WTAP Is a Novel Oncogenic Protein in Acute Myeloid Leukemia
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Letters to the Editor 1171 WTAP is a novel oncogenic protein in acute myeloid leukemia Leukemia (2014) 28, 1171–1174; doi:10.1038/leu.2014.16 understanding of leukemia biology in order to identify novel therapeutic targets. The Wilms’ tumor 1 (WT1) gene has an oncogenic role in leukemogenesis and its overexpression correlates with a poor The biology of acute myelogenous leukemia (AML) is character- prognosis.3 WTAP is a highly conserved protein that partners ized by a block in differentiation, increase in proliferation with WT14,5 to function as a switch gene, regulating the balance and inhibition of apoptosis, all of which when combined lead between quiescence and proliferation.6 WTAP-null mice exhibit to an expansion of leukemic blasts.1 AML therapy cures B20% embryonic lethality.7 WTAP has been recently described as an of those affected,2 highlighting the need for a better oncogenic factor in gliomas.8 We hypothesized that alterations in a b c 50 G-Primed CD34 NS shRNA Normal CD34 K562 WTAP shRNA HL-60 40 Normal PBL shRNA Primary AML cells 2.5 shRNA APL NS WTAP ) ) 2.5 NL AMLNL AML 6 NS WTAP 6 30 2 WTAP * 2 WTAP WTAP * 1.5 β-actin 20 β-actin 1.5 β-actin Frequency 1 * 1 * 10 0.5 0.5 Cell number(x 10 Cell number(x Cell number(x 10 Cell number(x 0 0 0 0 246 0 246 -4 -3 -2 -1 012 Time (d) Time (d) Log2 Concentration d e f shRNA 120 ** NS shRNA 40 NS WTAP 2300 WTAP shRNA 100 35 – Etoposide 30 + Etoposide 1800 ** 80 25 1300 60 20 ** 800 40 15 ** % Apoptosis 10 20 300 ** 5 % Colony formation % Colony 0 0 (mm3) volume Tumor -200 0 4 8 12 16 NS WTAP shRNA NS WTAP Time (d) shRNA g h i Isotype Isotype Control Control shRNA Control 120 WTAP-KD WTAP-KD NS WTAP NS WTAP WTAP #1 100 WTAP #2 pmTOR 80 Control WTAP #1 WTAP WTAP #2 WTAP ** mTOR 60 WTAP pS6K 40 ** β-actin 20 S6K % Viable % Viable cells β 0 1 2 3 4 5 1 2 3 4 5 -actin 0 h 48 h 72 h 10 10 10 10 10 10 10 10 10 10 CD11b-PE CD14-PE K562 HL-60 Figure 1. Expression of WTAP in AML and effect of WTAP silencing on AML cell behavior. (a) Peripheral blood mononuclear cells from normal donors (NL) and AML patients (AML) were obtained by Ficoll–Paque density centrifugation, and protein extracts were examined for WTAP expression by western blotting. (b) Histogram of WTAP expression measured by RPPA in bulk AML cells relative to normal CD34 þ cells. WTAP levels were above normal CD34 þ cells in 32% of AML patients and below normal in 9%. Expression is shown in log 2 scale. The genetic and clinical information on AML patients is described in Supplementary Table 1. (c) K562 and HL-60 cells were transfected with scrambled (NS) or WTAP shRNA, and selected by puromycin. The degree of WTAP knockdown was analyzed by western blotting (inset). Stable downregulation of WTAP expression by shRNA inhibited proliferation of K562 and HL-60 cells. *Pp0.05. (d) HL-60 cells stably expressing NS or WTAP shRNA were plated in methylcellulose medium, and colony formation was scored after 10 days. The values were expressed as % of colonies formed by WTAP- shRNA cells compared with NS shRNA cells taken as 100%. **Pp0.01. (e) Stable K562 cells with NS or WTAP shRNA were untreated or treated with etoposide (10 mM) for 48 h. The percentage of apoptotic cells was measured by the flow cytometric annexin-V/PI staining method. **Pp0.01. (f) Control (NS shRNA) or WTAP-shRNA K562 cells were subcutaneously implanted into flanks of nude mice and tumor formation and progression was monitored. Tumors originating from the control and WTAP-knockdown cells were excised and photographed. WTAP knockdown significantly reduced tumor growth in vivo.**Pp0.01. (g) Ba/F3 cells were transfected with WTAP or an empty vector and G418- selected stable clones (#1 and #2) were analyzed for WTAP overexpression by western blotting (left panel). Control and stable WTAP-expressing Ba/F3 cells were grown in an IL-3-free medium for indicated time and viable cells were assessed by annexin-V/PI staining followed by FACS analysis (right panel). **Pp0.01. (h) Control and stable WTAP knockdown (WTAP-KD) HL-60 cells were treated with 20 nM of PMA, and 96 h later, cells were labeled with anti-CD11b, anti-CD14 or an isotype-matched control. The expression of myeloid surface markers CD11b and CD14 was analyzed by flow cytometry. (i) WTAP knockdown inhibits the mTOR pathway. Control (NS) and stable WTAP knockdown K562 and HL-60 cell extracts were analyzed by western blot with the indicated antibodies. Accepted article preview online 13 January 2014; advance online publication, 11 February 2014 & 2014 Macmillan Publishers Limited Leukemia (2014) 1129 – 1174 Letters to the Editor 1172 Figure 2. Hsp90 associates with WTAP and is necessary for its stability. (a) The K562 cells were treated with vehicle ( À ) or Hsp90 inhibitor, ganetespib (1 mM for 6 h) followed by immunoprecipitation (IP) with IgG and WTAP antibody. The immunoprecipitates and cell lysates were immunoblotted (IB) with Hsp90, Hsp70 and WTAP antibodies. (b) The GST pull-down assay. In vitro-translated and 35S-methionine-labeled full- length WTAP was incubated with GST or GST-Hsp90 protein immobilized on glutathione–sepharose beads, and bound WTAP was detected by fluorography. Twenty percent of the in vitro-translated protein was used for pull-downs. (c) K562, MV4-11 and Kasumi-1 leukemia cells were treated with the Hsp90 inhibitor ganetespib (1 mM) for 24 h and analyzed for WTAP expression by western blotting. (d) AML blasts were isolated from peripheral blood or bone marrow of AML patients using Ficoll gradient separation followed by treatment with ganetespib (1 mM) for 24 h; WTAP expression was analyzed by western blotting. (e) MV4-11 tumor-bearing animals were given single doses of vehicle and ganetespib as described previously,14 and after 6 h, tumors were removed for western blot analysis using WTAP and b-actin antibody. (f) K562 cells were treated with vehicle ( À ) or proteasomal inhibitor (bortezomib; 100 nM) for 2 h and then treated with additional vehicle or ganetespib (1 mM) for 24 h, and cell lysates were subjected to western blot analysis using anti-WTAP and anti-b-actin antibodies. (g) K562 cells were treated as above for 6 h, and proteins extracts were immunoprecipitated (IP) with anti-WTAP followed by immunoblotting with anti- ubiquitin (top panel) and anti-WTAP (bottom panel) to detect the ubiquitinated WTAP (Ub-WTAP) and WTAP protein levels, respectively. WTAP, whose role in leukemogenesis is unknown, might provide WTAP knockdown alone did not induce apoptosis but markedly an alternate means of modulating the WT1 pathway in AML. increased (Pp0.01) the extent of apoptosis following etoposide We performed a series of experiments to test this possibility. First, treatment (Figure 1e). These results provide evidence for an we examined WTAP expression in several primary AML samples association between the increased expression of WTAP and observing increased levels of WTAP in AML compared with normal chemoresistance in AML. peripheral blood mononuclear cells (Figure 1a) as well as in AML To examine the role of WTAP in AML progression in vivo,we cell lines (data not shown). Next, the WTAP expression levels were performed tumor xenograft experiments in nude mice. As shown determined in 511 newly diagnosed AML patients using the in Figure 1f, the growth rates and masses of tumors derived from reverse-phase protein array (RPPA) technique. In comparison with WTAP-knockdown cells were significantly reduced (Pp0.01) normal bone marrow CD34 þ cells, WTAP expression in bulk AML compared with control. To complement this analysis, the in vitro cells was above normal levels in 32% of patients (Figure 1b). transforming activity of WTAP was examined by investigating its Although WTAP levels were not associated with individual effects on growth of the Ba/F3 cell line. This line depends on cytogenetic abnormalities, some specific molecular mutations interleukin 3 (IL-3) for survival and proliferation, but this such as NPM1 and FLT3-ITD were found to have significant dependence can be released by the transgenic expression of correlation (Pp0.05) with WTAP expression (Supplementary suitable oncogenes.9 Whereas control Ba/F3 cells were not viable Figure 1A). In addition, RPPA analysis showed that WTAP levels in the absence of IL-3 at 72 h, WTAP-expressing Ba/F3 cells were were positively correlated (R4|0.2|) with levels of various cell able to maintain growth factor-independent proliferation, as proliferation-related proteins (cyclins and Hsp90), antiapoptotic demonstrated by significantly higher (Pp0.01) number of viable proteins (Bcl-2 and Bax), oncoproteins (FLI1) and proteins cells (Figure 1g), suggesting that WTAP harbors oncogenic activity. important for stem cell functions such as Myc and Ash2L The aberrant cellular proliferation and terminal differentiation (Supplementary Figure 1B). To assess the functional significance block of myeloid cells are two hallmarks of AML.10 Having shown of increased WTAP expression, its expression was silenced in K562 that WTAP regulates growth and survival, we investigated whether and HL-60 cells, leading to a significant reduction (Pp0.05) in WTAP has a role in myeloid cell differentiation. As shown in proliferation (Figure 1c), and clonogenic survival (Pp0.01) Figure 1h, knockdown of WTAP promoted phorbol 12-myristate (Figure 1d). Similar effects on proliferation were observed in the 13-acetate (PMA)-induced myeloid differentiation, as revealed by AML cell line OCI-AML3 and in primary AML cells (Supplementary an increase in the expression of myeloid differentiation markers Figure 1C), suggesting a pro-proliferative role for WTAP in AML.