STAT3 Is a Substrate of SYK Tyrosine Kinase in B-Lineage Leukemia/Lymphoma Cells Exposed to Oxidative Stress

STAT3 is a substrate of SYK tyrosine kinase in B-lineage leukemia/lymphoma cells exposed to oxidative stress

Fatih M. Uckuna,b,1, Sanjive Qazic,d, Hong Maa,b, Lisa Tuel-Ahlgrenb, and Zahide Ozera,b

aDivision of Hematology–Oncology, Department of Pediatrics, University of Southern California Keck School of Medicine and Developmental Therapeutics Program, Childrens Hospital, P.O. Box 27367, Los Angeles, CA 90027-0367; bMolecular Oncology and Drug Discovery Program and cDepartment of Bioinformatics, Parker Hughes Institute, P.O. Box 130366, St. Paul, MN 55113-0004; and dGustavus Adolphus College, 800 W. College Avenue, St. Peter, MN 56082

Edited* by James E. Darnell, The Rockefeller University, New York, NY, and approved January 4, 2010 (received for review August 12, 2009)

We provide unprecedented genetic and biochemical evidence that cells (18). SYK has been demonstrated to play a crucial role the antiapoptotic transcription factor STAT3 serves as a substrate in OS signaling in B-cells (9, 17). Here we provide unprecedented for SYK tyrosine kinase both in vitro and in vivo. Induction of SYK genetic and biochemical evidence that SYK plays a mandatory in an ecdysone-inducible mammalian expression system results in role in OS-induced activation of STAT3 in B-lineage leukemia/ STAT3 activation, as documented by tyrosine phosphorylation and lymphoma (BLL) cells. Notably, inhibition of SYK with a small nuclear translocation of STAT3, as well as amplified expression of molecule drug candidate prevents OS-induced activation of several STAT3 target genes. STAT3 activation after oxidative stress STAT3 and overcomes the resistance of human BLL cells to (OS) is strongly diminished in DT40 chicken B-lineage lymphoma OS-induced apoptosis. cells rendered SYK-deficient by targeted disruption of the syk gene. Introduction of a wild-type, C-terminal or N-terminal SH2 Results and Discussion domain-mutated, but not a kinase domain-mutated, syk gene into STAT3 Is a PTK Substrate for SYK. Far Western blot analyses (WBA) SYK-deficient DT40 cells restores OS-induced enhancement of STAT- showed that purified recombinant SYK and STAT3 bind each BIOCHEMISTRY 3 activity. Thus, SYK plays an important and indispensable role in other in vitro in a concentration-dependent manner (Fig. S1A OS-induced STAT3 activation and its catalytic SH1 domain is critical and B). Recombinant SYK phosphorylates recombinant STAT3 for this previously unknown regulatory function. These results during in vitro kinase assays (Fig. S1C and D). Phosphoamino provide evidence for the existence of a novel mode of cytokine- acid analysis of the SYK-phosphorylated STAT3 confirmed that independent cross-talk that operates between SYK and STAT3 SYK phosphorylates STAT3 exclusively on tyrosine (Y) residues pathways and regulates apoptosis during OS. We further provide (Fig. S1E). Evaluation of STAT3/SYK interactions in a heterolo- experimental evidence that SYK is capable of associating with and gous baculovirus expression system documented that ectopically phosphorylating STAT3 in human B-lineage leukemia/lymphoma expressed recombinant STAT3 physically associates with cells challenged with OS. In agreement with a prerequisite role (Fig. S2A and B) and serves as an in vivo PTK substrate for of SYK in OS-induced STAT3 activation, OS does not induce tyrosine coexpressed recombinant SYK (Fig. S2C and D). SYK-mediated phosphorylation of STAT3 in SYK-deficient human proB leukemia Y-phosphorylation of ectopically expressed STAT3 enhances its cells. Notably, inhibition of SYK with a small molecule drug candi- DNA binding activity (Fig. S2E). We next sought to determine if date prevents OS-induced activation of STAT3 and overcomes the native STAT3 in human B-lineage lymphoid cells is also capable resistance of human B-lineage leukemia/lymphoma cells to OS- of physically interacting with native SYK. We found that STAT3 induced apoptosis. associates with SYK in BCL-1 cells challenged with pervanadate (PV)-induced (15) OS (Fig. S2F and G). apoptosis ∣ cancer ∣ radiation These results prompted us to further examine the regulatory role of SYK in STAT3 activation using an ecdysone-inducible -lineage acute lymphoblastic leukemia (ALL) is the most mammalian expression system (19). Exposure of SYK-deficient U373 cells stably transfected with wild-type syk gene to Pon-A Bcommon form of cancer in children and adolescents (1). A Resistance of B-lineage ALL cells to the proapoptotic effects induced expression of SYK in a time-dependent fashion (Fig. 1 and B). Induction of SYK resulted in Y-phosphorylation (Fig. 1C of radiation-induced oxidative stress (OS) hampers the attempts D E F to improve the survival outcome of relapsed ALL patients under- and ) and nuclear translocation (Fig. 1 and ) of STAT3. going total body irradiation (TBI) and stem cell transplantation Exposure of transfected but uninduced control U373 cells to (SCT) and only <20% of patients become long-term leukemia- PV did not result in STAT3 activation, showing that, in the free survivors after SCT (1–3). In recent years, intense research absence of SYK, exposure to oxidative stress is unable to cause efforts have therefore concentrated on elucidating the compo- STAT3 activation via stimulation of another PTK. SYK induction nents of the cellular signal transduction pathways controlling without any exposure to PV was sufficient for activating Y-phos- the apoptotic response vs. resistance to OS (4–9). phorylation of STAT3 and exposure of SYK-expressing U373 cells to PV did not substantially alter the Y-phosphorylation level The signal transducer and activator of transcription (STAT)3 C syk protein, has recently been identified as an important regulator of of STAT3 (Fig. 1 ). Treatment of -transfected U373 cells with the pan-JAK inhibitors AG-490 (100 μM) and Pyridone 6 (5 nM) cell survival after exposure to apoptotic signals, including OS (10–14). The protective effect of STAT3 against apoptosis has been explained partly by upregulation of antiapoptotic proteins Author contributions: F.M.U. designed research; F.M.U., S.Q., H.M., L.T.-A., and Z.O. such as Bcl-XL and survivin as well as inactivation of caspases performed research; F.M.U. and S.Q. analyzed data; and F.M.U. and S.Q. wrote the paper. (12–14). It has been well established that OS induces the activa- The authors declare no conflict of interest. tion of various protein tyrosine kinases (PTK) but the PTK This Direct Submission article had a prearranged editor. responsible for OS-mediated activation of STAT3 has not yet 1To whom correspondence should be addressed. E-mail: [email protected]. – been identified (7, 9, 15 17). SYK is a cytoplasmic PTK with This article contains supporting information online at www.pnas.org/cgi/content/full/ multiple important regulatory functions in B-lineage lymphoid 0909086107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0909086107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 25, 2021 Fig. 1. Activation of STAT3 in an ecdysone-inducible mammalian expression system for SYK. (A) AntiSYK WBA of SYK immune complexes from whole cell lysates of untransfected vs. transfected U373 cells before and after exposure to the ecdysone-analogue Pon-A (10 μM). (B) Antiactin WBA of actin IC from the same lysates used in (A). (C) Anti-phospho-STAT3Y705 WBA of STAT3 immune complexes from whole cell lysates of transfected uninduced vs. induced U373 cells. Lysates from syk-transfected U373 cells were prepared before (CON) and at various time points after addition of Pon-A (10 μM) as indicated. Some samples were exposed to PV as well where indicated. (D) Anti-STAT3 WBA of the STAT3 immune complexes shown in (C). (E) Confocal image depicting the cytoplasmic localization of native STAT3 in a representative syk-transfected U373 cell before exposure to Pon-A (10 μM). (F) Confocal image showing substantial nuclear (in addition to cytoplasmic) localization of native STAT3 in a representative syk-transfected U373 cell 6-h after exposure to Pon-A (10 μM). (G) Pon-A (10 μM)- induced SYK induction leads to upregulation of STAT3-responsive genes. The heat map represents the color-coded expression value reported as fold change relative to the average expression levels in the control samples (key shows from 0.6- to 3.6-fold change, Blue to Red, respectively). The two-way dendrogram depicts the similarity of expression pattern for all probes (39 probesets for 29 genes) across the 6 treatments and the 6 treatments across the 39 probesets arranged in rows and columns respectively.

plus the JAK3 inhibitor JANEX-1/WHI-P131 (100 μM) during induction-associated increase in expression levels that did not the Pon-A exposure resulted in abrogation of JAK activity, as reach statistical significance (Fig. 1G, SI Text). None of the documented by the absence of autophosphorylation in the remaining 20 STAT3 target genes were down-regulated after anti-phosphotyrosine (APT) Western blots of JAK kinases immu- SYK induction (Fig. 1G). Thus, SYK is capable of causing noprecipitated with a cocktail of antibodies to JAK1, JAK2, and Y-phosphorylation, nuclear translocation, and activation of tran- JAK3 (Fig. S3A and B). However, JAK inhibition did not prevent scription factor function of STAT3 in mammalian cells. the Y-phosphorylation of STAT3 after Pon-A induced SYK expression (Fig. S3C and D), which excludes any direct or indirect SYK Plays a Pivotal Role in OS-Induced Activation of STAT3 in B-lineage involvement of JAK kinases in SYK-mediated STAT3 acti- Lymphoid Cells. We next examined the role of SYK in OS-induced vation. We next used the U95Av2 GeneChip microarrays from STAT3 activation in B-lineage lymphoid cells using SYK- Affymetrix to interrogate the expression levels of validated deficient DT40 clones that were established by homologous STAT3 target genes (20) before and after induction of SYK ex- recombination knockout (Fig. S4A–C). In these experiments pression in the U373 expression platform (Fig. 1G, SI Text). Six of we used PV, a strong oxidizing agent that triply oxidizes the the 29 interrogated STAT3 targets belonging to 3 subclusters of catalytic cysteine of protein tyrosine phosphatases (21). EMSAs genes showed marked upregulation following SYK induction of nuclear proteins from wild-type DT40 cells treated with PV (Fig. 1G). In addition to these 6 genes, 3 additional genes revealed significant mobility shifts in complexes binding the belonging to the same 3 subclusters also showed a SYK SIE probe especially after 30–60 min of treatment (Fig. 2). This

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0909086107 Uckun et al. Downloaded by guest on September 25, 2021 Fig. 2. Role of SYK in oxidative stress-induced activation of STAT3 in DT40 chicken lymphoma B-cells (7, 9). EMSAs in (A) and (B) were performed with end-labeled SIE probe and nuclear extracts prepared from wild-type DT40 cells, SYK- deficient DT40 cells (SYK−), SYK-deficient DT40 cells reconstituted with wild-type SYK (SYK−, rSYK [WT]), catalytic kinase domain-mutant of SYK (SYK−, rSYK [K−], and SH2 domain mutants of SYK (SYK−, rSYK [mSH2-C] and SYK−, rSYK [mSH2-N]). Cells were left untreated (no PV) or treated with 400 μM PV for 15 min, 30 min, or 60 min, as indicated. The nuclear extracts were preincubated and then the labeled probe was added (A, Lanes 2–13). In unlabeled com- BIOCHEMISTRY petition reactions, 100-fold excess unlabeled homologous SIE (A, Lanes 14 and 16) or nonhomologous AP-1 probe (A, Lanes 15 and 17) was added prior to the preincubation. Controls included samples containing only the SIE probe without any nuclear extract (A, Lane 1; B, Lane 7). Mobility shifts were determined by electrophoresis as described in Materials and Methods. Following electrophoresis, gels were dried and subjected to autoradiography on film. Shifted bands are indicated by arrows.

binding was specific to the SIE probe, as indicated by homologous but does not inhibit EGF-R, IRβ, BTK, HCK, JAK1, JAK2, or versus nonhomologous competition with 100x excess cold probe. JAK3 kinases (Fig. S6A–I). Incubation of NALM-6 B-lineage The shifted bands, previously shown to be STAT3 homo- and ALL cells with SYKINH-61 (but not WHI-P131, the multifunc- heterodimers (8), were not detectable in nuclear proteins from tional ATP site inhibitor of JAK3/EGF-R) results in inhibition of SYK-deficient (SYK−) DT40 cells (Fig. 2). The diminished constitutively active native SYK without any change in SYK pro- STAT3 signal in the SYK-deficient DT40 cells was due to the lack tein expression levels (Fig. S6J and K). SYKINH-61 prevented − of SYK because SYK DT40 cells reconstituted with wild-type PV-induced as well as H2O2-induced Y-phosphorylation of SYK showed a rapid and strong STAT3 response to PV. By con- SYK in RAMOS (Fig. 3A and B) and BCL-1 cells (Fig. 3C trast, SYK− DT40 cells reconstituted with the kinase domain- and D), respectively. As shown in Fig. 3E and F, the SYK inhib- mutant of SYK failed to show any restoration of the PV-induced itors SYKINH-61 and Piceattanol (PCT) inhibited H2O2-induced STAT3 signal (Fig. 2). Thus, SYK plays an important and indis- Y-phosphorylation of STAT3 in RAMOS cells. In contrast to pensable role in OS-induced STAT3 activation and its catalytic SYKINH-61 and PCT, tyrphostin AG-490 inhibiting JAK1, domain is critical for this previously unknown regulatory func- JAK2, and JAK3 (22, 23), JANEX-1 inhibiting JAK3 (24) or tion. Both the C-terminal SH2 mutant and N-terminal SH2 mu- BTK inhibitors CP-1/HI-12 and CP-2/HI-86 (25) did not prevent tant were effective in mediating SYK function in triggering PV-induced Y-phosphorylation of STAT3. STAT3 activation (Fig. 2), demonstrating that the two tandem We next compared the PV-responsiveness of SYKþ RAMOS SH2 domains of SYK are not necessary for optimal STAT3 sig- (26) cells vs. SYK-deficient cells from a proB ALL patient naling during OS. In agreement with the EMSA data, comparison (ProB#4) with a missplicing at the 5′ end of syk exon 5 leading − − 865 868 of H2O2-treated SYK DT40 cells vs. SYK DT40 cells recon- to a 4 bp deletion (Δ½G -G ) in the syk coding sequence that stituted with wild-type SYK by confocal microscopy showed that causes a frameshift starting after Trp239 with absent SYK protein only the nuclei of the SYK-reconstituted DT40 cells showed the expression and SYK kinase activity (27). Notably, PV [but not presence of translocated active phospho-STAT3 protein CD19 receptor engagement with an anti-CD19 homoconjugate (Fig. S4D). (26)] failed to induce Y-phosphorylation of the STAT3(Y705) phosphoepitope in SYK-deficient ProB#4 cells (Fig. 3G and Inhibition or Deficiency of SYK Prevents OS-Induced, SYK-Mediated H). Thus, STAT3 in SYK-deficient ProB#4 cells is capable of Y-Phosphorylation of STAT3 in Human B-lineage Lymphoid Cells. We undergoing Y-phosphorylation and the inability of PV to induce next used SYKINH-61 as a potent and selective SYK inhibitor STAT3 phosphorylation in ProB#4 cells is not due to a defective to examine the role of SYK in OS-induced Y-phosphorylation STAT3 protein. We next examined the effects of SYKINH-61 on of STAT3 in human B-lineage lymphoid cells. SYKINH-61 is a DNA binding activity of STAT3. In accord with our previous pentapeptide mimic targeting the substrate-binding site of studies (8), PV-induced OS triggered STAT3 activation in all SYK (Fig. S5), that inhibits SYK at nanomolar concentrations, human B-lineage lymphoid cell lines tested, and this activation

Uckun et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 25, 2021 Fig. 3. Role of SYK in oxidative stress-induced tyrosine phosphorylation of STAT3 in human B-lineage lymphoid cells. (A and B) RAMOS Burkitt’s leukemia/lymphoma cells were either left untreated or treated with the oxidative agent PV at a 400 μM concentration in the presence or absence of SYKINH-61 (1 nM, 10 nM, 50 nM, 100 nM) or 100 μM PCT. Cells were lysed using Nonidet-P40 buffer after 30 min exposure to PV, PV þ SYKINH-61,orPVþ PCT and lysates were immunoprecipitated with antiSYK antibodies, as indicated. The SYK immune complexes were resolved by SDS-PAGE and examined by APT (A) or antiSYK (B) Western blot analysis. (C and D) BCL-1 cells were either left untreated or treated with 100 mM H2O2 as the oxidative agent in the presence or absence of 100 nM SYKINH-61. Cells were lysed using Nonidet-P40 buffer after 1 min, 5 min, or 10 min exposure to H2O2 or H2O2 þ SYKINH-61 and lysates were immunoprecipitated with APT antibodies. The immune complexes were resolved by SDS-PAGE and examined by APT (C) or antiSYK (D) WBA. (E and F) RAMOS cells were left untreated (CON, -H2O2)(Lane 4) or treated with 100 mM H2O2 for 10 min in the absence (Lane 5)or presence of SYK kinase inhibitors SYKINH-61 (100 nM) (Lane 6) and PCT (100 μM) (Lane 8), JAK1,2,3 inhibitor AG-490 (100 μM) (Lane 1), JAK3 kinase inhibitor JANEX-1 (100 μM), or BTK inhibitors compound 1 (CP-1)/HI-12 (100 μM) (Lane 3), and compound 2 (CP-2/HI-86) (100 μM) (Lane 7). STAT3 immune complexes from whole cell lysates of these cells were subjected to WBA with antiphospho STAT3Y705 (E) or anti-STAT3 (F) antibodies. (G and H) RAMOS Burkitt’s leukemia/lymphoma cell and ProB#4 cells (27) were left untreated (CON) (Lanes 1 and 2) or treated with 400 μM PV for 30 min (PV) (Lanes 3 and 4). Controls were treated with an anti-CD19 antibody homoconjugate (1 μg∕mL) (Lanes 5 and 6) to stimulate the CD19-linked signaling pathway. STAT3 immune complexes from whole cell lysates were subjected to WBA with antiphospho STAT3Y705 (G) or anti-STAT3 (H) antibodies.

was abrogated by SYK kinase inhibitors PCT and SYKINH-61, (29–34). Compelling evidence from gene profiling studies indi- but not by inhibitors of JAK3, (24), BTK (25), or tubulin cates that abundant STAT3 expression in ALL is associated with polymerization (28) (Fig. S7). chemotherapy and steroid resistance (35–37). Our experimental findings presented herein provide unprecedented genetic and Inhibition of SYK Promotes OS-Induced Apoptosis in Radiation-Resis- biochemical evidence that SYK plays an important and indispens- tant Human B-lineage Leukemia/Lymphoma Cells. In agreement with able role in OS-induced activation of STAT3 and its catalytic do- the antiapoptotic function of the SYK-regulated STAT3 response main is critical for this previously unknown survival-promoting to OS, the sensitivity of DT40 cells to PV was remarkably function in B-lineage leukemia/lymphoma cells. SYK is the first enhanced when they were rendered SYK-deficient (Fig. S8). cytoplasmic nonJAK PTK to be identified as a positive regulator As shown in Fig. 4A, SYKINH-61 markedly enhanced both of STAT3 in B-lineage lymphoid cells exposed to OS. Several stu- H2O2-induced (83.3% vs. 9.5%, P < 0.001) and PV-induced dies have independently demonstrated that cytokine-mediated apoptosis (98.8% vs. 20.6%, P < 0.001) in human BLL cells, as (38) as well as B-cell antigen receptor (BCR) mediated (39) ac- measured by in vitro TUNEL assays, including primary leukemic tivation of STAT3 bypasses JAKs in B-lineage lymphoid cells. cells from two B-lineage ALL patients, who had relapsed after Several nonJanus family PTK, including TEC family kinase TBI and allogeneic SCT. Examination of each treatment versus ETK/BMX (40, 41), FES (42), SRC (43), SRC family kinases control mean values showed significant effects of SYKINH-61 LCK (44) and LYN (39), and breast tumor kinase BRK (45) have (Dunnett’s post hoc, p ¼ 0.004)(“*”), SYKINH-61 þ H2O2 been shown to be capable of activating STAT3 by phosphorylating (p < 0.0001)(“**”), and SYKINH-61 þ PV (p < 0.0001) STAT3(Y705) phosphoepitope independent of JAKs and without (“**”) (Fig. 4A). Fig. 4B depicts the confocal images of leukemic inducing activation of endogenous JAKs. Thus, it appears that cells from a radiation-resistant relapsed ALL patient after treat- STAT3 licenses different cytoplasmic PTK in distinct activation ment with H2O2, SYKINH-61, and H2O2 þ SYKINH-61. pathways. Whereas H2O2-treated leukemic cells maintained their viability, In the present study, we documented that OS induces rapid virtually all of the cells treated with H2O2 þ SYKINH-61 showed stimulation of Y-phosphorylation and nuclear translocation of morphologic signs of advanced apoptosis, including shrinkage, STAT3 in B-lineage lymphoid cells, which differs from the pub- nuclear fragmentation, loss of cytoplasmic, and nuclear integrity lished reports of STAT3 responses that follow BCR engagement (Fig. 4B). Thus, inhibition of SYK with SYKINH-61 promotes (39, 46, 47). BCR stimulation of murine B-cells has been shown OS-induced apoptosis against human BLL cells. to result in a delayed accumulation of Y-phosphorylated STAT3 SYK has recently emerged as a potential new molecular target via activation of a lymphokine pathway mediated by interleukins for the treatment of B-lineage leukemias and lymphomas 6 and 10 produced by the stimulated B-cells (46). Although BCR

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0909086107 Uckun et al. Downloaded by guest on September 25, 2021 tion of SYK under suboptimal OS. Notably, BLNK deficiency plays an important role in the leukemogenesis of B-lineage ALL (52) and a significant portion of B-lineage ALL cases are BLNK-negative (53). The remarkable radiation resistance of relapsed B-lineage ALL patients (pre-pre-B immunophenotype) (6) with abundant expression of SYK (27) indicates that the balance of SYK-linked proapoptotic vs. antiapoptotic signals triggered by OS is an antiapoptotic signal, which may in part be related to their underlying BLNK deficiency. It is noteworthy that SYK also has been reported to have an antiapoptotic function in DT40 cells in the context of ceramide responses that is independent of its enzymatic activity (54). However, in the context of STAT3 activation after OS, the kinase activity of SYK is clearly required for tyrosine phosphorylation of STAT3, as PV did not activate STAT3 in SYK− DT40 cells reconstituted with a kinase domain-mutant SYK and inhibition of the kinase activity of SYK with small molecule inhibitors SYKINH-61 or PCT prevented OS-induced STAT3 activation in the absence of any alteration in SYK protein levels. Likewise, the kinase activity of SYK has been shown to be essential for its reported antiapoptotic activity in the context of OS (9), tonic or ligand-mediated BCR signaling (31, 33, 34), and cytokine signaling (55). The identification of SYK as a regulator of the antiapoptotic STAT-3 response to OS prompts the hypothesis that PTK inhibitors targeting SYK may overcome the resistance to OS-induced apoptosis and thereby provide the foundation for more effective

multimodality treatment regimens for poor prognosis B-lineage BIOCHEMISTRY ALL patients. This hypothesis is strongly supported in this study by the documented ability of SYK inhibitor SYKINH-61 to markedly enhance OS-induced apoptosis in primary leukemic cells from radiation-resistant ALL patients. In a recent study, constitutive STAT3 phosphorylation in CLL cells appeared to correlate with their SYK expression levels and was reduced by SYK inhibitors (34). Therefore, the antiapoptotic transcription factor STAT3 may also serve as a PTK substrate for SYK in multiple survival-promoting signaling pathways, including the tonic Fig. 4. SYKINH-61 promotes oxidative stress-induced apoptosis in primary BCR signaling in mature B-cell neoplasms. B-lineage ALL cells. (A) Cells from two EBV-transformed lymphoblastoid cell lines BCL-1 and BCL-2, Burkitt’s leukemia/lymphoma cell line RAMOS, as well Materials and Methods as primary leukemic cells from two B-lineage ALL patients were either Recombinant Baculovirus Construction and Protein Expression. The gene encoding wild-type SYK was ligated into pFastBac1 (PFB) (Gibco-BRL) (8, 56). left untreated or treated with 100 mM H2O2, 50 nM SYKINH-61, or 100 mM H2O2 þ 50 nM SYKINH-61 for 30 min at 37 °C. TUNEL assays were The resulting vector, PFB-syk, was then used to generate the recombinant used after 24 hr to determine the percentage of apoptotic cells after treat- baculovirus by site-specific transposition in E. coli DH10Bac cells (Gibco- ment. (B) Cells from a B-lineage ALL patient in postSCT relapse were either BRL), which harbor a baculovirus shuttle vector (bacmid), bMON14272. left untreated or treated with 100 mM H2O2, 50 nM SYKINH-61, or The resulting recombinant bacmid DNA was introduced into Sf21 insect cells 100 mM H2O2 þ 50 nM SYKINH-61 for 30 min at 37 °C. After 24 hr of culture, (Invitrogen) by transfection as reported (8, 56). Recombinant baculovirus con- cells were costained with a rabbit polyclonal antitubulin antibody (Green taining the gene encoding murine STAT3 was a kind gift from J. Feng and J.N. Fluorescence) and the DNA-specific dye Toto-3 (Blue Fluorescence), and Ihle (St. Jude Children’s Hospital). Coexpression experiments were carried out examined by laser scanning confocal microscopy (7, 8, 48). to analyze potential interactions between SYK and STAT3 using previously published procedures (8, 56).

stimulation in DT40 cells has been shown to rapidly activate Biochemical Assays. Immunoprecipitations, immune complex kinase assays STAT3 Y-phosphorylation through LYN in a JAK independent (KA), cell-free KA, phosphoamino acid analysis, and WBA were performed pathway (39), others have reported that BCR stimulation of as previously reported (7, 8, 15, 16, 26, 27, 56–58). Antibodies against phos- murine B-cells induces rapid serine phosphorylation of STAT3 pho-STAT3 (anti-phospho-STAT3[pY705) were obtained from Sigma–Aldrich. without any Y-phosphorylation (47). SYK has been reported to participate in both upregulation as Electrophoretic Mobility Shift Assays (EMSAs). EMSAs were performed with a double-stranded m67-SIE probe (top strand: ttgcATTTCCCGTAAATcttgtcta- well as downregulation of apoptosis in B-lineage lymphoid cells ga), end-labeled with 32P using T4 polynucleotide kinase and γ32P-ATP, as (17, 18). SYK is required for BCR-independent calcium induced previously described (8). apoptosis as well as BCR-mediated apoptosis (48, 49). SYK is not required for radiation or OS-induced apoptosis (7, 50). To the Molecular Modeling. Fixed docking in the Affinity program within Insight II contrary, SYK has been reported to have an antiapoptotic func- (Molecular Simulation Inc.) was used for docking SYKINH-61 to the ATP vs. tion in the context of OS (17). However, SYK may also play a substrate-binding sites of SYK, which was built, based on the crystal structure of IR kinase ternary complex with an ATP analog, a peptide, and Mg ions proapoptotic role via activation of PLCγ2 when oxidative stress using the homology module of InsightII. The final docked position of the is caused by low doses of H2O2 (17, 51). Han et al. (51) reported molecule was chosen based on both the lowest energy estimation, as we that the presence of the B-cell linker protein (BLNK) (also previously reported for other PTK inhibitors (24, 56, 59–61). known as SLP-65) is required for this apoptosis-accelerating func- For more information, see SI Materials and Methods.

Uckun et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 25, 2021 ACKNOWLEDGMENTS. DT40 and its subclones were obtained from T. Kurosaki microscopy, and molecular modeling. This work was funded by Parker (Yale University School of Medicine). The authors thank the members of the Hughes Trust and Hughes Chair in Molecular Oncology at Parker Hughes Uckun Lab from Parker Hughes Institute for providing the PTK inhibitors, Institute (F.M.U.). technical assistance, and immunoprecipitations/immunoblotting, confocal

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