A Pathway from Leukemogenic Oncogenes and Stem Cell Chemokines to RNA Processing Via THOC5

ORIGINAL ARTICLE A pathway from leukemogenic oncogenes and stem cell chemokines to RNA processing via THOC5

F Griaud1,4, A Pierce1,4, MB Gonzalez Sanchez1, M Scott2, SA Abraham2, TL Holyoake2, DDH Tran3, T Tamura3 and AD Whetton1

THOC5 is a member of the THO complex that is involved in processing and transport of mRNA. We have shown previously that hematopoietic stem cells have an absolute requirement for THOC5 for survival and that THOC5 is phosphorylated on tyrosine 225 as a consequence of leukemogenic protein tyrosine kinase (PTK) action. We have investigated pathways for THOC5 phosphorylation to develop an understanding of THO complex modulation by tyrosine kinase (TK) oncogenes in leukemias. We demonstrate that THOC5 phosphorylation is mediated by Src PTK and CD45 protein tyrosine phosphatase action and that this event is sensitive to oxidative status. We show that THOC5 phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia (CML) and that this phosphorylation is sensitive to the frontline drugs used in CML treatment. Further we show that THOC5 Y225 phosphorylation governs mRNA binding. In addition, CXCL12 is shown to induce THOC5 Y225 phosphorylation, and site-directed mutagenesis demonstrates that this modulates motile response. In conclusion, we delineate a signaling pathway stimulated by leukemogenic PTKs, chemokines and oxidative stress that can affect THO complex mediation of gene expression describing mechanisms for post-transcriptional regulation of protein levels.

Leukemia (2013) 27, 932–940; doi:10.1038/leu.2012.283 Keywords: mRNA export complex THOC; motility; CML

INTRODUCTION activation of the macrophage-colony-stimulating factor receptor THOC5/Fms-interacting protein is a member of the THO complex, TK. It is also tyrosine phosphorylated on Y225 as a consequence 8 which in turn is a part of the TREX (transcription/export) complex. of leukemogenic protein TK (PTK) action. TREX has been conserved from yeast to man and has been shown THOC5 knockouts are embryonically lethal in the mouse, and to be required for coupled transcription elongation and nuclear we have shown that after induction of THOC5 knockdown, mice export of mRNAs.1 In Drosophila melanogaster, decreases in the die from rapid hematopoietic failure due to primitive hemato- 9 THO complex reduce overall protein synthesis by approximately poietic cell depletion. This collapse is far swifter than observed 10 half, although there is an increase in specific proteins, including with ATM-null mice, where self-renewal is compromised. Thus in those involved in DNA repair.2 In yeast, a similar effect is observed, hematopoietic terms, we have a protein implicated in stem cell suggesting a relationship between the THO complex and response survival, which is a target for downstream action of receptor PTKs to adverse changes in the cellular environment.3 The THO and leukemogenic PTKs and which modulates hematopoietic complex is associated with the messenger ribonucleoparticle, transcription factor action. the nascent RNA and the (nuclear) exported RNA. Mutations in the How these effects are achieved is not clear. Subcellular THO complex can lead to decreased polyadenylation of RNAs and localization of THOC5 has been shown to be in a dynamic their enhanced rate of degradation. Heat-shock protein 70 is one equilibrium with the protein shuttling between the cytosol and 11 gene product that can be regulated by THO complex; specifically, nucleus. This raises the possibility that THOC5 protein could be THOC5 binds heat-shock protein 70 messenger ribonucleoparticle critical in conveying survival/development signals, for example, and associates with the Tap-p15 bulk mRNA nuclear export from the macrophage-colony-stimulating factor receptor to the receptor to facilitate export.4,5 THOC5 has been implicated in spliceosome/mRNA export complex. We have investigated pathways the regulation of differentiation and development via modulation for THOC5 phosphorylation to determine its biological role. We of transcription factor C/EBPa,6 a gene often mutated in human report that THOC5 phosphorylation is sensitive to cellular oxidative leukemias. This protein regulates adipocyte and neutrophil/ status, an event mediated by Src PTK and CD45 protein tyrosine macrophage development.6 Indeed, THOC5 was originally phosphataseandthestemcellchemokineCXCL12.Theresultant named Fms-interacting protein and identified as a substrate and phosphorylation governs mRNA binding and may modulate motility. binding partner of the macrophage-colony-stimulating factor We have elucidated a receptor-mediated activation of CD45/Src receptor.7 THOC5 is a 75 kDa phosphoprotein found both in the kinases/THOC5 phosphorylation that is affected by leukemogenic nucleus and cytoplasm and is phosphorylated on tyrosine via oncogenes and found to occur in leukemia stem cells.

1Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Wolfson Molecular Imaging Centre, Withington, Manchester, UK; 2Paul O’Gorman Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, Gartnavel General Hospital, Glasgow, UK and 3Institut fuer Biochemie, OE4310, Medizinische Hochschule Hannover, Hannover, Germany. Correspondence: Dr A Pierce, Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Wolfson Molecular Imaging Centre, 27 Palatine Road, Withington Manchester M20 3LJ, UK. E-mail: [email protected] 4The first two authors contributed equally to this work. Received 28 August 2012; revised 24 September 2012; accepted 26 September 2012; accepted article preview online 3 October 2012; advance online publication, 23 October 2012 A role for THOC5 in response to oxidative stress F Griaud et al 933 MATERIALS AND METHODS Immunocytochemistry Cell lines K562 cells and CML CD34 þ cells were left untreated or treated with the Ba/F3 cells were transfected with either MSCV retroviral vector or MSCV indicated drugs for 24 h. Cells were harvested and spotted onto glass containing BCR/ABL, NPM/ALK, THOC5 and phosphomutants (Y to F) of NPM/ microscope slides coated with poly-L-lysine and were subsequently fixed ALK and THOC5 as described previously.12 K562 cells were maintained in RPMI with 3.7% (w/v) formaldehyde for 15 min and permeablized using a 0.25% (Gibco, Paisley, UK) with 10% (v/v) horse serum (Biowest, Nuaille´,France). (w/v) Triton-phosphate-buffered saline solution for 20 min. Cells were blocked with 5% (w/v) bovine serum albumin-phosphate-buffered saline and stained with the indicated primary antibodies and appropriate Patient samples secondary antibodies. Cells were concurrently stained with 4’, 6-diamidino- Human samples were leukapheresis products taken at the time of 2-phenylindole (Vectashield, Vector Laboratories Ltd, Peterborough, UK). diagnosis from patients with chronic phase chronic myeloid leukemia Cells were imaged using a Zeiss Imager M1 AX10 fluorescence microscope (CML), with informed consent in accordance with the Declaration of (Carl Zeiss, Birmingham, UK) and subjected to deconvolution (AxioVision Helsinki and approval of the National Health Service Greater Glasgow Software; Carl Zeiss) for image manipulation. Fluorescent signal was Institutional Review Board. The CD34 þ population was enriched using measured in three dimension by Image Processing and Analysis in Java CliniMACS (Miltenyi Biotec, Bisley, UK) according to standard protocols, (Image J) program. before storage as aliquots at À 150 1C. CML CD34 þ samples were cultured in serum-free media consisting of Iscove’s modified Dulbecco’s Transient transfection medium (Sigma-Aldrich Company Ltd, Gillingham, UK) supplemented with serum substitute (bovine serum albumin/insulin/transferrin, BIT9500; Transient transfection was achieved using the Amaxa Cell Line Nucleo- StemCell Technologies, Grenoble, France), 2 mML-glutamine, 105 U/ml fector II device (Lonza, Verviers, Belgium) following the manufacturer’s M protocols optimized for Ba/F3 cells (Kit V and Program X-001, Lonza). penicillin, 100 mg/ml streptomycin, 0.1 m 2-mercaptoethanol and 0.8 mg/ 21 ml low-density lipoprotein (Sigma-Aldrich). Serum-free medium was Constructs used contained wild-type Src or viral (activated) Src. supplemented with a growth factor cocktail of 0.20 ng/ml recombinant Transfection efficiency was measured with the vector pmaxGFP. Cells human stem cell factor, 1 ng/ml recombinant human interleukin-6, 0.20 ng/ were lysed 24 h post-electroporation and the phosphorylation of THOC5 ml recombinant human granulocyte colony-stimulating factor, 0.05 ng/ml assessed by western blot analysis. leukemia inhibitory factor, 0.2 ng/ml MIPa (macrophage inflammatory protein) (StemCell Technologies). Non-CML samples were CD34 þ - Chemotaxis and cell motility assays enriched leukapheresis products maintained and used as described for Chemotaxis assays were performed as described previously22 using a CML CD34 samples. þ Boyden chamber assay (Fisher Scientific, Loughborough, UK). The number of cells migrating through a 5 mm filter in response to 200 ng/ml CXCL12 Preparation of cellular nuclei and western blotting was assessed over 6 h. Nuclear proteins were enriched using a nuclear isolation kit from Active motif (la Hulpe, Belgium) with some modifications as described 13 THOC5–mRNA complex isolation previously. Western blotting was performed using standard protocols. Ba/F3 cells expressing NTAP-THOC5 and NTAP-THOC5 Y225F in MSCV were Antibodies used were actin (Sigma-Aldrich; A5060), a-tubulin (Santa produced as described previously.23 The THOC5–mRNA complexes were Cruz, Heidelberg, Germany; sc-5286), phospho-CRK-L (Cell Signalling isolated and reverse transcription-polymerase chain reaction performed as Technology, Hitchin, UK; no. 3181), GAPDH (Cell Signalling Technology; 4 described previously. Briefly, cells were lysed in lysis buffer (10 mM Tris, no. 5174), heat-shock protein 70 (Cell Signalling Technology; no. 4875), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 0.4% NP-40) by freeze lamin A/C (Cell Signalling Technology; no. 2032), myc (Cell Signalling thawing and complexes isolated by incubation with streptavidin sepharose Technology; no. 2276) and Src and phosphotyrosine 416 Src (Cell (GE Healthcare, Amersham, UK) in lysis buffer with 10 mM mercaptoethanol Signalling Technology; nos. 2109 and 2101). Antibodies to THOC5 (F6D/ and 0.5 mM EDTA. Reverse transcription was carried out using oligo(dT) 11) were produced by the Leukemia and Lymphoma Research-funded primers and the Omniscript reverse transcriptase kit from Qiagen (Crawley, Immunodiagnostics Unit (Oxford, UK) and the anti-phospho-Y225 THOC5 UK). Primer pairs for Gigyf2 were 50-CGCCGACTGGAAGAGAACC-30 and by Eurogentec (Seraing, Belgium). 50-TTGCTGTGTTAGACTGCTGAC-30 (235 bp).

Inhibitor treatments Cells were treated with ATM inhibitor KU-55933 (ref. 14) (Tocris Bioscience, RESULTS Bristol, UK) at 10 mM for 4 h, MEK1/2 inhibitor U0126 (ref. 15) (Cell Signalling Structure function analysis on NPM/ALK reveals the key Technology) at 10 mM for 1 h, Src inhibitor SU6656 (ref. 16) at 20 mM for 1 h residues for activation of tyrosine phosphorylation and phosphatidylinositol-3 kinase inhibitor LY294002 (ref. 17) at 10 mM for We have recently demonstrated that the THOC5 protein 4 h (Calbiochem, Nottingham, UK). Etoposide VP-16 (PCH Pharmachemie, is a downstream target of the leukemogenic PTKs BCR/ABL, Haarlem, The Netherlands) was used at 20 mM for 20 min, unless stated FIP1L/PDGFRa, TEL/PDGFR and NPM/ALK.8 To discover the otherwise, hydrogen peroxide (H2O2) (Sigma-Aldrich) at 50 mM for 20 min and 4-hydroxynonenal (HNE) at 10 mM for 20 min (Cambridge Bioscience, potential pathways lying downstream from the oncogenic PTKs, Cambridge, UK). Imatinib mesylate (Selleck Chemicals, Newmarket, UK) was NPM/ALK mutants were expressed in Ba/F3 cells, and the effect of used at 5 mM for 4 h and at 5 mM overnight on human primary cells. site-directed mutagenesis on the ability to promote phosphory- 18 Dasatinib (Bristol-Myers Squibb, Uxbridge, UK) was used at 50 nM on K562 lation on tyrosine 225 of THOC5 assessed. Mutation of NPM/ALK to 19 and 150 nM overnight for human primary cells. Nilotinib (Novartis, create a kinase-dead version showed that the kinase activity of Camberley, UK) was used at 5 mM overnight for human primary cells. NPM/ALK is required for the observed increase in THOC5 phosphorylation (Figure 1a). Flow cytometry Initial analysis of point-mutated NPM/ALK identified Y461F, For cell surface staining, CD34 þ cells were incubated with the appropriate Y567F and Y644F triple mutant as deficient in the ability antibodies for 20 min in the dark in 2% fetal calf serum/phosphate- to promote Y225 THOC5 phosphorylation (Figure 1a). Single buffered saline. Antibodies used were anti-CD34-APC, CD38-PerCP, CD90- mutation analysis then identified Y567F, but not Y461F, Y644F PE (BD Biosciences, Oxford Science Park, Oxford, UK). For intracellular NPM/ALK as deficient in this respect (Figure 1a). The Y567 staining, CD34 þ and K562 cells were re-suspended in ‘Fix and Perm’ phosphoresidue of NPM/ALK is known to bind ShcA.24 All three following the manufacturer’s instructions (Merck Chemicals Ltd, Notting- isoforms of the ShcA family are phosphorylated by PTKs, including ham, UK). Primary antibody was added at room temperature for 1 h at 1/100 dilution and anti-rabbit FITC (Sigma-Aldrich) secondary for 30 min, p66 Shc, which can enhance reactive oxygen production by and also at 1/100 dilution. Samples were analyzed by flow cytometry mitochondria. using the FACSCanto Flow Cytometer (Becton Dickinson, Oxford, UK). Data Based on these data and the known ability of leukemogenic analysis was performed with FACSDiva (Becton Dickinson) and FlowJo PTKs to stimulate reactive oxygen production, we assessed the 20 (Tree Star, Olten, Switzerland) software. ability of H2O2 to promote THOC5 Y225 phosphorylation. Addition

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Figure 1. THOC5 Y225 phosphorylation. (a) Cells expressing different tyrosine mutants of NPM/ALK were lysed and their ability to phosphorylate THOC5 on Y225 assessed by western blot analysis with a phospho-Y225 (pY225) phospho-speciﬁc antibody. Cells were treated with 50 mM H2O2 for the times shown (b) before being lysed and the levels of Y225-phosphorylated THOC5 assessed by western blot analysis. (c) Following isolation, 10 mg of nuclear and 30 mg of cytoplasmic lysates from Ba/F3 cells were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and the distribution of THOC5 and pY225 THOC5 assessed by western blot analysis. Tubulin and histone H3 are used as loading controls and markers of fractionation quality. (d) Control MSCV cells and myc-tagged THOC5 Y225F mutant-transfected cells were subject to nuclear and cytoplasmic fractionation. Cellular distribution of endogenous and Y225F mutant THOC5 was then assessed by western blot analysis with anti-THOC5 and anti-myc antibodies on 10 mg of nuclear and 30 mg of cytoplasmic lysates. Tubulin and lamin are used as loading controls and markers of fractionation quality.

27 of H2O2 to Ba/F3 cells led to a transient increase in THOC5 Y225 overexpression, which leads to opposite response to oxidative phosphorylation (Figure 1b). stress as compared with ATM.28 This could account for the different THOC5 is part of the THO complex that is critical for the consequences of their inhibition on THOC5 phosphorylation. With processing and transport of RNA. Under normal conditions, THOC5 respect to kinases downstream of stress-induced signaling (eto- has a nuclear:cytoplasmic location and has been shown to shuttle poside or H2O2), Src inhibitor completely abrogated THOC5 Y225 between these compartments. We have previously shown THOC5 phosphorylation. Unexpectedly, MEK inhibition led to an increase in not only to be phosphorylated as a consequence of macrophage- Y225 phosphorylation. Further studies treating the cells with the colony-stimulating factor stimulation but also indeed to interact MEK and Src inhibitors, while simultaneously subjecting the cells to with its receptor c-fms.11 We therefore hypothesized that the DNA damage by etoposide treatment, showed that Src activity is phosphorylation of THOC5 at Y225 was a cytosolic event. Western required for the etoposide-induced increase in THOC5 phosphoryla- blot analysis of nuclear and cytosolic fractions showed phospho- tion (Figure 2c). The MEK inhibitor again resulted in an increase in Y225 to reside predominantly in the cytoplasm (Figure 1c). We phosphorylation. As already stated, we have previously shown that also generated an myc-tagged phosphomutant Y225F THOC5 to leukemogenic oncogenes lead to an increase in Y225 phosphoryla- determine if phosphorylation inﬂuenced subcellular localization. tion. As these oncogenes are known to activate Src, we asked the This was not the case as the proportion of Y225F THOC5 in the question whether inhibition of Src in oncogene-transfected cells nucleus was the same as native THOC5 (Figure 1d). would lead to a decrease in Y225 phosphorylation. This was indeed As cellular stress can be caused by chemotherapeutic agents the case indicating that Y225 phosphorylation is downstream of Src that can promote oxidative stress,25 we also assessed the effects following DNA damage and oncogene action (Figure 2d). Further- of etoposide on THOC5 phosphorylation. We have previously more, the induction of THOC5 Y225 phosphorylation following published that the appropriate concentration of etoposide to H2O2 treatment was prevented when Src was inhibited (Figure 2e). induce double-strand breaks in DNA (as a measure of biological It has been reported that oxidative stress is linked to Src activation 26 29 response) in Ba/F3 cells is 20 mM. Figure 2a demonstrates that via lipid peroxidation. As HNE is produced during oxidative stress, treatment of cells with etoposide (20 mM) led to a transient we asked whether HNE could lead to THOC5 Y225 phosphorylation increase in Y225 phosphorylation. THOC5 has been shown to be a and whether this was via Src activation. Treatment of cells with HNE target for the ATM kinase in respect of serine phosphorylation. led to an increase in phospho-Y225 (Figure 2f), which was inhibited ATM is a DNA damage-associated protein kinase as is the PTK if Src inhibitors were present (Figure 2g). Transient transfection of c-ABL. To investigate the potential kinases that may be involved in Ba/F3 cells with both active and inactive Src mutants further the phosphorylation of THOC5, we undertook a study using demonstrated that THOC5 Y225 is phosphorylated as a conse- protein kinase inhibitors implicated in downstream signaling from quence of Src activity (Figure 2h). Transfection of only the active Src DNA damage. Figure 2b illustrates that while the inhibition of abl led to an increase in Y225 phosphorylation. had no effect on Y225 phosphorylation, ATM inhibition increased THOC5 phosphorylation. Although one cannot discard the possibility, the differences are due to off-target effects. THOC5 phosphorylation is elevated in primary CML stem cells ATM inhibition has been shown to increase reactive oxygen Having established the fact that THOC5 Y225 was a target for species,10 whereas Abl deﬁciency results in antioxidant gene numerous leukemogenic PTKs, we were keen to demonstrate that

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Figure 2. Mechanism of THOC5 phosphorylation. (a) Ba/F3 cells were treated with 20 mM etoposide for the times indicated before being lysed and the levels of THOC5 phosphorylation assessed by western blot analysis. (b) Western blot analysis of THOC5 phosphorylation in Ba/F3 cells following treatment with 5 mM imatinib (ABLi), 10 mM KU-55933 (ATMi), 10 mM LY294002 (phosphatidylinositol-3 kinase inhibitor PI3Ki)) for 4 h or 10 mM U0126 (MEK1/2i) and 20 mM SU6656 (SFKi) for 1 h. (c) Cells were treated with 20 mM etoposide with or without a 1 h preincubation with 20 mM SU6656 (SFKi) or 10 mM U0126 (MEK1/2i). Levels of phospho-Y225 THOC5 were then assessed by western blot analysis. (d) Cells expressing the leukemogenic oncogenes BCR/ABL, NPM/ALK and Tel/PDGFRb were treated with 20 mM SU6656 (SFKi) for 1 h and the level of phospho-Y225 THOC5 assessed by western blot analysis. Cells were treated with 50 mM H2O2 (e)or10mM HNE (g) for 20 min with or without a 1 h preincubation with 20 mM SU6656 (SFKi) or with 10 mM HNE alone for the times indicated (f) and the level of phospho-Y225 THOC5 assessed by western blot analysis. (h) Cells were transfected with pcDNA constructs containing either GFP, activated viral or wild-type Src and the affect on Y225 THOC5 phosphorylation assessed by western blot.

THOC5 phosphorylation was present in primary cells. To confirm we patients with CML. We then compared phospho-Y225 levels from were seeing human leukemia-associated effects, we firstly confirmed CML and healthy individuals by flow cytometry not only in the that BCR/ABL led to an increase in THOC5 phosphorylation in a CML bulk CD34 þ cell population but also in the more primitive patient-derived cell line. K562 cells were treated with the BCR/ABL CD34 þ CD38 À cells (Figure 4c). All three cell populations showed inhibitor dasatinib and then subjected to immunofluorescence higher levels of expression of phospho-Y225 THOC5 in CML than microscopy using the phospho-Y225 THOC5 antibody (Figure 3a): normal controls. the loss of phospho-Y225 observed following dasatinib treatment Dasatinib, in addition to inhibiting BCR/ABL, also inhibits Src. We was further confirmed by western blot (Figure 3b) and phospho- were therefore interested whether the other two drugs commonly CRKL assay demonstrated successful inhibition of BCR/ABL kinase used in the treatment of CML, namely imatinib, and nilotinib, activity. Flow cytometry measurements also demonstrated the which have no Src inhibitory activity, also inhibited THOC5 Y225 sensitivity of phospho-Y225 levels to dasatinib treatment (Figure 3c). phosphorylation. Figure 4d clearly shows that phospho-Y225 These observations were then repeated in primary cells. levels were reduced by all three drugs in all three cell populations. First, we demonstrated by immunofluorescence (Figure 4a) and The potential role of THOC5 phosphorylation and its regulation by western blot (Figure 4b) that phospho-Y225 levels were sensitive kinase inhibitors in primary leukemic stem cells led us to examine to dasatinib treatment in the bulk CD34 þ cell population from its function more closely.

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Figure 3. THOC5 Y225 phosphorylation is sensitive to dasatinib treatment in human CML cells. K562 cells were treated with 50 nM dasatinib for 24 h before THOC5 phospho-Y225 levels were assessed by immunofluorescence (a), western blot analysis (b) or flow cytometry (c). Western blot analysis of CRKL and phospho-CRKL were used as controls for the efficacy of dasatinib treatment and GAPDH as a loading control. Flow cytometry data are displayed as mean fluorescence intensity±s.e.m.

THOC5 Y225 involvement in stem cell homing chemokine Phosphorylation of Y225 modulates mRNA binding signaling pathways We have previously identiﬁed mRNAs that bind to THOC5 and rely There are a variety of cellular stimuli that can activate the Src on THOC5 for export from the nucleus.4 Here we tested whether kinase family. We therefore considered whether there were other mutation of tyrosine 225 had an effect on the ability of THOC5 to hematopoietic stem cell-relevant pathways to THOC5 phosphor- bind to just such an mRNA. THOC5 and THOC5 Y225F fused ylation. CXCL12 is less able to promote chemotaxis in the presence to streptavidin-inducing peptide (pNTAP) were expressed in Ba/F3 of BCR/ABL.30 This suggests some overlap in CXCL12 and BCR/ABL cells and the THOC5 containing messenger ribonucleoparticle signaling in hematopoietic cells. As BCR/ABL induces THOC5 enriched with streptavidin conjugated to sepharose. Western blot phosphorylation8 and CXCL12 can promote activation of Src, we analysis of the cell lysates (input) and enriched samples (bound) therefore investigated its effects on Y225 THOC5 phosphorylation. demonstrate the isolation of THOC5–messenger ribonucleoparti- This would also demonstrate whether G-protein-coupled receptor cle complex (Figure 5d, upper panels). Reverse transcription- agonists can affect THOC5 phosphorylation. We found that polymerase chain reaction analysis (Figure 5d, lower panels) CXCL12 stimulation led to a transient increase in THOC5 clearly showed that mutation of Y225 abolishes THOC5’s ability to phosphorylation (Figure 5a). Further evidence that this was a bind to the mRNA for Gigyf2. This implicates the phosphorylation direct consequence of CXCL12-mediated Src activation was of tyrosine 225 of THOC5 via leukemogenic kinases in the obtained by investigating THOC5 phosphorylation status in modulation of mRNA export and hence translational control. CD45-null mast cells. In CD45 wild-type cells, Src was activated Of note is the larger input of endogenous THOC5 in the lysate of as illustrated by Src autophosphorylation on Y416, and THOC5 THOC5 Y225F-expressing cells to ensure normalization of the phosphorylated. In the absence of CD45, Src was not activated two TAP-THOC constructs and subsequent co-enrichment of and THOC5 was not phosphorylated, indicating that Src and CD45 endogenous THOC5. are upstream of THOC5 phosphorylation (Figure 5b). We next investigated whether the phosphorylation of THOC5 had an effect on CXCL12-induced motility. Motility assays were performed on DISCUSSION cells transfected with THOC5 and Y225F mutant THOC5. Figure 5c We have previously demonstrated that leukemogenic PTKs clearly shows that the presence of the Y–F mutation increases the promote the phosphorylation of THOC5 on tyrosine 2258 cellular response to CXCL12. Thus, the data infer the Y225 site has and that THOC5 is a key protein in the maintenance of a role in motile regulation via an as yet unidentiﬁed mechanism. hemopoietic stem cells in vivo.9 The aims of this study were to

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Figure 4. THOC5 Y225 phosphorylation levels are higher in primary CML patients when compared with normal controls and is sensitive to BCR/ABL kinase inhibitors. CD34 þ cells from patients with CML were treated with 50 nM dasatinib for 24 h before THOC5 phospho-Y225 levels were assessed by immunofluorescence (a) and western blot analysis (b). Heat-shock protein 70 (HSP90) was used as a loading control in the western blot analysis. (c) Flow cytometric comparison of THOC5 phospho-Y225 expression in CML (n ¼ 4) and normal (n ¼ 3) CD34 þ , CD34 þ CD38 þ and CD34 þ CD38 À cells. Results are displayed as mean fluorescence intensity±s.e.m. Student’s t-test indicates that the CD34 þ and the CD34 þ CD38 À samples are significantly different between normal and CML cells (P ¼ 0.019 and 0.018, respectively). (d) THOC5 phospho-Y225 levels were assessed in the populations shown by flow cytometry following 24 h treatment with 5 mM imatinib, 150 nM dasatinib or 5 mM nilotinib. Results are displayed as mean fluorescence intensity (n ¼ 4)±s.e.m. identify the mechanism and consequences of THOC5 tyrosine non-transformed Ba/F3 cells that express c-Abl, this elevated phosphorylation. THOC5 phosphorylation in primary CML cells was sensitive to Abl Inferences from structure/function analysis of NPM/ALK impli- inhibitors when the active PTK BCR/ABL was present. The fact that cated oxidative phosphorylation in the induction of THOC5 imatinib and nilotinib are as effective as dasatinib (BCR/ABL and phosphorylation. Oxidative stress did indeed lead to an increase Src inhibitor) in reducing THOC5 phosphorylation suggests that in THOC5 Y225 phosphorylation and we were able to further BCR/ABL kinase inhibition may be sufficient to decrease THOC5 demonstrate that genotoxic stress increased THOC5 phosphoryla- phosphorylation. Other leukemogenic PTKs enhance production tion via oxidative stress including lipid peroxidation, and also of reactive oxygen species and this effect can be reversed by activation of Src kinase (see Figure 6). Src family kinases such as TK inhibitors.33 Foxo3a can protect the CML stem cell via increased Lyn have been shown to have a role in CML progression to blast expression of antioxidative stress proteins.34,35 Nonetheless, crisis as well as being a direct source of imatinib and nilotinib there is a BCR/ABL-associated oxidative stress-related protein resistance in CML,31,32 suggesting that THOC5 phosphorylation phosphorylation event seen in the CML stem cells, THOC5 might be involved in the pathogenesis of leukemias. We showed phosphorylation. that THOC5 tyrosine phosphorylation is higher in primary human Src activation has been shown to be regulated by CD45 tyrosine CML CD34 þ cells than in normal cells and is expressed in the phosphatase, a key transmembrane glycoprotein involved primitive CML CD34 þ CD38 À CD90 þ leukemia stem cells. While in hematopoiesis and stem cell motility.36 CD45 is able to Abl inhibition had no effect on THOC5 phosphorylation in the dephosphorylate Src on either inhibitory tyrosine 527 or on the

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Figure 5. CXCL12 induced phosphorylation of THOC5. (a) Ba/F3 cells were treated with 200 ng/ml of CXCL12 for the times indicated before the cells were lysed and phospho-Y225 THOC5 levels assessed by western blot analysis. (b) Mast cells isolated from CD45-null and CD45 wild-type (wt) mice were lysed and expression of the proteins indicated assessed by western blot analysis. Actin was included as a loading control. (c) The ability of cells to migrate in response to 200 ng/ml CXCL12 was assessed in Boydon chamber assay. The number of cells migrating in the presence and absence of CXCL12 are shown. (d) THOC5–mRNA complexes were isolated from Ba/F3 NPM/ALK cells expressing NTAP wtTHOC5 or NTAP Y225F THOC5 by binding to streptavidin sepharose. Cell lysates (input) and puriﬁed samples (bound) were analyzed by western blot for THOC5 expression and by reverse transcription-polymerase chain reaction (RT-PCR) for Gigyf2 expression.

activating tyrosine 416, thus potentially having antagonistic action residual disease.41 We are currently assessing the role of THOC5 in on different Src family kinase members and at different stages of motility and how this inﬂuences leukemic cell behavior. development.37 Using CD45-null/wild-type mast and Lin À cells, Preliminary proteomic data we have generated on the THOC5 we have shown that CD45 phosphatase is required for Src interactome suggests that in addition to members of the THO activation (as determined by its autophosphorylation on tyrosine complex, THOC5 interacts with numerous proteins involved 416) and THOC5 phosphorylation on tyrosine 225. As CD45 also in motility (T Tamura and AD Whetton, unpublished data). Thus, regulates motility, a process impaired in leukemia, we further it may be a multifunctional protein. investigated the connection between THOC5 and CD45 with THOC5 is required for primitive cell survival9 and has been stimulation of CD45 by CXCL12/CXCR4. CXCL12 is a chemokine shown to modulate the speciﬁc nuclear export of mRNAs involved expressed by the bone marrow stromal cells, which has a key role in cell development and hematopoiesis.4 Given the role of reactive in the migration of hematopoietic stem/progenitor cells to bone oxygen species in stem cell biology and THOC5 phosphorylation, marrow during fetal development and then their retention we asked whether tyrosine 225 phosphorylation has any part in during adult life.38,39 Our data showed that THOC5 tyrosine mRNA binding and export. Using a known THOC5 binding mRNA, phosphorylation is mediated by the CXCL12/CXCR4 pathway in we were able to demonstrate that indeed THOC5 phosphorylation such a way as to suggest that THOC5 phosphorylation participates on tyrosine 225 affected the mRNA binding properties of THOC5. in a negative feedback loop. CXCL12 leads to the phosphorylation Tyrosine 225 is actually within the mRNA binding domain of of THOC5, whereas cells expressing the Y–F mutant of tyrosine 225 THOC5 (T Tamura, unpublished observations) and as we show that display an enhanced response to CXCL12. These observations are phosphorylation of this residue affects mRNA binding. Given that consistent with the fact that BCR/ABL induces THOC5 phosphor- Y225 is a downstream target of numerous leukemogenic PTKs, it ylation and display a reduced ability to respond to CXCL12.23 It can be hypothesized that this phosphorylation event modulates could be hypothesized that inhibition of CXCR4 with perixafor, for mRNA processing and contributes to the pathogenesis of example, in the presence of CXCL12, would inhibit the leukemia and as such offers a novel pathway for intervention in phosphorylation of THOC5. disease treatment. Our results are thus developing an understanding on THO In conclusion, we propose that THOC5 phosphorylation is part complex modulation by TK oncogenes in leukemias and of a mechanism to inform the transcription/translation machinery chemotactic factors that control stem cell retention in the marrow. of a change in environment requiring modulation of translation. This may, in part, be an explanation for the loss of stem cells so Leukemogenic PTKs and receptors modulate the pathway by rapidly in THOC5-inducible knockout mice.9 The CXCL12/CXCR4 affecting THOC5 phosphorylation via reactive oxygen species pathway is perturbed in CML and has recently been reported to production and Src TK activation (summary depicted in Figure 6). contribute to non-pharmacological drug resistance.40 Indeed, it This in turn modulates THOC5 mRNA binding and expression has been proposed that CXCR4 inhibition in conjunction with TK of transcripts for key entities in transformation, such as Myc inhibition could over-ride drug resistance in CML and reduce (T Tamura, unpublished observations).

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Leukemia (2013) 932 – 940 & 2013 Macmillan Publishers Limited