Gene Therapy (2009) 16, 570–573 & 2009 Macmillan Publishers Limited All rights reserved 0969-7128/09 $32.00 www.nature.com/gt SHORT COMMUNICATION Reduced proliferation of CD34+ cells from patients with acute myeloid leukemia after transfer of INPP5D

A Metzner1, C Precht1, B Fehse2, W Fiedler3, C Stocking4,AGu¨ nther5, GW Mayr1 and M Ju¨ cker1 1Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology I, Cellular Signal Transduction, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 2Clinic for Stem Cell Transplantation, Hamburg, Germany; 3Department of Medicine II, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 4Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany and 5Division of Stem Cell Transplantation and Immunotherapy, Second Department of Medicine, University Hospital Schleswig-Holstein, Kiel, Germany

Acute myeloid leukemia (AML) is a malignant disease factor (GM-CSF)-dependent proliferation was reduced in all characterized by deregulated proliferation of immature samples analyzed (average 86%; range 72–93%). An myeloid cells. Constitutive activation of the phosphatidylino- enzymatically inactive form of SHIP1 (D672A) had no effect. sitol 3-kinase (PI3K)/AKT signaling pathway is frequently In addition, SHIP1 reduced the autonomous proliferation of detected in approximately 50–70% of AML patients. CD34+ cells from a patient with a secondary AML who had a The gene INPP5D encodes the SH2-containing inositol very high peripheral blast count (300 Â 10 9 lÀ1). These data 5- 1 (SHIP1), which is a negative regulator show that SHIP1 can effectively block GM-CSF-dependent of PI3K/AKT signaling. After lentiviral-mediated gene transfer and autonomous proliferation of AML cells. of INPP5D into CD34+ cells derived from AML patients Gene Therapy (2009) 16, 570–573; doi:10.1038/gt.2008.184; (n ¼ 12) the granulocyte macrophage-colony stimulating published online 15 January 2009

Keywords: acute myeloid leukemia; SHIP1; inositol 5-phosphatase

Introduction autonomous proliferation of CD34+ cells from AML patients. The INPP5D gene encodes the SH2-containing inositol 5-phosphatase 1 (SHIP1), which is an important negative 1 regulator of cytokine signaling. Missense mutations in Results and discussion the INPP5D gene have been detected in several patients with acute myeloid leukemia (AML) implicating mu- In this study, the effect of SHIP1 on the proliferation of tated SHIP1 in the pathogenesis of AML.2,3 One of the CD34+ cells purified from the peripheral blood of 12 mutations detected in a patient with AML caused a patients with AML was determined. First, we deter- reduction in SHIP1 activity suggesting a possible tumor mined the levels of endogenous SHIP1 protein, which suppressor function of SHIP1.4 Restoration of SHIP1 were highly variable in the AML samples analyzed activity in a human SHIP1 deficient leukemia cell line (Figure 1). Interestingly, two AML samples, that is, (Jurkat) resulted in a reduced proliferation owing to an AML5 and AML8, showed a very low expression of increased transit time through the G1 phase of the cell SHIP1 suggesting a downregulation of SHIP1 in the cycle.5 Gene transfer of INPP5D in CD34+ cells from leukemia cells from these patients. A downregulation of patients with juvenile myeloid leukemia carrying muta- SHIP1 has been detected in primary samples of patients tions in KRAS2 or PTPN11 reduced their hypersensitivity with Philadelphia -positive chronic myeloid for granulocyte macrophage-colony stimulating factor leukemia and can be experimentally induced in cells by (GM-CSF).6 In this report, we characterized the inhibi- overexpression of BCR/ABL.7 In AML, such a down- tory effects of SHIP1 on the GM-CSF-dependent and regulation of SHIP1 may be mediated by other tyrosine kinases like c-Kit or Flt3. Interestingly, the patient AML8 with very low expression of SHIP1 showed mutations in both, c-Kit (M536I) and Flt3 (internal tandem duplica- Correspondence: Dr M Ju¨cker, Center of Experimental Medicine, tion, amino acids 591–607; data not shown). Institute of Biochemistry and Molecular Biology I, Cellular Signal Because SHIP1 is a negative regulator of the phospha- Transduction, University Hospital Hamburg-Eppendorf, Martinis- tidylinositol 3-kinase (PI3K)/AKT signaling pathway, we trasse 52, D-20246 Hamburg, Germany. E-mail: [email protected] expected an increased activation of PI3K/AKT signaling Received 22 May 2008; revised 17 October 2008; accepted 3 in AML samples with low expression of SHIP1. In 8 of November 2008; published online 15 January 2009 the 12 AMLs (67%) analyzed, we observed a constitutive Effects of SHIP1 on acute myeloid leukemia cells A Metzner et al 571 phosphorylation of AKT at 473, however, there AML patients (Figure 2b). An enzymatic inactive form of was no inverse correlation between the levels of SHIP1 (D672A) had no effect on the proliferation of AML phosphoserine AKT (pAKT) and SHIP1 (Figure 1). These cells, indicating that the inhibitory effect of SHIP1 is data indicate that even a very low expression of SHIP1 as dependent on its enzymatic activity (Figure 2b). observed in AML8 does not automatically result in a We further analyzed the effect of SHIP1 on the constitutive activation of AKT (Figure 1). autonomous proliferation of the CD34+ cells from one Next, the effect of overexpression of SHIP1 on the AML patient (AML12), who had developed a secondary in vitro proliferation of AML cells was determined. The AML after a myeloproliferative syndrome and showed a GM-CSF-dependent proliferation of the transduced cells very high peripheral blast count (300 Â 109 lÀ1). SHIP1 was reduced by 72–93% in all AML samples analyzed was able to reduce the autonomous proliferation of the (Figure 2a). On an average, SHIP1 caused an 86±8% CD34+ cells from this patient by 45±2% in comparison to reduction of the proliferation of the CD34+ cells from 12 cells expressing enhanced green fluorescent protein (EGFP; EGFP+), only, or in comparison to nontransduced cells (EGFPÀ; Figure 2c). As observed before, an enzymatic inactive mutant of SHIP1 (D672A) had no effect, underlining the functional role of the enzymatic activity of SHIP1 to mediate its inhibitory function (Figure 2c). SHIP1, being an inositol 5-phosphatase, blocks the recruitment and activation of several pleckstrin homol- ogy containing proteins (VAV1, 2 and 3, PLC, etc.), in particular Akt, whose activation is strongly dependent on PI3K.8 The molecular mechanism by which SHIP1 inhibits the proliferation of AML cells is most likely

Figure 1 Endogenous levels of SH2-containing inositol 5-phos- mediated by reducing the levels of PtdIns(3,4,5)P3 and phatase 1 (SHIP1) and phosphoserine AKT (pAKT) in acute activated AKT, as observed after SHIP1 expression in myeloid leukemia (AML) patients. Total cellular lysates of periph- Jurkat cells.5 To address the effect of Akt on the eral blood cells from AML patients were prepared under denatur- + ing conditions and analyzed by immunoblotting with antibodies proliferation of AML cells directly, CD34 cells from a directed against SHIP1, pAKT (S473) and mitogen activated protein patient with AML (AML13) were incubated with the Akt kinase (MAPK; ERK1/ERK2) as a loading control. inhibitor triciribine and the autonomous proliferation

Figure 2 Overexpression of SH2-containing inositol 5-phosphatase 1 (SHIP1) inhibits the granulocyte macrophage-colony stimulating factor (GM-CSF)-dependent and autonomous proliferation of CD34+ cells from acute myeloid leukemia (AML) patients. (a) CD34+-cells were purified from the peripheral blood of 12 AML patients and transduced with lentiviral expression vectors containing enhanced green fluorescent protein (EGFP; black bars) or EGFP and SHIP1 (gray bars). The transduced cells were sorted and cultured in serum-free medium containing 10 ng mlÀ1 GM-CSF. Cell numbers were counted 10 days after plating. (b) The median±s.d. of cell counts obtained after 10 days are shown for AML cells expressing the vector control (EGFP), SHIP1 (SHIP1+EGFP) or an enzymatic inactive form of SHIP1 (D672A)+EGFP. (c) CD34+ cells from AML patient number12 was transduced with lentiviral vectors containing EGFP, SHIP1+EGFP or SHIP1D672A+EGFP. Transduced cells (EGFP+) and nontransduced cells (EGFPÀ) were separated and cultured in serum-free medium without cytokines. Cell numbers were counted 10 days after plating. Asterisks indicate statistically significant differences between cells expressing EGFP versus EGFP+SHIP1 (*Pp0.001).

Gene Therapy Effects of SHIP1 on acute myeloid leukemia cells A Metzner et al 572 SHIP1 on the proliferation of AML cells suggests a potential role of SHIP1 for gene therapy of patients with AML. Overexpression of SHIP1 in AML cells may reduce leukemia cell load thereby improving survival and response in combination with standard chemotherapy or alternative therapy (that is, monoclonal antibodies or tyrosine kinase inhibitors). The severe side effects and limitations of conventional chemotherapy and hemato- poietic stem cell transplantation indicate the urgent need for any new treatment strategy for AML patients.

Figure 3 Reduced proliferation of CD34+ acute myeloid leukemia (AML) cells after treatment with an Akt inhibitor. CD34+-cells were Materials and methods purified from the peripheral blood of an AML patient (AML13). Sister cultures were incubated in dimethyl sulfoxide alone or with Patient samples the Akt inhibitor V (triciribine) at indicated concentrations for 3 Peripheral blood samples were obtained from 13 patients days. BrdU was added to the culture for 16 h. Cells were stained with anti-BrdU-Allophycocyanin (APC) antibodies and APC-posi- with AML after informed consent within a protocol tive cells were analyzed by flow cytometry. The relative percentage approved by the medical board of Hamburg (OB/7/01). of BrdU incorporating cells±s.d. in comparison to untreated cells is The AML patients were classified according to the indicated. Asterisks indicate statistically significant differences French–American–British classification as M1 (n ¼ 2, between cells incubated with the Akt inhibitor in comparison to AML10, 13), M2 (n ¼ 3, AML4–6), M3 (n ¼ 1, AML8), untreated cells (*Pp0.01), (**Pp0.001). M4 (n ¼ 4, AML2, 3, 7, 9), M5 (n ¼ 1, AML1), secondary AML after MDS (n ¼ 1, AML11) and secondary AML was analyzed by BrdU incorporation. The Akt inhibitor after MPS (n ¼ 1, AML12). caused a 92% (Po0.01) and 95% (Po0.001) reduction of BrdU incorporation after treatment with 1 and 10 mM Lentiviral vectors triciribine, respectively (Figure 3). These data indicate Human SHIP1 cDNA and an enzymatic inactive SHIP1 that both, expression of SHIP1 and inhibition of Akt can mutant (D672A)5,6 were cloned in the lentiviral vector partially inhibit the autonomous proliferation of AML pRRL.PPT.CMV.GFPpre. 293T cells were cultured in cells in vitro. As Akt is constitutively activated in several Dulbecco’s modified Eagle’s medium supplemented tumors including AML, phospholipids analogs such as with 10% fetal calf serum and 2 mM glutamine. Pseudo- perifosine have been used but with the specific aim to typed lentiviral viruses were generated by transient activate chemotherapy-dependent apoptosis in cancer lipofectamine-mediated cotransfection of the plasmids cells.9 Whether overexpression of SHIP1 can sensitize pRRL.PPT.CMV.GFPpre (5 mg), pHCMV-VSV-G (2 mg), AML cells for treatment with chemotherapeutic sub- pMDLg/p (12 mg) and of pRSV-Rev (5 mg). Virus contain- stances has to be shown in further experiments. ing supernatants were collected, filtered and stored at An interesting question is why the endogenous levels À80 1C until used. The titer was determined on human of SHIP1, especially in those AML samples with high HT1080 cells and ranged from 5 Â 105 to 1 Â106 infectious amounts of SHIP1, are not sufficient to control the particles per ml. constitutive activation of the PI3K/AKT pathway in the AML cells efficiently. One possibility is that the enzy- Transduction, cell sorting and proliferation assays matic activity of SHIP1 is reduced because of mutations of CD34+ cells in its catalytic domain, as described for the SHIP mutant Enriched CD34+ cells were cultured in serum-free V684E detected in a patient with AML.4 Alternatively, StemSpan medium (StemCell Technologies, St Kathar- mutations in its interacting domains, such as the SH2 inen, Germany) supplemented with Flt3 ligand domain (F28L) or the proline rich motifs at the C (100 ng mlÀ1), stem cell factor (100 ng mlÀ1), thrombo- terminus (P1039S, P1042L), which have been detected poietin (20 ng mlÀ1) and interleukin-6 (20 ng mlÀ1; Pe- in some AML patients, may interfere with the recruit- proTech, St Katharinen, Germany). After 48 h, 5 Â 105 of ment of SHIP to the cytoplasmic membrane were its the prestimulated CD34+ cells were transferred in 1 ml 2 substrate PtdIns(3,4,5)P3 is located. serum-free medium and 1 ml of viral supernatant was Beside SHIP, another phosphatase regulating the added. The transduction was repeated twice. Transduced PI3K/AKT pathway is phosphatase and tensin homolog cells expressing the EGFP were sorted with a fluores- (PTEN), which is dysregulated in several malignant cence-activated cell sorter (FACS)Aria (Becton Dickinson, cancer including AML.10 Downregulation of PTEN, but Heidelberg, Germany) and cultured at 1 Â104 cells per not SHIP1, has been described in some cases of AML.11 ml in serum-free expansion medium (StemCell Technol- We have detected very low levels of SHIP1 in 2 out of 12 ogies) supplemented with human recombinant GM-CSF AML patients, suggesting that SHIP1 is downregulated (10 ng mlÀ1). The number of cells was counted 10 days in some cases of AML. The exact frequency of down- after plating. The Akt inhibitor V (triciribine) was regulation of SHIP1 in AML has to be analyzed in a purchased from Merck/Calbiochem (Nottingham, UK) larger study. and used at a final concentration of 1 and 10 mM. In summary, our data demonstrate that overexpres- Proliferation assays were performed with a BrdU- sion of SHIP1 can reduce the GM-CSF-dependent and Allophycocyanin (APC) flow kit according to the proto- autonomous proliferation of CD34+ cells from patients col of the manufacturer (Becton Dickinson). The percen- with AML. This effect of SHIP1 is dependent on the tage of APC-positive cells was determined by FACS enzymatic activity of SHIP1. The inhibitory effect of analysis (Becton Dickinson).

Gene Therapy Effects of SHIP1 on acute myeloid leukemia cells A Metzner et al 573 Preparation of cell lysates and western blotting 3 Gilby DC, Goodeve AC, Winship PR, Valk PJ, Delwel R, Reilly SHIP1 can become degraded by proteolytic cleavage JT. Gene structure, expression profiling and mutation analysis of during cell lysis under nondenaturing conditions.12 the tumour suppressor SHIP1 in Caucasian acute myeloid Therefore, cell lysates were prepared under denaturing leukaemia. Leukemia 2007; 21: 2390–2393. conditions by adding 1 ml trichloric acid to a cell pellet of 4 Luo JM, Yoshida H, Komura S, Ohishi N, Pau L, Shigeno K et al. 1 Â107 cells. After incubation for 30 min on ice the pellet Possible dominant-negative mutation of the SHIP gene in acute was washed tree times with acetone and dried. The pellet myeloid leukemia. Leukemia 2003; 17: 1–8. was solved in loading buffer and western blotting was 5 Horn S, Endl E, Fehse B, Weck MM, Mayr GW, Ju¨cker M. Restoration of SHIP activity in a human leukemia cell line performed as described previously.5 Antibodies against downregulates constitutively activated phosphatidylinositol SHIP1 (P1C1), Erk1/Erk2 (K-23) and the secondary 3-kinase/Akt/GSK3b signaling and leads to an increased transit antibodies were purchased from Santa Cruz Technolo- time through the G1 phase of the cell cycle. Leukemia 2004; 18: gies, Heidelberg, Germany. Antibodies against pAKT 1839–1849. S473 were purchased from , NEB, Frank- 6 Metzner A, Horstmann MA, Fehse B, Ortmeyer G, Niemeyer furt, Germany. Protein expression was quantified in an CM, Stocking C et al. Gene transfer of SHIP-1 inhibits LAS-3000 Imager from Fuji (Raytest, Straubenhardt, proliferation of juvenile myelomonocytic leukemia cells carrying Germany). KRAS2 or PTPN11 mutations. Gene Therapy 2007; 14: 699–703. 7 Sattler M, Verma S, Byrne CH, Shrikhande G, Winkler T, Algate PA et al. BCR/ABL directly inhibits expression of SHIP, an Acknowledgements SH2-containing polyinositol-5-phosphatase involved in the regulation of hematopoiesis. Mol Cell Biol 1999; 19: 7473–7480. The excellent technical assistance of W Wegner, M Engel 8 Kalesnikoff J, Sly LM, Hughes MR, Bu¨ chse T, Rauh MJ, Cao LP and A Du¨ sedau is thankfully acknowledged. We thank et al. The role of SHIP in cytokine-induced signaling. Rev Physiol L Naldini for the lentiviral vector system including the Biochem Pharmacol 2003; 149: 87–103. plasmids pRRL.PPT.CMV.GFPpre, pRSV-Rev, pMDLg/p 9 Nyakern M, Cappelini A, Mantovani I, Martelli AM. Synergistic and W Beyer for the plasmid pHCMV-VSV-G. This induction of apoptosis in human leukemia T cells by the Akt work was supported by grants from the Deutsche- inhibitor perifosine and etoposide through activation of intrinsic Forschungsgemeinschaft to MJ and GWM (JU255/2-4 and Fas-mediated extrinsic cell death pathways. Mol Caner Ther 5 and JU255/2-5). 2006; : 1559–1570. 10 Steelman LS, Bertrand FE, McCubrey JA. The complexity of PTEN: mutation, marker and potential target for therapeutic intervention. Expert Opin Ther Targets 2004; 8: 537–550. References 11 Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute myeloid leukemia cells requires PI3 kinase activation. 1 Rohrschneider LR, Fuller JF, Wolf I, Liu Y, Lucas DM. Structure, Blood 2003; 102: 972–980. function and biology of SHIP proteins. Dev 2000; 14: 12 Horn S, Meyer J, Heukeshoven J, Schulze C, Li S, Frey J et al. The 505–520. inositol 5-phosphatase SHIP is expressed as 145 and 135 kDa 2 Luo JM, Liu ZL, Hao HL, Wang FX, Dong Z, Ohno R. Mutation proteins in blood and bone marrow cells in vivo, whereas analysis of SHIP gene in acute leukemia. J Exp Hematol 2004; 12: carboxyl-truncated forms of SHIP are generated by proteolytic 420–426. cleavage in vitro. Leukemia 2001; 15: 112–120.

Gene Therapy