Translocation-Generated ITK-FER and ITK-SYK Fusions Induce STAT3 Phosphorylation and CD69 Expression

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Biochemical and Biophysical Research Communications xxx (2018) 1e4

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Translocation-generated ITK-FER and ITK-SYK fusions induce STAT3 phosphorylation and CD69 expression

Narmeen N. Fathi a, b, Dara K. Mohammad a, c, AndreG orgens€ a, d, Samir EL. Andaloussi a, * ** Rula Zain a, e, Beston F. Nore a, f, , C.I. Edvard Smith a, a Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86, Huddinge, Sweden b Department of Microbiology, College of Medicine, University of Sulaimani, Sulaimaniyah, Kurdistan Region-Iraq, Iraq c Department of Biology, College of Science, Salahaddin University-Erbil, 44002, Erbil, Kurdistan Region-Iraq, Iraq d Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, 45122, Essen, Germany e Department of Clinical Genetics, Centre for Rare Diseases, Karolinska University Hospital, SE-171 76, Stockholm, Sweden f Department of Biochemistry, College of Medicine, University of Sulaimani, Sulaimaniyah, Kurdistan Region-Iraq, Iraq article info abstract

Article history: Many cancer types carry mutations in protein tyrosine kinase (PTK) and such alterations frequently drive Received 20 August 2018 tumor progression. One category is gene translocation of PTKs yielding chimeric proteins with trans- Accepted 4 September 2018 forming capacity. In this study, we characterized the role of ITK-FER [Interleukin-2-inducible T-cell Ki- Available online xxx nase (ITK) gene fused with Feline Encephalitis Virus-Related kinase (FER) gene] and ITK-SYK [Interleukin- 2-inducible T-cell Kinase (ITK) gene fused with the Spleen Tyrosine Kinase (SYK)] in Peripheral T Cell Keywords: Lymphoma (PTCL) signaling. We observed an induction of tyrosine phosphorylation events in the Chimeric tyrosine kinases presence of both ITK-FER and ITK-SYK. The downstream targets of ITK-FER and ITK-SYK were explored Fusion genes ITK and STAT3 was found to be highly phosphorylated by these fusion kinases. In addition, the CD69 T-cell ﬁ SYK activation marker was signi cantly elevated. Apart from tyrosine kinase inhibitors acting directly on the FER fusions, we believe that drugs acting on downstream targets could serve as alternative cancer therapies Oncogenes for fusion PTKs. © 2018 Elsevier Inc. All rights reserved.

1. Introduction protein [4]. Over the last years, Tyrosine Kinase Inhibitors (TKIs) have become a very attractive and effective treatment in many Oncogenic transformation alters signal transduction in multiple types of cancers [5e7] and have shown promising target selectively ways [1]. Mutations causing the formation of constitutively-active [8]. Although, the efficacy of such drugs is high, recurrent resistance or dominant-negative proteins, including the formation of constitutes a general problem. Therefore, cell-based characteriza- chimeric fusions of tyrosine kinases, represent underlying mecha- tion of the fusion proteins is essential to determine downstream nisms. Examples of fusions are the TEL gene (E26 Transforming- signaling molecules, which could serve as alternative targets for Specific [ETS] Translocation Variant gene 6, ETV6) fused with SYK intervention. (Spleen Tyrosine Kinase) forming the TEL-SYK protein [2], the The ITK-FER fusion was detected in Peripheral T-Cell Lymphoma interleukin-2-inducible T-cell Kinase (ITK) gene fused with the (PTCL) in one of eleven patients (9%). The fusion gene, comprised of SYK gene creating the ITK-SYK chimera [3] and the recently exons 1 to 8 of ITK and exons 11 to 20 of FER, resulted in the described fusion between ITK and the Feline Encephalitis abnormal expression of a chimeric kinase in the lymphoma cells virus-Related kinase (FER) gene creating the novel ITK-FER chimeric [4]. The ITK part contains 256 amino acids of PH (Pleckstrin homology), TH (Tec Homology), SH3 (Src Homology 3) domains with a portion of the SH2 domain, whereas the FER portion contains a * Corresponding author. Dept. of Laboratory Medicine, Clinical Research Center, stretch of 409 C-terminal amino acids (413-822 aa), encompassing Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86, Hud- part of FX, the SH2 and the tyrosine kinase domains (Fig. 1A). ITK dinge, Sweden. belongs to the TEC family of non-receptor tyrosine kinases, con- ** Corresponding author. fi E-mail addresses: [email protected] (B.F. Nore), [email protected] sisting of ve members: BTK, ITK, RLK/TXK, TEC, and BMX [9], ITK is (C.I.E. Smith). crucial for the function of T-lymphocytes, and, similar to BTK and https://doi.org/10.1016/j.bbrc.2018.09.019 0006-291X/© 2018 Elsevier Inc. All rights reserved.

Please cite this article in press as: N.N. Fathi, et al., Translocation-generated ITK-FER and ITK-SYK fusions induce STAT3 phosphorylation and CD69 expression, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.019 2 N.N. Fathi et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e4

regions of the mammalian expression plasmid pEF-GFP (Mock), a gift from Connie Cepko (Addgene plasmid #1154), and verified by Sanger sequencing. Myc and Flag tags were inserted through syn- thetic nucleotide linkers at the 30 ends in order to generate tagged proteins. Jurkat cells (human T lymphocyte cells), HEK293T (human embryonic kidney cells), COS 7 (African green monkey kidney cells) and NIH-3T3 (mouse embryonic fibroblasts) were used. Jurkat cells were cultured in RPMI 1640 medium supplemented with 10% heat- inactivated fetal bovine serum (FBS), and all other cells were cultured in DMEM supplemented with 10% FBS at 37 C in a hu- midified atmosphere with 5% CO2. Jurkat cells were transfected by electroporation using neon transfection system, and all other cells were transfected using polyethylenimine (PEI). The protein SDS- PAGE and Immunoblotting were carried out as previously described [3,17]. Monoclonal mouse anti-FER, anti-ITK, and anti- SYK (4D10), and polyclonal anti-STAT3 were purchased from Santa Cruze Biotechnology). Monoclonal anti-c-Myc 9E10 was from Roche. Monoclonal anti-phosphotyrosine 4G10 was from Upstate Biotechnology. Monoclonal anti-Flag M2, polyclonal anti- phosphotyrosine cortactin (pY421) and anti-actin were from Sigma Aldrich. Polyclonal anti-STAT3 (pY705) and polyclonal anti- SLP76 were from Cell Signaling Technology. Monoclonal anti-SLP- 76 (pY128) was from BD Biosciences. Secondary antibodies: goat anti-mouse-700CW, goat anti-mouse-800CW, goat anti-rabit- 700CW and goat anti-rabit-800CW were from LI-COR Biosciences. All membranes of western blots were scanned and quantified with Odyssey Imager systems (LI COR Biosciences).

2.2. CD69 analysis

CD69 surface levels on transfected or control Jurkat cells were measured following staining of cells with PE-conjugated anti-CD69 Fig. 1. A. Schematic representation of ITK, FER, ITK-FER, SYK and ITK-SYK domain antibodies (Beckman Coulter, clone TP1.55.3) by flow cytometric structure. B. Western blot analysis to assess the expression pattern and phosphory- analysis on a MACSQuant Analyzer 10 instrument (Miltenyi Biotec) lation status of ITK, FER, ITK-FER, ITK-SYK, SYK and pEF-GFP (Mock) in whole cell lysates (WCLs) of HEK293T cells. The membrane was probed with anti-pY monoclonal with gating on single cells. Dead cells were discriminated by DAPI antibody (4G10). The cells were starved for 18 h and activated with serum and per- staining. FlowJo software (v10, FlowJo LLC) was used to analyze vanadate for 5 min at room temperature then left on ice for 2 min. The presented data flow cytometric data. is a representative experiment from three independent results. C. Quantification analysis of tyrosine phosphorylation (4G10) intensity of ITK, FER, ITK-FER, ITK-SYK, SYK and Mock expressions. 2.3. Statistical analysis

Prism 7 (GraphPad Software, CA, USA) was used for data anal- TEC, carries PH-TH-SH3-SH2 domains in tandem, followed by the ysis. Mean and standard deviation were recorded for each group catalytic domain [9,10]. FER is a member of the FPS/FES family and from at least three independent data-set (experimental raw data). was essential in the seminal discovery of the SH2 domain by Tony One-way ANOVA was used to compare the results between each Pawson [11]. FER is ubiquitously expressed and its domain archi- group and Duncan test was carried out to compare the sets of mean tecture is from the N-terminus: F-BAR (FER/CIP4 homology-Bin/ in different groups. * ¼ P < 0.05 and ** ¼ P < 0.01 were considered amphiphysin/RVS), FX, SH2, followed by the catalytic tyrosine ki- to be significant and highly significant, respectively. nase entity [12,13]. FER signaling enhances actin polymerization [12,14,15] and promotes cell migration, and cell growth and survival in leukemia [13]. The SYK kinase carries two N-terminal, tandem 3. Results and discussion SH2 domains connected to each other by inter-domain A and to the C terminal kinase domain by inter-domain B [16]. The ITK-SYK 3.1. Experimental design fusion [3] was found in 17% of patients with unspecified PTCLs and is generated through chromosomal translocation between ITK We here report on the function of the novel ITK-FER fusion and and SYK genes t(5; 9)(q33; q22) [3,17]. The PTCLs form a hetero- compare it with the related ITK-SYK chimera, and with the corre- geneous group of mainly aggressive non-Hodgkin lymphomas with sponding parental kinases to assess any biochemical and signaling poor prognosis in most patients [18]. differences (Fig. 1A). For this purpose, we cloned these kinases (Fig. 1A) in frame with addition of C-terminal tandem Myc and Flag 2. Material and methods tags. Initially, we validated the expression pattern of each kinase by immunoblotting, determining the size and expression level of each 2.1. Constructs and cell lines construct with an anti-Flag antibody (Figure S1). We found that ITK, SYK, FER, ITK-SYK and ITK-FER protein expressions are comparable, The open reading frames for the genes/fusion genes ITK, FER, following normalization of anti-Flag bands with anti-actin bands, SYK, ITK-FER and ITK-SYK were cloned and replaced the GFP coding respectively (Fig. 1B and Figure S1).

Please cite this article in press as: N.N. Fathi, et al., Translocation-generated ITK-FER and ITK-SYK fusions induce STAT3 phosphorylation and CD69 expression, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.019 N.N. Fathi et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e4 3

3.2. Induction of tyrosine phosphorylation Furthermore, it has been shown that the C-terminal part of the FER-FX domain modulates the interaction with phosphatidic acid, We then sought to determine the phosphorylation status by promoting actin polymerization through phosphorylation of cor- transiently transfecting of starved and activated HEK 293T cells, tactin [12]. To this end, the ITK-FER fusion retains the SH2 domain followed by immunoblotting of whole cell lysates with anti- and C-terminal part of the FX domain from FER (Fig. 1A), potentially phosphotyrosine (4G10) antibody (Fig. 1B and C). We found that enabling intact downstream signaling. We therefore examined FER, ITK-FER, SYK and ITK-SYK auto-phosphorylated on tyrosine cortactin phosphorylation in Jurkat cells. Cortactin phosphorylation residues under serum starving conditions. However, the activated was induced, not only by the ITK-FER and ITK-SYK fusions, but also cells with serum plus pervanadate induced tyrosine phosphoryla- with parental kinases (Figure S4). Similarly, we found that SLP76 tion further with similar pattern to serum starving conditions. phosphorylation was induced by all kinases (data not shown). We Under heterologous conditions the ITK-FER and ITK-SYK fusions also performed a wound-healing assay in NIH-3T3 cells and observed to be constitutively active (Figure 1B and C). observed that transient expression of ITK-FER or FER did not signiﬁcantly shorten the healing time (Figure S5).

3.3. Downstream phosphorylation of STAT3 3.4. Elevation of CD69 expression To identify the downstream events in lymphoma, we first investigated three substrate candidates: Signal Transducer and To further address the function of ITK-FER, we assessed the Activator of Transcription 3 (STAT3), cortactin and Src homology 2 expression of CD69, a cell surface glycoprotein [23], as a general domain-containing Leukocyte Protein of 76 kDa (SLP76) in Jurkat marker for T-cell activation. CD69 is essentially undetectable in cells. STAT3 plays an important role in T cell activation, differenti- resting lymphocytes, but is expressed in chronic inflammatory in- ation, proliferation and survival [19,20]. Furthermore, constitutive filtrates and at the sites of active immune responses, and, impor- STAT3 activation is associated with malignancy [21] and the SH2 tantly, in chronic myeloid leukemia transformed by BCR-ABL [23]. domain of FER mediates an interaction with STAT3 [22]. Remark- Furthermore, CD69 is not present at high levels on Jurkat cells. ably, compared to the parental kinases FER and SYK, both ITK-FER Forty-eight hours post-transfection of the parental and fusion ki- and ITK-SYK significantly enhanced phosphorylation of the acti- nase, the CD69 surface expression level was assessed by flow vation marker, phosphotyrosine 705, in STAT3 (Fig. 2A, Figure S2), cytometry (Fig. 2B and Figure S3). We observed drastically elevated specifically following pervanadate activation. surface expression on ITK-SYKe and ITK-FER-transfectants. Importantly, the CD69 surface expression remained very low in Jurkat cells receiving the parental FER and ITK kinase constructs (Fig. 2B and Figure S3). Thus, in this work, the function of ITK-FER is characterized for the first time. This chimeric protein, recently identified in a patient with PTCL [4], was found to induce STAT3 signaling and the expression of the activation marker CD69. STAT3 and CD69 were also induced by ITK-SYK, suggesting that this pattern is common to different forms of PTCLs. Inhibitors of tyrosine kinases have become important in cancer treatment and the identification of downstream signaling components could yield an alternative comple- ment for drug target candidates. Thus, even if FER, or SYK, inhibitors would suppress the transforming activity of the ITK-FER and ITK- SYK fusions, respectively, resistance toward such inhibitors may emerge [6,24]. It remains to be seen whether the use of inhibitors for STAT3 could serve as alternative treatment for ITK-FER- or ITK- SYK-expressing lymphomas, but our findings suggest that they may.

4. Conclusion

The investigated chimeric tyrosine kinases, unlike their endog- enous counterparts, are constitutively active and both ITK-FER and ITK-SYK, induce STAT3 phosphorylation and elevation of CD69 expression. These ﬁndings indicate common downstream path- ways in PTCL and suggest new treatment strategies.

Acknowledgements

Fig. 2. A. Graphic representation of STAT3 phosphorylation. Jurkat cells were transiently transfected with the indicated expression constructs, starved and following N.N.F. and B.F.N. are grateful to the Ministry Higher-Education in activation with serum plus pervanadate, immunoblotted with anti-STAT3-pY705 and Kurdistan region - Iraq for generous funding through split-site PhD Flag antibodies. ** ¼ p < 0.01, n.s. not significant. B. Quantification of CD69 surface program covering the living expenses and bench-fee cost for N.N.F. expression on Jurkat cells transfected with the respective constructs. Representative B.F.N. grateful for the research support fund by CRDF Global Vir- dot plots gated on single, viable cells show elevated CD69 surface expression in ITK- ginia (USA). This work was supported by the Swedish Cancer So- FER, ITK-SYK, and SYK transfected cells. Quantification of 3 independently performed experiments (mean ± SEM), see supplementary data - Figure S3, for full dataset. ciety - Sweden to C.I.E.S. We would like to thank Dr. Anders € * ¼ p < 0.05, ** ¼ p < 0.01, n.s. not significant. Osterborg for reading the text and providing feedback.

Please cite this article in press as: N.N. Fathi, et al., Translocation-generated ITK-FER and ITK-SYK fusions induce STAT3 phosphorylation and CD69 expression, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.019 4 N.N. Fathi et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e4

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