The NPM-RAR fusion protein associated with the t(5;17) variant of APL does not interact with PML

Leukemia Research 30 (2006) 979–986

The NPM-RAR fusion protein associated with the t(5;17) variant of APL does not interact with PML Elizabeth A. Rush a , Kimberly W. Schlesinger a , Simon C. Watkins b , Robert L. Redner a,∗ a b

Department of Medicine, University of Pittsburgh Medical Center and the University of Pittsburgh Cancer Institute, United States Department of Cell Biology, University of Pittsburgh Medical Center and the University of Pittsburgh Cancer Institute, United States Received 22 September 2005; received in revised form 22 December 2005; accepted 23 December 2005 Available online 28 February 2006

Abstract The PML protein localizes to regions of the nucleus known as nuclear bodies or PODs. However, in t(15;17) Acute Promyelocytic Leukemia (APL) blasts, PML is found in a micro-punctate pattern. In order to test the hypothesis that delocalization of PML from PODs is necessary for APL, we investigated the interaction of the t(5;17) APL fusion protein NPM-RAR with PML. NPM-RAR localizes diffusely throughout the nucleoplasm. NPM-RAR does not alter the localization of PML in transfected HeLa cells, and does not associate with PML in vitro. These studies suggest that NPM-RAR does not interact with PML. © 2006 Elsevier Ltd. All rights reserved. Keywords: Acute Promyelocytic Leukemia; PML-RAR; Nuclear bodies; t(15 ;17)

1. Introduction Acute Promyelocytic Leukemia (APL, FAB M3) is characterized by a maturational blockade of myeloid precursors at the promyelocytic stage [1]. Treatment with all-trans retinoic acid overcomes this arrest and induces differentiation of APL leukemic blasts [2–4]. Most patients with APL have a t(15;17)(q22;q12–21) chromosomal translocation, which results in the production of a PML-retinoic acid receptor alpha (RAR) fusion [5,6]. Ectopic expression of PML-RAR in cell culture [7,8] and animal models [9–12] reproduces the APL phenotype, implicating this fusion protein in the pathogenesis of APL. Wild-type RAR is a ligand dependent transcriptional activator [13]. The PML-RAR fusion protein contains the DNA binding, dimerization, ligand binding, nuclear localization, and C-terminal activation domains of RAR [5,6]. The PML protein was first identified by virtue of its involvement as the partner in the t(15;17) translocation. It contains an N∗ Corresponding author at: 2.18A Hillman Research Pavilion, 5117 Centre Ave., Pittsburgh, PA 15213, United States. Tel.: +1 412 623 3257; fax: +1 412 623 7768. E-mail address: [email protected] (R.L. Redner).

0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.12.029

terminal proline rich domain, a ring finger, and two variant Zn-finger B-boxes, followed by a leucine zipper, nuclear localization signal, and C-terminal serine and proline rich domain [5,6]. The most common t(15;17) breakpoints produce PML-RAR fusion proteins that retain the ring-finger, B-boxes, and leucine zipper motifs of PML. Numerous cellular functions have been attributed to the wild-type PML protein [14]. It has been suggested that the PML leucine zipper mediates dimerization, and that the ring finger functions as a protein interaction interface [15]. PML expression can be upregulated by interferon, though its role in mediating the effects of interferon remains controversial [16–20]. A variety of evidence suggests that PML functions as a tumor suppressor: co-transfected PML inhibits neu-induced transformation of NIH-3T3 cells [21,22]; PML overexpression in HeLa cells is accompanied by growth inhibition [23]; PML is pro-apoptotic [23–26]; PML expression is decreased in a variety of malignancies [27]; PML nullmutant animals are more susceptible to carcinogenic agents [25,28]. PML has been shown to interact with the apoptotic protein DAXX [29], and modulate p53 activity [14]. Several groups have suggested that PML function relates to its localization to discrete regions of the nucleus [30–32], alternatively called nuclear bodies (NBs), PML

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oncogenic domains (PODs), ND10, or Kr bodies. Within these 0.3–0.5 ␮m spherical or doughnut-shaped particles, the PML protein associates with up to 20 other proteins, including SP100 [31,33], NDP52 [34], PIC 1 [35], NDP55 [36], and ISG20 [37]. In addition, PLZF [38] and dephosphorylated retinoblastoma protein [39] have also been reported to co-localize with PML, as do a number of viral proteins [40–44]. The PML-RAR fusion protein heterodimerizes with wild type PML through its PML leucine zipper domain [5,6]. As a consequence, wild-type PML becomes de-localized from PODs to give a microspeculated nuclear appearance in fluorescent microscopy [30–32]. Retinoic acid reverses this redistribution: PML dissociates from PML-RAR and reaggregates into PODs as APL blasts differentiate. The recovery of PODs occurs 24–36 h after retinoic acid treatment, and is dependent upon cAMP [45]. It has been postulated that PML-RAR interaction with wild-type PML and de-localization from PODs is a key step in APL pathogenesis [30–32]. Conversely, a critical step in APL differentiation is recovery of normal POD structure and wild-type PML function. In this set of experiments we test this hypothesis through investigation of the t(5;17)(q32;q21) APL variant [46,47]. t(5;17) blasts are morphologically similar to t(15;17) cells, and like t(15;17) blasts, t(5;17) leukemic cells differentiate in vitro when cultured with 1 ␮M all-trans retinoic acid [48]. These phenotypic similarities make t(5;17) a unique model system in which to identify the common molecular pathways that underlie APL. t(5;17) blasts express a fusion of RAR with the N-terminus of nucleophosmin (NPM) [47], a nucleolar phosphoprotein that is also frequently mutated in AML with normal cytogenetics [49,50]. It has been postulated that the C-terminal mutations of NPM result in cytoplasmic localization of NPM, causing it to relocate binding partners out of the nucleus or nucleolus [49,50]. In this context, studies of the localization and binding partners of NPM-RAR gain significance. Prior reports of NPM-RAR localization have been somewhat inconsistent: Hummel et al. described a diffuse nuclear pattern [51], with limited nucleolar localization. In a series of follow-up experiments, Hummel et al. [52] reported that overexpression of NPM-RAR in COS cells gave a mixed nuclear and nucleolar pattern of expression. Melnick and Licht [53] cite NPM-RAR as appearing in a microspeculated pattern. We demonstrate here that NPM-RAR localizes diffusely throughout the nucleus. Furthermore, NPM-RAR does not alter PML localization, nor does it directly interact with PML.

erate a FLAG-PML or Maltose Binding Protein (MBP)-PML fusion protein, respectively. Bacterially expressed FLAGPML was purified over an M2 anti-FLAG immunoaffinity column according to the manufacturer’s protocol (Kodak). Purified FLAG-PML protein was used to immunize New Zealand White rabbits using Complete Freund’s Adjuvant (Sigma, St. Louis, MO) for the initial immunization, and Incomplete Freund’s Adjuvant (Sigma) for subsequent boosts. Bacterially expressed MBP-PML was affinity purified over amylose resin, using the manufacturer’s protocol (New England Biolabs). Immune serum was affinity purified over a MBP-PML sepharose column that was generated by reaction of purified MBP-PML protein with CNBr-sepharose, using the manufacturer’s protocol (Amersham Pharmacia, Piscataway, NJ); PML-specific antibodies were eluted with glycine–HCl (pH 2.5) and brought to neutral pH with 1 M Tris pH 8 buffer. 2.2. Constructs FLAG epitope-tagged NPM-RAR constructs were generated by PCR to fuse the sequence encoding the FLAG peptide at either the N-terminus or C-terminus of NPM-RAR, using as PCR primers the overlapping sequences 5 -TACAAGGACGACGATGACAAGGAAGATTCGATGGACATGGAC3 and 5 -GCGGGATCCACCATGGACTACAAGGACGACGATGAC-3 for the N-terminal tag, and 5 -CTTGTCATCGTCGTCCTTGTAGTCCGGGGAGTGGGTGGCCGGGCTGCT-3 and 5 -CGCGGATCCTCACTTGTCATCGTCGTCCTTGTAGTC-3 for the C-terminal tag. All amplified sequences were verified by sequencing using dideoxy chain termination (Sequenase, USBio, Cleveland, OH) and subcloned into pSG5 expression vectors (Stratagene, La Jolla, CA). 2.3. Cells HeLa and COS cells were obtained from the ATCC (Bethesda, MD), and grown in DMEM supplemented with 10% Fetal Calf Serum (Gibco, Grand Island, NY), 2 mM glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. HeLa cells were co-transfected with pSV2Neo and epitopetagged-NPM-RAR expression vectors (or pSG5 vector as control) at a 1:10 molar ratio using CaPO4 co-precipitation [54]. Stable transformants were selected in medium containing 1 mg/ml G418 (Gibco). 2.4. Immunoblotting

2. Methods and materials 2.1. PML antiserum PML cDNA was subcloned into pFLAG1 (Eastman Kodak, Rochester, NY) or a pMAL vector (New England Biolabs, Beverly, MA) in the appropriate reading frame to gen-

CaPO4 precipitation was used to transfect COS cells with 20 ␮g of a B19-LN expression vector [55,56] encoding PMLRAR cDNA, or a pSG5 expression vector encoding PML. At 48 h after transfection cells were lysed and immunoblots prepared as previously described [47]. Membranes were probed with 1 ␮g/ml PML antiserum, and developed using the

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Enhanced Chemiluminescence Protocol (Amersham Pharmacia). 2.5. Immunofluorescence Cells were grown on glass coverslips, fixed in 2% paraformaldehyde/0.1% Triton X-100, washed first with PBS and then with PBS/0.5% BSA/20 mM glycine, and incubated with 1:20 dilution of normal goat serum. Primary antibody was diluted in PBS/0.5% BSA/20 mM glycine/1:20 normal goat serum, and applied to the coverslips for 1 h at room temperature. Cells were washed with PBS/BSA/glycine buffer, and secondary antibody applied for 1 h at room temperature. Cells were washed extensively, and visualized with an immunofluorescent microscope. One microgram per milliliter affinity-purified rabbit polyclonal anti-PML antiserum or 1 ␮g/ml mouse M1 anti-FLAG (IBI) monoclonal antibody were used as primary antibodies; normal mouse or normal rabbit sera were used as controls. FITC-labeled goat anti-rabbit or Cy3-labeled goat anti-mouse antiserum (Jackson Research Laboratory, Westgrove, PA) were used as secondary antibodies. Cells were visualized using either a Leica TCS-NT scanning laser confocal microscope or an Olympus Provis microscope. Images were captured on a digital camera. 2.6. In vitro association The coding sequence for NPM-RAR or PML was subcloned into a malE expression vector (New England Biolabs) in the appropriate reading frame to encode a MBPfusion. MBP-PML, MBP-NPM-RAR, or control MBP protein was affinity purified from bacterial lysates over an amylose resin column (New England Biolabs). 35 S-methionine labeled NPM-RAR, PML, or PML-RAR proteins were generated by in vitro translation (TNT Coupled Reticulocyte Lysate System, Promega, Madison, WI) of the cDNA subcloned in pBluescript KS II plasmids (Stratagene). For association studies, 30 ␮g MBP-fusion protein and 20 ␮l of the 35 SNPM-RAR, 35 S-PML, or 35 S-PML-RAR in vitro translation products were incubated for 4 h at 4 ◦ C in 20 mM Tris pH 8, 110 mM NaCl, 1 mM EDTA, and 0.5% NP-40, and then allowed to bind to amylose resin. After extensive washing with the incubation buffer, the resin was boiled and the eluted products separated by SDS-PAGE. The gels were dried and the radiolabeled proteins imaged by autoradiography.

3. Results 3.1. Specificity of antiserum The affinity purified anti-PML antiserum recognized the appropriately sized PML or PML-RAR protein in lysates of transiently transfected COS cells (Fig. 1). A faint band of the same molecular weight as the overexpressed PML protein

Fig. 1. Specificity of anti-PML antiserum. An immunoblot of COS cells expressing PML-RAR (lane 1), PML (lane 2), or empty vector (lane 3) was developed with the anti-PML polyclonal antiserum.

was detected in COS cells transfected with the empty vector; this presumably represents endogenous PML protein. Immunofluorescence microscopy with the PML antiserum identified characteristic PML containing nuclear structures in HeLa cells (Fig. 3) as well as in HL60 and U937 cells (not shown).

3.2. NPM-RAR shows a diffuse pattern of nuclear localization To investigate the functional consequences of NPM-RAR expression, we stably transfected HeLa cells with an expression vector encoding an epitope-tagged (FLAG) NPM-RAR. Concern that the hydrophilic FLAG epitope might alter NPMRAR localization or protein interaction led us to construct fusion proteins that express the epitope tag at either the C-terminus (designated NPM-RAR-FLAG) or N-terminus (designated FLAG-NPM-RAR). Expression of appropriately sized FLAG-tagged proteins in the transfected HeLa subclones was confirmed by Western blots using both anti-FLAG and anti-RAR antisera (data not shown). Three independent subclones expressing N-terminal tagged NPM-RAR and five independent subclones expressing C-terminal tagged NPMRAR were analyzed. Fig. 2 shows a representative confocal micrograph of stably transfected HeLa cells. The NPM-RAR protein localizes diffusely throughout the nucleoplasm, excluding the nucleolus. The same diffuse nuclear pattern is seen in clones expressing the fusion proteins epitope-tagged at either the Cterminus (Panel C) or the N-terminus (Panel D), discounting the possibility that the pattern of localization might represent an artifact introduced by the epitope-tag. Analysis of two other independent FLAG-NPM-RAR HeLa clones and four NPM-RAR-FLAG HeLa clones revealed similar immunofluorescence patterns. Parental HeLa (Fig. 2, Panel A) and cells transfected with the empty vector (Fig. 2, Panel B) showed levels of immunofluorescence equivalent to background.

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Fig. 2. Localization of NPM-RAR. Confocal micrograph of localization of epitope-tagged NPM-RAR-FLAG (Panels E and F) and FLAG-NPM-RAR (Panels G and H) in transfected HeLa cells. Panels A and B show non-transfected parental cells; Panels C and D show a HeLa subclone transfected with an empty pSG5 vector. Panels A, C, E, and G show immunofluorescence after incubation with anti-FLAG primary antibody and a FITC-labeled secondary antibody; Panels B, D, F, and H were incubated with the negative control primary antibody before incubation with FITC-labeled secondary antibody.

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Fig. 3. Localization of PML in NPM-RAR expressing clones. Immunofluorescent micrographs double-labeled with antibodies against PML (green-FITC signal) and FLAG (red-Cy3 signal). HeLa clones were transfected with: nothing (Panels A–C), empty pSG5 vector (Panels D–F), NPM-RAR-FLAG (Panels G–I), or FLAG-NPM-RAR (Panels J–L). Cells were treated with media alone (Panels A, D, G, and J), 1 ␮M retinoic acid for 1 day (Panels B, E, H, and K), or 1 ␮M retinoic acid for 7 days (Panels C, F, I, and L).

3.3. NPM-RAR does not alter PML distribution To determine whether NPM-RAR expression induces redistribution of PML protein, we examined the localization of endogenous PML in the NPM-RAR-expressing HeLa clones. Fig. 3 reveals that PML localizes to typical POD structures of similar size and number in the NPM-RAR expressing subclones (Panels G and J) as in non-transfected HeLa (Panel A) or control-transfected clones (Panel D). No relocalization of PML protein to a microspeculated or cytoplasmic location was seen in scanning more than two hundred cells observed in three independent experiments. Treatment of the cultures with 1 ␮M all-trans retinoic acid had no effect on NPM-RAR distribution or PML distribution when cells were assayed

after 1 day (Fig. 3, Panels B, E, H, and K) or 7 days (Fig. 3 Panels C, F, I, and L). Identical results were obtained in NPMRAR subclones expressing the fusion protein tagged at either the C-teminus (Panels G–I) or N-terminus (Panels J–L). 3.4. NPM-RAR and PML do not interact in vitro The lack of co-localization of NPM-RAR and PML suggest that these proteins do not interact in vivo. Nevertheless, since NPM-RAR localizes diffusely throughout the nucleoplasm, microscopic analysis does not exclude the possibility that NPM-RAR might interact with PML protein without grossly changing its localization. We directly tested the ability of PML and NPM-RAR proteins to interact using an in

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Fig. 4. In vitro association assay. Panel A: 35 S-PML (lanes 1–3) or 35 SNPM-RAR (lanes 4–6) was incubated with MBP (lanes 2 and 5) or MBPNPM-RAR (lanes 3 and 6). Complexes were captured on amylose resin, and run on SDS-PAGE. Lanes 1 and 4 represent input material. Panel B: 35 SNPM-RAR (lanes 1 and 2) or 35 S-PML-RAR (lanes 3 and 4) was incubated with MBP-PML (lanes 1 and 3) or MBP (lanes 2 and 4).

vitro association assay. As depicted in Fig. 4, we did not detect interaction between MBP-NPM-RAR and radiolabeled PML (Fig. 4A), nor conversely between MBP-PML and radiolabeled NPM-RAR (Fig. 4B). However, in positive control experiments, we were able to detect association between MBP-PML and PML-RAR (Fig. 4B), and MBP-NPM-RAR and NPM-RAR (Fig. 4A).

4. Discussion We have shown that NPM-RAR protein localizes diffusely throughout the nucleoplasm. We find no direct interaction between PML and NPM-RAR. Retinoic acid treatment had no effect on the localization of either NPM-RAR or PML. Confirming the prior report of Hummel et al. [51], we find no alteration in the localization of PML from PODs in NPMRAR expressing cells. Despite containing the same nucleophosmin sequence that directs the NPM-ALK protein to the nucleolus [57], NPMRAR localizes throughout the nucleus but excludes the nucleolus. NPM-RAR thus shows a pattern on immunofluorescence that is similar to wild-type RARa [32]. This distribution is different than that of PML-RAR or PLZF-RAR, which localize in both APL blasts and transfected tissue culture models to microspeculated regions of the nucleus [31,32,38]. Recent reports have identified C-terminal NPM mutations in the majority of patients with AML having otherwise normal cytogenetics [49,50]. These mutations relocalize the mutated NPM to the cytoplasm, presumably relocalizing

NPM-binding proteins as well. The localization of NPMRAR takes on importance in any hypothesis that seeks to unify the mechanism underlying the NPMc and NPM-RAR mutations in AML. There have been two prior reports on NPM-RAR localization. In the initial report studying t(5;17) leukemic cells Hummel et al. described a diffuse nuclear pattern [51], with limited nucleolar localization. In a series of follow-up experiments, Hummel et al. [52] reported that overexpression of NPM-RAR in COS cells gave a mixed nuclear and nucleolar pattern of expression. Melnick and Licht [53], in their extensive review of the molecular basis of APL, allude to a microspeculated distribution of NPM-RAR. Our results would tend to agree with Hummel’s initial set of observations, though it is possible that discrepancies between Hummel’s later work and our findings relate to differences in cell lines used for study. Evidence indicating that PML functions as a tumor suppressor gene [14,21,23,27,28], as well as evidence suggesting that PML plays a role in myeloid maturation [30], have been used to support the hypothesis that PML-RAR dominant negative action on wild-type PML contributes to the APL phenotype. The observation that PML relocalization to PODs occurs early in the course of retinoic acid-induced differentiation also supports this hypothesis [32,45]. The hypothesis suggests that localization of PML to PODs is critical for PML function, and that delocalization is the mechanism of the dominant negative action of PML-RAR. This model would predict that fusion proteins expressed in APL variants would also alter PML localization. As previously reported by Hummel et al. [51], we found no such redistribution of PML protein in cells expressing NPM-RAR. Furthermore, we have shown that retinoic acid exposure does not change the distribution of NPM-RAR, nor PML. Other findings support our conclusion that delocalization of PML from PODs is not essential for the APL phenotype. t(11;17)(q23;q21) APL blasts, which have a somewhat different phenotype [58] than t(15;17) or t(5;17) APL, also show normal PML localization to PODs [58], as do blasts from a patient expressing the t(11;17)(q13;q21) NUMA-RAR protein [59]. Our conclusions are also supported by the identification of PML-RAR deletion mutants that retain biological activity yet fail to dimerize with PML and do not delocalize PML from PODs [60]. Conversely, adenovirus E1A and E4ORF3 [42], HSV-1 [61], and Arenavirus RING proteins [41] delocalize PML from the POD structure: none of these viruses have been implicated in APL or aberrant myelopoiesis. Does disruption of PML function contribute to the APL phenotype in the t(15;17) blasts? As cited above, wild-type PML may modulate cell growth or apoptosis, and may function as a tumor suppressor gene. PML is necessary for retinoic acid induced expression of p21/WAF1/CIP1, and PML may be a part of the RXR/RAR transcriptional complex [28]. PML may also have a role in apoptotic pathways [24,25]. PML might therefore have an important biological role independent of its interaction with other POD proteins, and hence disruption of wild-type PML, or binding of PML to PML-

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RAR, might indeed contribute to the t(15;17) phenotype. However, our data suggest that fusion protein interaction with PML and delocalization of PML from PODs is not an essential mechanism underlying APL. Acknowledgements The authors thank Daniel Johnson and Richard Steinman for helpful discussions, and Eric Loeffert for technical assistance. PML and PML-RAR cDNAs were gifts from Ronald Evans. Supported by grants from the National Institutes of Health Grant No. R29 CA67346, and the American Institute for Cancer Research Grant No. 96B057. References [1] Warrell Jr R, de The H, Wang ZY, Degos L. Acute promyelocytic leukemia. N Engl J Med 1993;329(3):177–89. [2] Castaigne S, Chomienne C, Daniel MT, Ballerini P, Berger R, Fenaux P, et al. All-trans retinoic acid as a differentiation therapy for acute promyelocytic leukaemia. I. Clinical results. Blood 1990;76(9):1704–9. [3] Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L, et al. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 1988;72(2):567–72. [4] Warrell RJ, Frankel SR, Miller WJ, Scheinberg DA, Itri LM, Hittelman WN, et al. Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid). N Engl J Med 1991;324(20):1385–93. [5] de The H, Lavau C, Marchio A, Chomienne C, Degos L, Dejean A. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell 1991;66(4):675–84. [6] Kakizuka A, Miller WJ, Umesono K, Warrell RJ, Frankel SR, Murty VV, et al. Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor. PML Cell 1991;66(4):663–74. [7] Grignani F, Ferrucci PF, Testa U, Talamo G, Fagioli M, Alcalay M, et al. The acute promyelocytic leukemia-specific PML-RARa fusion protein inhibits differentiation and promotes survival of myeloid precursor cells. Cell 1993;74:424–31. [8] Grignani F, Testa U, Fagioli M, Barberi T, Masciulli R, Mariani G, et al. Promyelocytic leukemia-specific PML-retinoic acid alpha receptor fusion protein interferes with erythroid differentiation of human erythroleukemia K562 cells. Cancer Res 1995;55(2):440–3. [9] Brown D, Kogan S, Lagasse E, Weissman I, Alcalay M, Pelicci PG, et al. A PMLRARalpha transgene initiates murine acute promyelocytic leukemia. Proc Natl Acad Sci USA 1997;94(6):2551–6. [10] Early E, Moore MA, Kakizuka A, Nason-Burchenal K, Martin P, Evans RM, et al. Transgenic expression of PML/RARalpha impairs myelopoiesis. Proc Natl Acad Sci USA 1996;93(15):7900–4. [11] Grisolano JL, Wesselschmidt RL, Pelicci PG, Ley TJ. Altered myeloid development and acute leukemia in transgenic mice expressing PML-RAR alpha under control of cathepsin G regulatory sequences. Blood 1997;89(2):376–87. [12] He LZ, Tribioli C, Rivi R, Peruzzi D, Pelicci PG, Soares V, et al. Acute leukemia with promyelocytic features in PML/RARalpha transgenic mice. Proc Natl Acad Sci USA 1997;94(10):5302–7. [13] Evans R. The steroid and thyroid hormone receptor superfamily. Nature 1988;240:889–95. [14] Salomoni P, Pandolfi PP. The role of PML in tumor suppression. Cell 2002;108(2):165–70.

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