragment, which includes exon 2, was ligated 5′ of the STOP cassette. The targeting vector was linearized by digestion with Kpn I and electroporated into W4 ES cells (Taconic). Proper integration of the short arm was confirmed by Southern blotting using an exon 2 probe, which detected a 2.3-kb EcoN I fragment in the WT genomic locus and a 5.6-kb fragment in the targeted allele. Confirmation of long arm integration was performed with an exon 4 probe, which detects a 7.5-kb Nde I-Age I fragment in the WT locus and a 5.7-kb fragment of the mutant allele. LSL-Ptpn11D61Y mice are healthy, fertile and undistinguishable from wild-type control littermates. Phosphatase assay
ES cells were lysed in buffer containing 50 mM Tris-HCl (pH7.4), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, and a mixture of protease inhibitors (5 µg/ml leupeptin, 5 µg/ml aproptinin, 1 µg/ml pepstatin A and 1 mM phenylmethylsulfonyl fluoride). Shp2 was immunoprecipitated by polyclonal anti-Shp2 antibodies (C-18, Santa Cruz) coupled to protein A-Sepharose, and immune complexes were washed three times in lysis buffer, and once in buffer containing 20 mM HEPES (pH 7.4) and 150 mM NaCl. Phosphatase assays were carried out at 37°C in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM dithiothreitol and 20 mM para-nitrophenyl phosphate (pNpp, Sigma). Phosphate release was determined by measuring A410. Flow cytometry
Single cell suspensions from bone marrow or spleen were resuspended in PBS with 2% FBS and stained with directly conjugated monoclonal antibodies specific for Mac1 (M1/70), Gr1(Ly-6G), CD71 (R17217), Ter119, CD3 (145-2C11), CD19 (6D5), CD45/B220 (RA3-6B2), CD45.1 (A20) and/or CD45.2 (104). For HSC/progenitor analysis, cells were stained with antibodies against c-Kit (2B8), Sca1 (E13-161.7), CD127 (A7R24), CD34 (RAM34), CD16/32 (93), CD135 (A2F10.1), and antibodies against lineage (Lin) markers including CD45/B220, CD3, CD4 (RM4-5), CD8α (53–6.7), CD19, Gr1, Ter119. For cell cycle analyses, sorted cells were stained with Hoechst 33342 (Molecular Probes) and Pyronin Y (Sigma) at 37°C in the presence of Verapamil (Sigma) before flow cytometry. Antibodies were purchased from BD Pharmingen, eBioscience, BioLegend or Cell Signaling Technology. Relevant cell populations were purified using a FACSAria (Becton-Dickinson, Mountain View, CA). Flow cytometry was performed with an LSRII (Becton-Dickinson) and analyzed with FlowJo software (TreeStar, Ashland, OR). Intracellular flow cytometric analysis
GMP and CMP were purified by FACS, and starved for 1 hour before they were either left unstimulated or stimulated with GM-CSF (5 ng/ml) for 5 or 10 minutes, fixed with 2% paraformaldehyde, permeabilized with methanol and then stained with anti-pERK pT202/pY204 (E10) or anti-pSTAT5 pY694 (47). To analyze signaling in LK and LSK cells, Lin− cells were FACS-purified and starved for 1 hour before stimulation with SCF (50 ng/ml) for 5 or 10 minutes. Cells were fixed with 1.5% paraformaldehyde, permeabilized with acetone, and then stained with anti-cKit, anti-Sca1 and anti-pERK, anti-pAKT pS473 (193H12). Phospho-specific antibodies were purchased from Cell Signaling Technology; cytokines were from Peprotech. Discussion
PTPN11 mutants have not been reported in human T-ALL.1–3 However, T-ALL was frequently observed in the transplant setting described herein and in our previous retroviral over-expression/BMT study.4 Furthermore, recent data show activated mutants of Ras collaborate with Notch mutations to evoke T-ALL.5,6,7 Our data indicate that activated Shp2 can also initiate T-lymphomagenesis in mice, but unlike in KrasG12D mice,8 T-ALL only is seen in the transplant setting and not in primary Ptpn11D61Y animals. It will be interesting to see how myelo-ablation/transplantation facilitates transformation of T-lymphoid cells expressing leukemogenic allele of Ptpn11, and determine whether Notch mutants are also seen in this setting. KrasG12D mice also develop squamous papillomas and lung adenomas,8 but these are not present in Ptpn11D61Y animals. Together, our data and those from human mutation surveys3,9 suggest that activated Ptpn11 produces a more restricted disease profile compared to that of Ras family members. REFERENCES 1. Tartaglia M, Martinelli S, Cazzaniga G, et al. Genetic evidence for lineage-related and differentiation stage-related contribution of somatic PTPN11 mutations to leukemogenesis in childhood acute leukemia. Blood. 2004;104:307–313.
2. Yamamoto T, Isomura M, Xu Y, et al. PTPN11, RAS and FLT3 mutations in childhood acute lymphoblastic leukemia. Leuk Res. 2006;30:1085–1089.
3. Bentires-Alj M, Paez JG, David FS, et al. Activating mutations of the Noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia. Cancer Res. 2004;64:8816–8820.
4. Mohi MG, Williams IR, Dearolf CR, et al. Prognostic, therapeutic, and mechanistic implications of a mouse model of leukemia evoked by Shp2 (PTPN11) mutations. Cancer Cell. 2005;7:179–191.
5. Dupuy AJ, Akagi K, Largaespada DA, Copeland NG, Jenkins NA. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature. 2005;436:221–226.
6. Kindler T, Cornejo MG, Scholl C, et al. K-RasG12D–induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to {gamma}-secretase inhibitors. Blood. 2008.
7. Chiang MY, Xu L, Shestova O, et al. Leukemia-associated NOTCH1 alleles are weak tumor initiators but accelerate K-ras–initiated leukemia. J Clin Invest. 2008;118:3181–3194.
8. Chan IT, Kutok JL, Williams IR, et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J Clin Invest. 2004;113:528–538.
9. Martinelli S, Carta C, Flex E, et al. Activating PTPN11 mutations play a minor role in pediatric and adult solid tumors. Cancer Genet Cytogenet. 2006;166:124–129.
Files in this Data Supplement:
- Figure S1. Generation of LSL-Ptpn11D61Y mice (JPG, 132 KB)
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(A) Schematic of WT Ptpn11 locus, targeting vector, LSL-Ptpn11D61Y allele with floxed STOP cassette and the D61Y mutation, and the activated Ptpn11D61Y knock-in allele after Cre-mediated excision. Locations of exon 2 and exon 4 probes for Southern blots are indicated by black bars over the respective exons. (B) Southern blots of WT and Ptpn11D61Y (DY) ES clones. An exon 2 probe detected a 2.3-kb EcoN I fragment in the WT genomic locus and a 5.6-kb fragment representing the targeted allele (Left). Proper integration was confirmed by an exon 4 probe that revealed the 7.5-kb Nde I-Age I fragment of the WT locus and the 5.7-kb of the mutant allele (Right). (C) In the absence of co-expressed Cre recombinase, the Ptpn11D61Y allele in properly targeted ES clones was transcriptionally silent, as expected. Transfection of such clones with a vector directing Cre-expression (MSCV-Cre) caused STOP cassette excision, expression of the mutant Ptpn11D61Y transcripts (arrow). (D) Shp2 immunoprecipitates from Cre-expressing Ptpn11D61Y ES clones show 6–7 fold higher PTP activity. (E) Properly targeted ES clones were injected into blastocysts, and chimeric mice were crossed with C57BL/6 mice to generate F1 animals. PCR typing of tail DNA indicates that germ line transmission of the inducible Ptpn11D61Y (LSL-Ptpn11D61Y) allele was obtained. (F) Ptpn11+∕+ (+∕+), LSL-Ptpn11D61Y (LS) and Mx1-Cre; LSL-Ptpn11D61Y mice (DY) were injected with three doses (300 µg) of plpC. After 3.5 months, cells from peripheral blood, BM and spleen were harvested and assessed for deletion of the STOP cassette by PCR using primers located outside of the 5′ and 3′ lox sites. The presence of the wild type (wt) and deleted (Δ) alleles are shown; the latter indicates complete deletion of the entire STOP cassette. (G) LSK, CMP, GMP and MEP from control and Ptpn11D61Y mice were purified by FACS and assessed for deletion of the STOP cassette by PCR.
- Figure S2. Colony forming ability of Ptpn11D61Y progenitor cells in the presence of SCF (JPG, 26 KB)
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LK and LSK cells from control (n=2) and Ptpn11D61Y (n=2) mice were purified and seeded in methylcellulose media in the presence of SCF for 8 days before colonies were enumerated.
- Figure S3. Adoptive transfer of Ptpn11D61Y BM and spleen cells (JPG, 147 KB)
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(A) BM cells from control and Ptpn11D61Y mice were loaded with CSFE and transplanted into lethally irradiated CD45.1 recipients as described in Figure 4. BM cells from recipients of control (n=4) and mutant (n=5) cells were analyzed 16 hours post transplantation for the presence of Lin−CFSE+ labelled cells. (B) BM cells (BM) and spleen cells (SPL) of recipients of mutant BM (#7 and #21) or spleen cells (#5) were harvested and subjected to RT-PCR, as described in Figure S1C. #7 and #21 developed T-ALL at ~24 weeks. #5 was phenotypically normal when necropsy was performed at ~25 weeks. All samples show presence of the Ptpn11D61Y RNA. (C) Flow cytometric analysis of BM and spleen cells from recipients that developed donor-derived (CD45.2) T-ALL. Note the relative homogeneity in light-scattering properties (FSC, forward scatter; SSC, side scatter) (middle panels) and high levels of staining with CD4 and CD8 (bottom panel), but not B220, antibodies. Shown is a contour plot from a diseased animal, with the percent of parental gate indicated. (D) Representative histopathology indicating abundance of lymphoid cells in the spleen and liver from a transplanted mouse. (E) PCR performed on DNA purified from PBL of recipients 24 weeks after transplant showing deletion of the STOP cassette.
- Figure S4. Colony forming ability of Ptpn11D61Y cells in the presence of GM-CSF or M-CSF (JPG, 20.9 KB)
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BM cells from control (n=2) and Ptpn11D61Y mice (n=2) were purified and seeded in methylcellulose media in the presence of GM-CSF and M-CSF for 8 days before colonies were enumerated.
- Figure S5. Colony forming ability of Ptpn11D61Y cells in the presence or absence of growth factors (JPG, 118 KB)
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(A) Control and Ptpn11D61Y mice were injected with pIpC, and BM progenitors were harvested after 2 weeks and plated into methylcellulose media in the absence of cytokines. (B) GMP from control (n=2) and Ptpn11D61Y mice (n=2) were purified by FACS and IL-3, GM-CSF, and M-CSF RNA levels were determined by quantitative real-time PCR. Cells from mix lymphocyte reaction (MLR) were used as positive controls for IL-3 and GM-CSF RNA. (N.D., not detectable). Note that both control and Ptpn11D61Y GMP produce M-CSF, but at comparable levels. (C) CMP and GMP from ER-Cre and ER-Cre;Ptpn11D61Y mice were purified and cultured for 16 hours in media containing SCF and IL-11 in the presence or the absence of Tam. Live cells were replated on M3434 methylcellulose medium, and colonies were counted 7–9 days later.
- Figure S6. Signaling aberrations in Ptpn11D61Y cells (JPG, 98.5 KB)
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(A, B) Lin− BM cells from control and Ptpn11D61Y mice were purified by FACS and treated as described in Figure 7. Cells were fixed, permeabilized and stained with anti-cKit and anti-Sca1 and antibodies against pErk (B) or pAkt (C). The levels of phospho-specific antigens on the LK population were determined with by flow cytometry. (C, D) CMP from control and Ptpn11D61Y mice were purified and treated as described above. Cells were fixed, permeabilized and stained with antibodies against pErk (C) and pStat5 (D). Phosphospecific antigens were quantified by flow cytometry (± SEM; *, p<0.05, by Wilcoxon-Mann-Whitney test).
- Figure S7. Ptpn11D61Y affects multiple stages of hematopoiesis (JPG, 34.2 KB)
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Ptpn11D61Y expression decreases self-renewal in LT-HSC (dashed curved arrow), and increases production (large arrow) of ST-HSC and MPP, CMP and GMP (represented by more circles). This expansion is amplified by cell-autonomous cytokine-independence of the CMP and GMP compartment (represented by gray), and ultimately creates surplus numbers of granulocytes and monocytes/macrophages. In MEP compartment, there is a bias towards production of BFU-E in Ptpn11D61Y mice, but erythroid differentiation at or distal to EP/CFU-E stage is partially blocked.
- Figure S8. Models of Ptpn11D61Y-evoked MPD (JPG, 29 KB)
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(Top) HSC are a homogenous cell population in which Ptpn11D61Y expression (DY) is insufficient to evoke disease. MPD develops after a HSC expressing Ptpn11D61Y acquires a secondary mutation. (Bottom) HSC comprise a heterogeneous population in which only rare cells can undergo transformation upon expression of Ptpn11D61Y and produce MPD; whereas the expression of the mutant allele in other HSC does not evoke leukemia.
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