TOSO Interacts with SYK and Enhances BCR Pathway Activation in Chronic Lymphocytic Leukemia
Chronic lymphocytic leukemia (CLL) is the most common leukemic disorder in the western hemisphere, characterized by the accumulation of mature B cells in the blood, bone marrow, and lymphoid organs. CLL is considered a prototypic antigen-driven leukemia/lymphoma, with chronic antigen stimulation playing a crucial role in its pathogenesis. The B-cell receptor (BCR) is the main receptor for transmitting extrinsic stimulation, and the BCR signaling pathway has been reported to be constitutively activated in CLL. Strong evidence indicates that signaling via the BCR plays a major role in the development of CLL and determines variable clinical behaviors. Patients with immunoglobulin heavy variable (IGHV) somatic mutations exhibit more indolent disease and longer overall survival than those with IGHV germline mutation or “unmutated” configurations. CLL cells display many characteristics of antigen-experienced B cells, including a skewed IGHV gene repertoire and the expression of BCRs with a structurally similar antigen-binding pocket, defined as a conserved “stereotypic” pattern of an immunoglobulin (Ig) variable region. The antigen-antibody reaction mediated by immunoglobulin is the main defense against intruding pathogens or autoantigens, achieved by the binding of immunoglobulins to the antigen via their variable amino-terminal regions and to effector molecules, such as Fc receptors (FcRs), via their constant carboxyl-terminal regions. The interactions between FcRs and antibodies initiate a broad spectrum of effector functions important in host defense. Distinct FcRs have been identified for immunoglobulin G (IgG; FcgRI, FcgRII, and FcgRIII), IgE (FceR), and IgA (FcaR). IgM is the first antibody isotype to appear in the immune response to pathogens and self-antigens. However, the IgM Fc receptor (FcmR) was not identified until recently and was found to be identical to TOSO, also known as the Fas inhibitory molecule 3 (FAIM3). We and others previously reported that TOSO/FcmR was selectively and highly expressed in CLL cells compared to the expression in normal B cells and other B-cell lymphomas. Nevertheless, its function in CLL has not been well defined. The structural analysis of TOSO/FcmR indicates that it has a long cytoplasmic tail (118 amino acid residues) containing conserved residues, which makes it different from other FcRs. The binding of IgM to TOSO/FcmR on natural killer (NK) cells can initiate intracellular signaling, inducing the phosphorylation of PLCg and Erk1/2. We hypothesized that TOSO interacts with some proteins involved in important pathways upregulated in B-cell hematologic malignancies. This study aimed to identify new partners of TOSO and uncover the role of TOSO in the oncogenesis of CLL.
Methods
Ethical approval was obtained, and written informed consent was provided by all patients in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Institute of Hematology and Blood Disease Hospital. Human non-Hodgkin lymphoma B-cell lines (Granta-519 and Z138) and primary B lymphocytes isolated from nine CLL patients were used in this study. Freshly isolated peripheral blood mononuclear cells were subject to CD19+ B-cell enrichment (>98%) by standard positive selection using magnetic beads conjugated with a specific anti-CD19 antibody. The human non-Hodgkin lymphoma B-cell line Z138 was propagated and maintained in Iscove modified Dulbecco medium supplemented with 10% fetal bovine serum. The human non-Hodgkin lymphoma B-cell line Granta-519 and primary B lymphocytes isolated from CLL patients were propagated and maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. All cells were cultured at 37°C with 5% CO2 in a humidified atmosphere. BCR stimulation was performed by adding goat F(ab’)2 anti-human IgM (m-chain-specific) at a final concentration of 10 mg/mL for 6 or 24 hours, as described previously.
Full-length human TOSO cDNA was amplified and cloned into the NheI and AscI sites of the pLenti6.3_MCS_IRES2-EGFP plasmid to generate the pLenti6.3_TOSO_IRES2-EGFP plasmid. This recombinant vector was subsequently transfected into HEK293 cells with Lipofectamine 2000 transfection reagent. Viral supernatants were collected 48 hours after transfection, filtered through a 0.45 µm nitrocellulose filter, and concentrated. The Granta-519 and Z138 cells were transfected by the viral supernatants with polybrene at a final concentration of 8 mg/mL. The viral medium was removed and replaced with fresh, normal culture medium 12 hours post-transduction.
Fas inhibitory molecule 3 (TOSO) siRNA and control non-targeting siRNA were transfected into primary B lymphocytes from CLL patients by nucleofection, following the manufacturer’s instructions. Total RNA from transfected and non-transfected Granta-519 and Z138 cells was extracted using TRIzol reagent according to the manufacturer’s instructions. Total RNA (2 µg) was reverse transcribed to cDNA with the PrimeScript RT reagent kit. Real-time quantitative polymerase chain reaction (PCR) was performed using SYBR Green PCR MasterMix according to the manufacturer’s instructions.
The whole-cell lysate was generated with radio-immunoprecipitation assay (RIPA) lysis buffer, and the protein concentration was measured by Bio-Rad protein assay following the manufacturer’s instructions. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride (PVDF) membranes, and the blots were blocked and incubated overnight at 4°C with the appropriate primary antibody. The blots were then incubated with a specific HRP-conjugated secondary antibody. Immunodetection was performed by enhanced chemiluminescence.
Protein pellets were solubilized and digested by trypsin. Protein constituents were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The RAW data files collected on the mass spectrometer were converted to mzXML and MGF files by use of MassMatrix data conversion tools. The resulting MGF files were searched using Mascot Daemon by Matrix Science, and the data files were searched against the full SwissProt database or NCBI database. Protein identifications were checked manually, and proteins with a Mascot score of 50 or higher with a minimum of two unique peptides from one protein having a b or y ion sequence tag of five residues or better were accepted.
Apoptosis was assessed by Annexin V reagents according to the manufacturer’s instructions. Samples were analyzed on a FACS-Calibur flow cytometer with CellQuest software. A total of 500 µg of extracted cellular proteins was incubated with anti-Toso/anti-SYK/anti-phosphor-SYK and anti-IgG at 4°C overnight. The protein-antibody complexes were captured by protein A/G plus-agarose and pelleted with the agarose beads after the final wash. Western blotting of each protein was then performed.
Results
TOSO was successfully over-expressed in the transfected cells at both RNA and protein levels. By TOSO co-immunoprecipitation (Co-IP) and subsequent mass spectrometric analysis, we identified TOSO-binding candidates, among which was spleen tyrosine kinase (SYK), a core target because of its well-established role in BCR signaling transduction and the previously identified correlation between increased expression of TOSO and enhanced autoreactive BCR signaling pathways as well as unmutated IGVH gene status.
We confirmed the interaction between TOSO and SYK by Co-IP in the TOSO-over-expressing Granta-519 and Z138 cells and the primary B lymphocytes from three CLL patients. Using a siRNA strategy, primary CD19+ B lymphocytes with TOSO over-expression isolated from CLL patients were successfully down-regulated at both RNA and protein levels.
After 1 hour treatment with the SYK inhibitor fostamatinib disodium (R788) at a concentration of 10 mmol/L, the TOSO protein in the over-expressed Granta-519 and Z138 cells was collected by Co-IP and was found to have a low-level interaction with SYK, while the B lymphocytes isolated from CLL patients had a notable reduction in the interaction of TOSO and SYK after SYK inhibitor treatment. These observations suggested that TOSO and SYK interacted directly in CLL cells.
High levels of phosphorylated SYK (p-SYK) were detected in the TOSO-over-expressing Granta-519 and Z138 cells, and lower p-SYK status was observed in the TOSO-down-regulated primary CLL cells, which indicated that TOSO increased the phosphorylation of SYK.
After BCR stimulation, we screened the activated downstream components of the BCR signaling pathway and found lower phosphorylation levels of IkBa, p65, ERK, and p38 in the primary CLL cells with down-regulated TOSO and higher phosphorylation levels of IkBa, p65, ERK, p38 in the Granta-519 and Z138 cells over-expressing TOSO.
After treating with the SYK inhibitor fostamatinib disodium (R788), the levels of p-SYK, p-p65, p-IkBa, p-ERK, and p-p38 were reduced in the TOSO-over-expressing Granta-519 and Z138 cells. These evidences demonstrated that TOSO regulated the components downstream of the BCR signaling pathway, including the NF-kB and MAPK pathways, in CLL.
TOSO has been observed to play a role in anti-apoptosis. We sought to verify this finding in CLL cells, as apoptosis resistance is the main characteristic of CLL cells. After stimulation with anti-IgM, the percentage of apoptotic cells of TOSO-over-expressing Granta-519 and Z138 was (8.46 ± 2.90)% and (4.20 ± 1.21)%, respectively, which was significantly lower than that of the control groups, (25.20 ± 4.60)% and (19.72 ± 1.10)% (P < 0.05 in both). Besides, TOSO siRNA treatment in primary CLL cells from three patients significantly increased the apoptosis.
B-cell lymphoma 2 (BCL-2) over-expression is a known mechanism for the intrinsic apoptosis resistance of CLL, and there was a positive correction between TOSO and BCL-2 expression. Here we found that the BCL-2 expression level was decreased in the primary cells from the three CLL patients after TOSO knockdown and that it was increased when TOSO was over-expressed in the two cell lines (Granta-519 and Z138 cells). This finding indicated that TOSO might induce apoptosis resistance by up-regulating BCL-2 expression.
Discussion
Compared to the level in other B-cell lymphomas, TOSO is over-expressed in CLL cells, but its role in the pathogenesis of CLL has not been determined. Our group and others have reported that TOSO over-expression is associated with poor survival and progressive CLL. However, the mechanism of TOSO over-expression in CLL cells remains unknown. Vire et al reported that TOSO/FcmR localized to the cell membrane and could be internalized upon IgM binding and shuttled to the lysosome for degradation. TOSO over-expression could also be down-regulated in response to TLR activation. Nguyen et al demonstrated that TOSO mediates the balance between apoptotic and non-apoptotic death receptor signaling by facilitating RIP1 ubiquitination. In this study, we showed that TOSO interacted with the SYK protein and activated the BCR signaling pathway.
Previous clinical observations discovered that TOSO was over-expressed in patients with unmutated IGHV, indicating an association between TOSO over-expression and the BCR signaling pathway. Ouchida previously found that TOSO contributed to events downstream of the BCR signaling pathway when triggered by BCR cross-linking in mice. BCR is a trans-membrane complex located on the outer surface of B cells and composed of a heterodimer with heavy chain and light-chain Igs, Iga/CD79A, and Igb/CD79B. The interaction between antigens and the BCR antigen-binding site triggers intrinsic downstream signaling, involving proteins such as LYN, SYK, BLNK, BTK, PLC-g, and PI3K, and determines the fate of a BCR-bearing B cell. Because the BCR signaling pathway plays an important role in the pathogenesis of CLL and mediates the communication between CLL cells and the environment, we hypothesized that TOSO might contribute to the pathogenesis of CLL through the BCR pathway. Indeed, TOSO was found to interact with SYK. Moreover, the SYK phosphorylation and subsequent activation of the BCR signaling pathway were enhanced when TOSO was over-expressed and could be blocked by the SYK inhibitor or by siRNA knockdown of TOSO in primary CLL cells. These results showed that the TOSO interacted with SYK enhanced its phosphorylation and downstream BCR signaling pathway.
Our results provide evidence to explain why the BCR signaling pathway is constitutively activated in CLL. Cytogenetic aberrations of the components in the BCR signaling pathway are rare in CLL cells compared with those in other B-cell malignancies, such as the mutation of CD79A/B and CRAD11 in diffuse large B-cell lymphoma and the mutation of TCF3 and ID3 in Burkitt lymphoma, which contribute to the activation of the BCR signaling pathway in these lymphomas. Additional elements cooperate with the BCR complex to trigger or enhance this pathway, which may explain its constitutive activation in CLL. In the lymph node and/or bone marrow environment, where the IgM level is higher than it is in the serum, antigens stimulate CLL cells through the BCR, while IgM interacts with TOSO/FcmR to enhance the activation of the downstream BCR signaling pathway. When pathogens are involved, IgM recognizes them and produces IgM-antigen immune complexes, which may crosslink the BCR complex (antigens) and TOSO (IgM Fc fragment) together, leading to the constitutive activation of BCR.
Binding of IgM to TOSO to initiate intracellular signaling in NK cells has also been reported by other groups. Murakami et al reported that anti-Toso mAb can pull down PLC-g2 and activate ERK signaling after IL-2 stimulation. Therefore, Toso can generate intracellular signals and regulate cellular processes. In liver cells, TOSO can promote the activation of MAPK and NF-kB signaling pathways in response to CD95L and TNFa stimulation. These results are in accordance with our conclusion that TOSO can sense outer stimulation and trigger the intracellular responses.
The evasion of apoptosis is a hallmark of CLL. Both the intrinsic and extrinsic apoptosis pathways were dys-regulated in CLL, which led to apoptosis resistance. BCL-2 is the critical protein of the intrinsic apoptosis pathway, the level of which is increased in more than 80% of CLL cases. Although there is controversy about the role of TOSO in the Fas-mediated apoptosis pathway (extrinsic pathway), TOSO might be involved in the anti-apoptotic activity. Based on the positive correlation observed between TOSO and BCL-2 expression, TOSO may induce apoptosis resistance through the BCL-2 pathway. In the study, TOSO over-expression led to apoptosis resistance in B-cell lymphoma cell lines and down-regulating the expression of TOSO increased the apoptosis sensitivity of primary CLL cells, suggesting that TOSO is involved in apoptosis resistance.
TOSO is a good candidate for target therapy because it is highly expressed in CLL cells and not expressed in normal blood components. Besides, blocking its function induces cell apoptosis, as shown in this study. Faitschuk et al demonstrated that TOSO was a good target for chimeric antigen receptor T cell therapy in experiments conducted in vitro and in vivo.
Although we have identified that TOSO can interact with SYK and activate BCR downstream components after stimulation and induce apoptosis resistance, the mechanism by which TOSO interacts with SYK remains unclear and it has not been discovered how TOSO influences BCR signaling downstream. In addition, the mechanism by which TOSO up-regulated BCL-2 expression still requires further investigation.
In summary, the over-expression of TOSO is involved in the pathogenesis of CLL by enhancing the BCR signaling pathway through interactions with SYK and elevation of its phosphorylation. TOSO up-regulates BCL-2 expression and participates in the apoptosis resistance of CLL.
doi.org/10.1097/CM9.0000000000000999
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