Figure 5 - uploaded by John G Gribben
Content may be subject to copyright.
Validation of SYK inhibitor specificity and SYK gene silencing. A, protein tyrosine kinase activity assay performed with human recombinant SYK ( rSYK ) and ZAP70 ( rZAP70 ) in the absence and presence of 4 A mol/L R406 or SYKII ( columns, mean of five independent experiments; bars, SE). For relative activity absorption without inhibition was defined as 1. B, primary CLL cells (7 Â 10 6 , respectively) were transfected with control siRNA or SYK-specific siRNA. After 72 h of culture, viability and SYK expression were determined by
Source publication
B-cell receptor signaling contributes to apoptosis resistance in chronic lymphocytic leukemia (CLL), limiting the efficacy of current therapeutic approaches. In this study, we investigated the expression of spleen tyrosine kinase (SYK), a key component of the B-cell receptor signaling pathway, in CLL and its role in apoptosis. Gene expression profi...
Contexts in source publication
Context 1
... B cells. Direct detection of phosphorylated SYK in whole cell lysates is hampered by the lack of suitable antibodies. We therefore performed phospho-tyrosine immunoprecipitation followed by total SYK immunoblotting. In parallel, we performed a total SYK immunoblot with equal protein amounts as used for immunoprecipitation. To rule out that any effects observed on phospho-SYK resulted from differential expression of total SYK, densitometric ratios were generated between phosphorylated and total SYK. In addition to the higher overall SYK expression levels (Fig. 1 C ), the phosphorylation of SYK in CLL was f 2-fold higher than in normal B cells (Fig. 2 A ). Finally, phosphorylation of PLC g , and signal transducers and activators of transcription 3 (STAT3), typical downstream intermediates of SYK, was significantly increased and p-ERK1/2 tended to be increased in CLL cells compared with healthy B cells (Fig. 2 B ). mediators in CLL cells. To test whether activation of these downstream targets is SYK dependent, we analyzed their phosphorylation pattern in the presence and absence of SYK inhibitors. All inhibitors significantly reduced phosphorylation of PLC g 2 , STAT3, and ERK1/2 in MEC-1 cells (Fig. 2 C ). Similar effects were observed in primary CLL cells (low expression levels prevented analogous analysis of p-PLC g 2 ; Fig. 2 D ). MEC-1 cells were analyzed after overnight treatment. Due to the common high rate of apoptosis in extended culture of primary CLL cells, phosphorylation was analyzed after 2 hours of SYK inhibitor treatment (4 A mol/L). Because increased SYK kinase activity might promote survival of CLL cells, we tested the effect of SYK inhibitors on the viability of CLL cells. Quantification of viable and necrotic cells by Guava Viacount revealed significant cytotoxicity for Curcumin, Piceatannol, SYKII, and SYK Inhibitor IV in primary CLL cells. SYKII showed the highest cytotoxic effect on primary CLL cells, whereas the effect on the viability of healthy B cells was minor (Fig. 3 A ). SYKII achieved a significant decrease in viability at a concentration of as low as 1 A mol/L (Fig. 3 B ). Reduction of viability by SYK inhibition predominantly resulted from increased apoptosis rather than cell necrosis both in CLL and healthy B cells as assessed by flow cyto- metric quantification of Annexin-V and 7-AAD staining (Fig. 3 C ). This might explain the overall low effects observed in Fig. 3 A (note that Guava Viacount analysis is unable to detect apoptotic/ Annexin-V + /7-AAD À cells). Because the concentration of 4 A mol/L has been described to be achievable in vivo for R406 (26), and SYKII and R406 share equal IC 50 values regarding SYK inhibition in vitro (41 nmol/L), we continued with a concentration of 4 A mol/L. Intracellular staining for active Caspase 3 suggested that induction of apoptosis was Caspase 3 dependent (Fig. 3 D ). Interestingly, SYKII-induced Caspase activation was more effective in unmutated CLL samples than mutated ones (Fig. 3 D, right ). Finally, we treated CLL cells from 38 CLL patients with SYKII (4 and 10 A mol/L) or R406 (4 A mol/L) for 48 h. SYK inhibitors reduced the relative viability of CLL cells to 76% and 44% for SYKII (4 and 10 A mol/L) and 66% for R406 (4 A mol/L), respectively, as assessed by Annexin- V/7AAD staining (Fig. 4 A ). factors and SYK protein expression. Given the differences in clinical course, the differential SYK expression pattern (Fig. 1 D ), and Caspase 3 activation (Fig. 3 D ) depending on mutational status, we separately analyzed the susceptibility of CLL cells of these patient subgroups to SYK inhibition. CLL cells from unmutated patients showed a significantly higher rate of SYK inhibitor– induced apoptosis corresponding to decreased viability compared with cells of mutated patients. Similar results were obtained when cells were stratified by ZAP70 expression status. These data suggest that particularly patients with a poor prognosis might benefit from SYK inhibition (Fig. 4 B ). In addition, SYK protein expression correlated with cytotoxic effects of SYK inhibition (Fig. 4 C ). These data show that high SYK expression correlates with better response to SYK inhibitor treatment, indicating that SYK expression might predict for SYK inhibitor responsiveness. apoptosis. Because ZAP70 and SYK belong to the same protein family and share a high homology, we performed a protein tyrosine kinase activity assay demonstrating that both inhibitors only decreased the kinase activity of SYK but not ZAP70 at the concentrations used (Fig. 5 A ). Still, SYK inhibitors might act via unspecific binding of other kinases. To verify that SYK regulates apoptosis in CLL, we performed SYK gene silencing experiments in CLL cells. Seventy-two hours after transfection, we observed a significant induction of apoptosis along with down-regulation of SYK protein expression in SYK siRNA-transfected compared with control siRNA-treated CLL cells as assessed by flow cytometry (Fig. 5 B ). In line with our previous findings, we observed a conco- mitant induction of Caspase 3 (Fig. 5 C ). Finally, in three of three analyzed samples, a reduction of Akt phosphorylation was observed, another SYK downstream signaling molecule (Fig. 5 D ). SYK inhibitors increase the efficacy of fludarabine. The combination of fludarabine, the most potent cytoreductive agent in CLL, with other drugs enhances its antileukemic effects (27). Therefore, we assessed the in vitro effects of SYK inhibitors in combination with the active fludarabine derivate F-ara-A. Cotreat- ment of F-ara-A with SYKII or R406 significantly decreased viability of CLL cells at low, in vivo achievable doses of R406 (Fig. 6 A ). The mean viability of F-ara-A–treated cells was reduced from 47% to 34% by SYKII and to 30% by R406, respectively. cells toward SYK inhibition. Because the microenvironment enhances the viability of CLL cells and thereby decreases their sensitivity toward chemotherapy (28), we assayed apoptosis rates of CLL cells incubated with SYKII in the presence and absence of the stromal cell line M2-10B4. Stromal coculture protected CLL cells against spontaneous apoptosis (data not shown). However, the presence of stromal cells did not limit SYK inhibitor-induced cytotoxicity of CLL cells (Fig. 6 B ). In proliferative centers, CD40L-expressing T cells represent an important feature of the CLL microenvironment (29). Therefore, we analyzed the effect of SYKII in combination with CD40 ligation. Stimulation with CD40L (100 ng/mL) significantly increased SYKII- mediated cytotoxicity (Fig. 6 C ), suggesting that SYK inhibitors might ensure potential targeting of both, CD40 stimulated, proliferating and resting CLL cells. The signal transducer SYK is a crucial factor for function and survival of B cells. In the absence of SYK, B-cell maturation is blocked at the early pro–B-cell state in murine knockout models (30). Furthermore, inducible depletion of Ig a , the linker between the BCR and its signaling cascade, results in rapid death of mature B cells in mice, revealing the importance of BCR signaling for B-cell survival independent of antigen contact (31). Moreover, SYK formally acts as a proto-oncogene and is suggested to be involved in tumorgenesis of diverse hematologic malignancies including mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), lymphoblastic leukemia, and follicular lymphoma (26, 32–34). Here, we hypothesized that SYK plays a crucial role in CLL by transmitting BCR-mediated signals to the dependent downstream signal transduction pathways. In contrast to a previous report claiming normal SYK expression in CLL compared with normal B cells (35), but lacking quantitative analysis, we show SYK overexpression in CLL compared with healthy B cells. SYK protein expression was enhanced in unmutated compared with mutated CLL, potentially reflecting the increased BCR signaling in unmutated CLL reported by others (6–9). In accordance with its role for tonic BCR signaling, active SYK is crucial for DLBCL cell survival (26). In line with these data, all tested SYK inhibitors resulted in apoptosis induction and reduction of phosphorylation of downstream targets in CLL in this study. SYK inhibitor treatment also affected normal B cells. However, we observed a nearly 2-fold higher expression along with an f 2-fold higher phosphorylation status of SYK in CLL compared with healthy B cells, suggesting that SYK inhibition, although not fully CLL specific, may be feasible in CLL. In fact, the prodrug of R406, R788 (Fostamatinib disodium, FosD), an orally available SYK inhibitor under development for rheumatoid arthritis, showed significant in vitro activity against BCR-dependent non–Hodgkin lymphomas (26) and, very recently, even high clinical activity in patients with relapsed/refractory CLL. CLL/SLL patients showed an overall response rate of 54%, whereas other B-cell malignancies included in the study such as DLBCL (21% overall response rate), follicular lymphoma (10%), and mantle cell lymphoma (11%) showed considerably lower response rates (36), lending direct in vivo evidence to the concept of SYK inhibition in CLL. Our data may contribute to the understanding of these effects. We observed a decrease of CLL cell viability along with an increase of Caspase 3 activation in CLL cells upon treatment with SYKII or R406. Importantly, these events correlated with prognostic factors suggesting that SYK targeting may be most effective in patients with worst prognosis. Recently, a similar correlation was reported for the treatment of CLL cells with the SRC and ABL kinase inhibitor Dasatinib (37). Other studies showed elevated levels of SYK in cases with high ZAP70 expression, arguing for a synergistic role of ZAP70 and SYK in initial BCR signaling (38). The enhanced sensitivity of ZAP70 + CLL samples and the homology of SYK and ZAP70 raise the possibility that SYK inhibitors also interact with ZAP70 kinase activity. ...
Context 2
... antibodies. We therefore performed phospho-tyrosine immunoprecipitation followed by total SYK immunoblotting. In parallel, we performed a total SYK immunoblot with equal protein amounts as used for immunoprecipitation. To rule out that any effects observed on phospho-SYK resulted from differential expression of total SYK, densitometric ratios were generated between phosphorylated and total SYK. In addition to the higher overall SYK expression levels (Fig. 1 C ), the phosphorylation of SYK in CLL was f 2-fold higher than in normal B cells (Fig. 2 A ). Finally, phosphorylation of PLC g , and signal transducers and activators of transcription 3 (STAT3), typical downstream intermediates of SYK, was significantly increased and p-ERK1/2 tended to be increased in CLL cells compared with healthy B cells (Fig. 2 B ). mediators in CLL cells. To test whether activation of these downstream targets is SYK dependent, we analyzed their phosphorylation pattern in the presence and absence of SYK inhibitors. All inhibitors significantly reduced phosphorylation of PLC g 2 , STAT3, and ERK1/2 in MEC-1 cells (Fig. 2 C ). Similar effects were observed in primary CLL cells (low expression levels prevented analogous analysis of p-PLC g 2 ; Fig. 2 D ). MEC-1 cells were analyzed after overnight treatment. Due to the common high rate of apoptosis in extended culture of primary CLL cells, phosphorylation was analyzed after 2 hours of SYK inhibitor treatment (4 A mol/L). Because increased SYK kinase activity might promote survival of CLL cells, we tested the effect of SYK inhibitors on the viability of CLL cells. Quantification of viable and necrotic cells by Guava Viacount revealed significant cytotoxicity for Curcumin, Piceatannol, SYKII, and SYK Inhibitor IV in primary CLL cells. SYKII showed the highest cytotoxic effect on primary CLL cells, whereas the effect on the viability of healthy B cells was minor (Fig. 3 A ). SYKII achieved a significant decrease in viability at a concentration of as low as 1 A mol/L (Fig. 3 B ). Reduction of viability by SYK inhibition predominantly resulted from increased apoptosis rather than cell necrosis both in CLL and healthy B cells as assessed by flow cyto- metric quantification of Annexin-V and 7-AAD staining (Fig. 3 C ). This might explain the overall low effects observed in Fig. 3 A (note that Guava Viacount analysis is unable to detect apoptotic/ Annexin-V + /7-AAD À cells). Because the concentration of 4 A mol/L has been described to be achievable in vivo for R406 (26), and SYKII and R406 share equal IC 50 values regarding SYK inhibition in vitro (41 nmol/L), we continued with a concentration of 4 A mol/L. Intracellular staining for active Caspase 3 suggested that induction of apoptosis was Caspase 3 dependent (Fig. 3 D ). Interestingly, SYKII-induced Caspase activation was more effective in unmutated CLL samples than mutated ones (Fig. 3 D, right ). Finally, we treated CLL cells from 38 CLL patients with SYKII (4 and 10 A mol/L) or R406 (4 A mol/L) for 48 h. SYK inhibitors reduced the relative viability of CLL cells to 76% and 44% for SYKII (4 and 10 A mol/L) and 66% for R406 (4 A mol/L), respectively, as assessed by Annexin- V/7AAD staining (Fig. 4 A ). factors and SYK protein expression. Given the differences in clinical course, the differential SYK expression pattern (Fig. 1 D ), and Caspase 3 activation (Fig. 3 D ) depending on mutational status, we separately analyzed the susceptibility of CLL cells of these patient subgroups to SYK inhibition. CLL cells from unmutated patients showed a significantly higher rate of SYK inhibitor– induced apoptosis corresponding to decreased viability compared with cells of mutated patients. Similar results were obtained when cells were stratified by ZAP70 expression status. These data suggest that particularly patients with a poor prognosis might benefit from SYK inhibition (Fig. 4 B ). In addition, SYK protein expression correlated with cytotoxic effects of SYK inhibition (Fig. 4 C ). These data show that high SYK expression correlates with better response to SYK inhibitor treatment, indicating that SYK expression might predict for SYK inhibitor responsiveness. apoptosis. Because ZAP70 and SYK belong to the same protein family and share a high homology, we performed a protein tyrosine kinase activity assay demonstrating that both inhibitors only decreased the kinase activity of SYK but not ZAP70 at the concentrations used (Fig. 5 A ). Still, SYK inhibitors might act via unspecific binding of other kinases. To verify that SYK regulates apoptosis in CLL, we performed SYK gene silencing experiments in CLL cells. Seventy-two hours after transfection, we observed a significant induction of apoptosis along with down-regulation of SYK protein expression in SYK siRNA-transfected compared with control siRNA-treated CLL cells as assessed by flow cytometry (Fig. 5 B ). In line with our previous findings, we observed a conco- mitant induction of Caspase 3 (Fig. 5 C ). Finally, in three of three analyzed samples, a reduction of Akt phosphorylation was observed, another SYK downstream signaling molecule (Fig. 5 D ). SYK inhibitors increase the efficacy of fludarabine. The combination of fludarabine, the most potent cytoreductive agent in CLL, with other drugs enhances its antileukemic effects (27). Therefore, we assessed the in vitro effects of SYK inhibitors in combination with the active fludarabine derivate F-ara-A. Cotreat- ment of F-ara-A with SYKII or R406 significantly decreased viability of CLL cells at low, in vivo achievable doses of R406 (Fig. 6 A ). The mean viability of F-ara-A–treated cells was reduced from 47% to 34% by SYKII and to 30% by R406, respectively. cells toward SYK inhibition. Because the microenvironment enhances the viability of CLL cells and thereby decreases their sensitivity toward chemotherapy (28), we assayed apoptosis rates of CLL cells incubated with SYKII in the presence and absence of the stromal cell line M2-10B4. Stromal coculture protected CLL cells against spontaneous apoptosis (data not shown). However, the presence of stromal cells did not limit SYK inhibitor-induced cytotoxicity of CLL cells (Fig. 6 B ). In proliferative centers, CD40L-expressing T cells represent an important feature of the CLL microenvironment (29). Therefore, we analyzed the effect of SYKII in combination with CD40 ligation. Stimulation with CD40L (100 ng/mL) significantly increased SYKII- mediated cytotoxicity (Fig. 6 C ), suggesting that SYK inhibitors might ensure potential targeting of both, CD40 stimulated, proliferating and resting CLL cells. The signal transducer SYK is a crucial factor for function and survival of B cells. In the absence of SYK, B-cell maturation is blocked at the early pro–B-cell state in murine knockout models (30). Furthermore, inducible depletion of Ig a , the linker between the BCR and its signaling cascade, results in rapid death of mature B cells in mice, revealing the importance of BCR signaling for B-cell survival independent of antigen contact (31). Moreover, SYK formally acts as a proto-oncogene and is suggested to be involved in tumorgenesis of diverse hematologic malignancies including mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), lymphoblastic leukemia, and follicular lymphoma (26, 32–34). Here, we hypothesized that SYK plays a crucial role in CLL by transmitting BCR-mediated signals to the dependent downstream signal transduction pathways. In contrast to a previous report claiming normal SYK expression in CLL compared with normal B cells (35), but lacking quantitative analysis, we show SYK overexpression in CLL compared with healthy B cells. SYK protein expression was enhanced in unmutated compared with mutated CLL, potentially reflecting the increased BCR signaling in unmutated CLL reported by others (6–9). In accordance with its role for tonic BCR signaling, active SYK is crucial for DLBCL cell survival (26). In line with these data, all tested SYK inhibitors resulted in apoptosis induction and reduction of phosphorylation of downstream targets in CLL in this study. SYK inhibitor treatment also affected normal B cells. However, we observed a nearly 2-fold higher expression along with an f 2-fold higher phosphorylation status of SYK in CLL compared with healthy B cells, suggesting that SYK inhibition, although not fully CLL specific, may be feasible in CLL. In fact, the prodrug of R406, R788 (Fostamatinib disodium, FosD), an orally available SYK inhibitor under development for rheumatoid arthritis, showed significant in vitro activity against BCR-dependent non–Hodgkin lymphomas (26) and, very recently, even high clinical activity in patients with relapsed/refractory CLL. CLL/SLL patients showed an overall response rate of 54%, whereas other B-cell malignancies included in the study such as DLBCL (21% overall response rate), follicular lymphoma (10%), and mantle cell lymphoma (11%) showed considerably lower response rates (36), lending direct in vivo evidence to the concept of SYK inhibition in CLL. Our data may contribute to the understanding of these effects. We observed a decrease of CLL cell viability along with an increase of Caspase 3 activation in CLL cells upon treatment with SYKII or R406. Importantly, these events correlated with prognostic factors suggesting that SYK targeting may be most effective in patients with worst prognosis. Recently, a similar correlation was reported for the treatment of CLL cells with the SRC and ABL kinase inhibitor Dasatinib (37). Other studies showed elevated levels of SYK in cases with high ZAP70 expression, arguing for a synergistic role of ZAP70 and SYK in initial BCR signaling (38). The enhanced sensitivity of ZAP70 + CLL samples and the homology of SYK and ZAP70 raise the possibility that SYK inhibitors also interact with ZAP70 kinase activity. Such cross- inhibition is, however, highly unlikely because SYK but not ZAP70 activity was ...
Context 3
... SYK immunoblot with equal protein amounts as used for immunoprecipitation. To rule out that any effects observed on phospho-SYK resulted from differential expression of total SYK, densitometric ratios were generated between phosphorylated and total SYK. In addition to the higher overall SYK expression levels (Fig. 1 C ), the phosphorylation of SYK in CLL was f 2-fold higher than in normal B cells (Fig. 2 A ). Finally, phosphorylation of PLC g , and signal transducers and activators of transcription 3 (STAT3), typical downstream intermediates of SYK, was significantly increased and p-ERK1/2 tended to be increased in CLL cells compared with healthy B cells (Fig. 2 B ). mediators in CLL cells. To test whether activation of these downstream targets is SYK dependent, we analyzed their phosphorylation pattern in the presence and absence of SYK inhibitors. All inhibitors significantly reduced phosphorylation of PLC g 2 , STAT3, and ERK1/2 in MEC-1 cells (Fig. 2 C ). Similar effects were observed in primary CLL cells (low expression levels prevented analogous analysis of p-PLC g 2 ; Fig. 2 D ). MEC-1 cells were analyzed after overnight treatment. Due to the common high rate of apoptosis in extended culture of primary CLL cells, phosphorylation was analyzed after 2 hours of SYK inhibitor treatment (4 A mol/L). Because increased SYK kinase activity might promote survival of CLL cells, we tested the effect of SYK inhibitors on the viability of CLL cells. Quantification of viable and necrotic cells by Guava Viacount revealed significant cytotoxicity for Curcumin, Piceatannol, SYKII, and SYK Inhibitor IV in primary CLL cells. SYKII showed the highest cytotoxic effect on primary CLL cells, whereas the effect on the viability of healthy B cells was minor (Fig. 3 A ). SYKII achieved a significant decrease in viability at a concentration of as low as 1 A mol/L (Fig. 3 B ). Reduction of viability by SYK inhibition predominantly resulted from increased apoptosis rather than cell necrosis both in CLL and healthy B cells as assessed by flow cyto- metric quantification of Annexin-V and 7-AAD staining (Fig. 3 C ). This might explain the overall low effects observed in Fig. 3 A (note that Guava Viacount analysis is unable to detect apoptotic/ Annexin-V + /7-AAD À cells). Because the concentration of 4 A mol/L has been described to be achievable in vivo for R406 (26), and SYKII and R406 share equal IC 50 values regarding SYK inhibition in vitro (41 nmol/L), we continued with a concentration of 4 A mol/L. Intracellular staining for active Caspase 3 suggested that induction of apoptosis was Caspase 3 dependent (Fig. 3 D ). Interestingly, SYKII-induced Caspase activation was more effective in unmutated CLL samples than mutated ones (Fig. 3 D, right ). Finally, we treated CLL cells from 38 CLL patients with SYKII (4 and 10 A mol/L) or R406 (4 A mol/L) for 48 h. SYK inhibitors reduced the relative viability of CLL cells to 76% and 44% for SYKII (4 and 10 A mol/L) and 66% for R406 (4 A mol/L), respectively, as assessed by Annexin- V/7AAD staining (Fig. 4 A ). factors and SYK protein expression. Given the differences in clinical course, the differential SYK expression pattern (Fig. 1 D ), and Caspase 3 activation (Fig. 3 D ) depending on mutational status, we separately analyzed the susceptibility of CLL cells of these patient subgroups to SYK inhibition. CLL cells from unmutated patients showed a significantly higher rate of SYK inhibitor– induced apoptosis corresponding to decreased viability compared with cells of mutated patients. Similar results were obtained when cells were stratified by ZAP70 expression status. These data suggest that particularly patients with a poor prognosis might benefit from SYK inhibition (Fig. 4 B ). In addition, SYK protein expression correlated with cytotoxic effects of SYK inhibition (Fig. 4 C ). These data show that high SYK expression correlates with better response to SYK inhibitor treatment, indicating that SYK expression might predict for SYK inhibitor responsiveness. apoptosis. Because ZAP70 and SYK belong to the same protein family and share a high homology, we performed a protein tyrosine kinase activity assay demonstrating that both inhibitors only decreased the kinase activity of SYK but not ZAP70 at the concentrations used (Fig. 5 A ). Still, SYK inhibitors might act via unspecific binding of other kinases. To verify that SYK regulates apoptosis in CLL, we performed SYK gene silencing experiments in CLL cells. Seventy-two hours after transfection, we observed a significant induction of apoptosis along with down-regulation of SYK protein expression in SYK siRNA-transfected compared with control siRNA-treated CLL cells as assessed by flow cytometry (Fig. 5 B ). In line with our previous findings, we observed a conco- mitant induction of Caspase 3 (Fig. 5 C ). Finally, in three of three analyzed samples, a reduction of Akt phosphorylation was observed, another SYK downstream signaling molecule (Fig. 5 D ). SYK inhibitors increase the efficacy of fludarabine. The combination of fludarabine, the most potent cytoreductive agent in CLL, with other drugs enhances its antileukemic effects (27). Therefore, we assessed the in vitro effects of SYK inhibitors in combination with the active fludarabine derivate F-ara-A. Cotreat- ment of F-ara-A with SYKII or R406 significantly decreased viability of CLL cells at low, in vivo achievable doses of R406 (Fig. 6 A ). The mean viability of F-ara-A–treated cells was reduced from 47% to 34% by SYKII and to 30% by R406, respectively. cells toward SYK inhibition. Because the microenvironment enhances the viability of CLL cells and thereby decreases their sensitivity toward chemotherapy (28), we assayed apoptosis rates of CLL cells incubated with SYKII in the presence and absence of the stromal cell line M2-10B4. Stromal coculture protected CLL cells against spontaneous apoptosis (data not shown). However, the presence of stromal cells did not limit SYK inhibitor-induced cytotoxicity of CLL cells (Fig. 6 B ). In proliferative centers, CD40L-expressing T cells represent an important feature of the CLL microenvironment (29). Therefore, we analyzed the effect of SYKII in combination with CD40 ligation. Stimulation with CD40L (100 ng/mL) significantly increased SYKII- mediated cytotoxicity (Fig. 6 C ), suggesting that SYK inhibitors might ensure potential targeting of both, CD40 stimulated, proliferating and resting CLL cells. The signal transducer SYK is a crucial factor for function and survival of B cells. In the absence of SYK, B-cell maturation is blocked at the early pro–B-cell state in murine knockout models (30). Furthermore, inducible depletion of Ig a , the linker between the BCR and its signaling cascade, results in rapid death of mature B cells in mice, revealing the importance of BCR signaling for B-cell survival independent of antigen contact (31). Moreover, SYK formally acts as a proto-oncogene and is suggested to be involved in tumorgenesis of diverse hematologic malignancies including mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), lymphoblastic leukemia, and follicular lymphoma (26, 32–34). Here, we hypothesized that SYK plays a crucial role in CLL by transmitting BCR-mediated signals to the dependent downstream signal transduction pathways. In contrast to a previous report claiming normal SYK expression in CLL compared with normal B cells (35), but lacking quantitative analysis, we show SYK overexpression in CLL compared with healthy B cells. SYK protein expression was enhanced in unmutated compared with mutated CLL, potentially reflecting the increased BCR signaling in unmutated CLL reported by others (6–9). In accordance with its role for tonic BCR signaling, active SYK is crucial for DLBCL cell survival (26). In line with these data, all tested SYK inhibitors resulted in apoptosis induction and reduction of phosphorylation of downstream targets in CLL in this study. SYK inhibitor treatment also affected normal B cells. However, we observed a nearly 2-fold higher expression along with an f 2-fold higher phosphorylation status of SYK in CLL compared with healthy B cells, suggesting that SYK inhibition, although not fully CLL specific, may be feasible in CLL. In fact, the prodrug of R406, R788 (Fostamatinib disodium, FosD), an orally available SYK inhibitor under development for rheumatoid arthritis, showed significant in vitro activity against BCR-dependent non–Hodgkin lymphomas (26) and, very recently, even high clinical activity in patients with relapsed/refractory CLL. CLL/SLL patients showed an overall response rate of 54%, whereas other B-cell malignancies included in the study such as DLBCL (21% overall response rate), follicular lymphoma (10%), and mantle cell lymphoma (11%) showed considerably lower response rates (36), lending direct in vivo evidence to the concept of SYK inhibition in CLL. Our data may contribute to the understanding of these effects. We observed a decrease of CLL cell viability along with an increase of Caspase 3 activation in CLL cells upon treatment with SYKII or R406. Importantly, these events correlated with prognostic factors suggesting that SYK targeting may be most effective in patients with worst prognosis. Recently, a similar correlation was reported for the treatment of CLL cells with the SRC and ABL kinase inhibitor Dasatinib (37). Other studies showed elevated levels of SYK in cases with high ZAP70 expression, arguing for a synergistic role of ZAP70 and SYK in initial BCR signaling (38). The enhanced sensitivity of ZAP70 + CLL samples and the homology of SYK and ZAP70 raise the possibility that SYK inhibitors also interact with ZAP70 kinase activity. Such cross- inhibition is, however, highly unlikely because SYK but not ZAP70 activity was significantly reduced by both SYK inhibitors in a protein tyrosine kinase activity assay. However, the inhibitors tested may interfere with other molecules. ...
Context 4
... expressed high SYK levels (Fig. 1 C, right ). CLL cells from unmutated patients revealed a significantly higher SYK protein expression than those from mutated cases (Fig. 1 D ), confirming the trend observed at mRNA level. targets in CLL cells compared with healthy B cells. Because expression of SYK does not provide information about its activity, we analyzed the phosphorylation status of SYK in CLL cells compared with healthy B cells. Direct detection of phosphorylated SYK in whole cell lysates is hampered by the lack of suitable antibodies. We therefore performed phospho-tyrosine immunoprecipitation followed by total SYK immunoblotting. In parallel, we performed a total SYK immunoblot with equal protein amounts as used for immunoprecipitation. To rule out that any effects observed on phospho-SYK resulted from differential expression of total SYK, densitometric ratios were generated between phosphorylated and total SYK. In addition to the higher overall SYK expression levels (Fig. 1 C ), the phosphorylation of SYK in CLL was f 2-fold higher than in normal B cells (Fig. 2 A ). Finally, phosphorylation of PLC g , and signal transducers and activators of transcription 3 (STAT3), typical downstream intermediates of SYK, was significantly increased and p-ERK1/2 tended to be increased in CLL cells compared with healthy B cells (Fig. 2 B ). mediators in CLL cells. To test whether activation of these downstream targets is SYK dependent, we analyzed their phosphorylation pattern in the presence and absence of SYK inhibitors. All inhibitors significantly reduced phosphorylation of PLC g 2 , STAT3, and ERK1/2 in MEC-1 cells (Fig. 2 C ). Similar effects were observed in primary CLL cells (low expression levels prevented analogous analysis of p-PLC g 2 ; Fig. 2 D ). MEC-1 cells were analyzed after overnight treatment. Due to the common high rate of apoptosis in extended culture of primary CLL cells, phosphorylation was analyzed after 2 hours of SYK inhibitor treatment (4 A mol/L). Because increased SYK kinase activity might promote survival of CLL cells, we tested the effect of SYK inhibitors on the viability of CLL cells. Quantification of viable and necrotic cells by Guava Viacount revealed significant cytotoxicity for Curcumin, Piceatannol, SYKII, and SYK Inhibitor IV in primary CLL cells. SYKII showed the highest cytotoxic effect on primary CLL cells, whereas the effect on the viability of healthy B cells was minor (Fig. 3 A ). SYKII achieved a significant decrease in viability at a concentration of as low as 1 A mol/L (Fig. 3 B ). Reduction of viability by SYK inhibition predominantly resulted from increased apoptosis rather than cell necrosis both in CLL and healthy B cells as assessed by flow cyto- metric quantification of Annexin-V and 7-AAD staining (Fig. 3 C ). This might explain the overall low effects observed in Fig. 3 A (note that Guava Viacount analysis is unable to detect apoptotic/ Annexin-V + /7-AAD À cells). Because the concentration of 4 A mol/L has been described to be achievable in vivo for R406 (26), and SYKII and R406 share equal IC 50 values regarding SYK inhibition in vitro (41 nmol/L), we continued with a concentration of 4 A mol/L. Intracellular staining for active Caspase 3 suggested that induction of apoptosis was Caspase 3 dependent (Fig. 3 D ). Interestingly, SYKII-induced Caspase activation was more effective in unmutated CLL samples than mutated ones (Fig. 3 D, right ). Finally, we treated CLL cells from 38 CLL patients with SYKII (4 and 10 A mol/L) or R406 (4 A mol/L) for 48 h. SYK inhibitors reduced the relative viability of CLL cells to 76% and 44% for SYKII (4 and 10 A mol/L) and 66% for R406 (4 A mol/L), respectively, as assessed by Annexin- V/7AAD staining (Fig. 4 A ). factors and SYK protein expression. Given the differences in clinical course, the differential SYK expression pattern (Fig. 1 D ), and Caspase 3 activation (Fig. 3 D ) depending on mutational status, we separately analyzed the susceptibility of CLL cells of these patient subgroups to SYK inhibition. CLL cells from unmutated patients showed a significantly higher rate of SYK inhibitor– induced apoptosis corresponding to decreased viability compared with cells of mutated patients. Similar results were obtained when cells were stratified by ZAP70 expression status. These data suggest that particularly patients with a poor prognosis might benefit from SYK inhibition (Fig. 4 B ). In addition, SYK protein expression correlated with cytotoxic effects of SYK inhibition (Fig. 4 C ). These data show that high SYK expression correlates with better response to SYK inhibitor treatment, indicating that SYK expression might predict for SYK inhibitor responsiveness. apoptosis. Because ZAP70 and SYK belong to the same protein family and share a high homology, we performed a protein tyrosine kinase activity assay demonstrating that both inhibitors only decreased the kinase activity of SYK but not ZAP70 at the concentrations used (Fig. 5 A ). Still, SYK inhibitors might act via unspecific binding of other kinases. To verify that SYK regulates apoptosis in CLL, we performed SYK gene silencing experiments in CLL cells. Seventy-two hours after transfection, we observed a significant induction of apoptosis along with down-regulation of SYK protein expression in SYK siRNA-transfected compared with control siRNA-treated CLL cells as assessed by flow cytometry (Fig. 5 B ). In line with our previous findings, we observed a conco- mitant induction of Caspase 3 (Fig. 5 C ). Finally, in three of three analyzed samples, a reduction of Akt phosphorylation was observed, another SYK downstream signaling molecule (Fig. 5 D ). SYK inhibitors increase the efficacy of fludarabine. The combination of fludarabine, the most potent cytoreductive agent in CLL, with other drugs enhances its antileukemic effects (27). Therefore, we assessed the in vitro effects of SYK inhibitors in combination with the active fludarabine derivate F-ara-A. Cotreat- ment of F-ara-A with SYKII or R406 significantly decreased viability of CLL cells at low, in vivo achievable doses of R406 (Fig. 6 A ). The mean viability of F-ara-A–treated cells was reduced from 47% to 34% by SYKII and to 30% by R406, respectively. cells toward SYK inhibition. Because the microenvironment enhances the viability of CLL cells and thereby decreases their sensitivity toward chemotherapy (28), we assayed apoptosis rates of CLL cells incubated with SYKII in the presence and absence of the stromal cell line M2-10B4. Stromal coculture protected CLL cells against spontaneous apoptosis (data not shown). However, the presence of stromal cells did not limit SYK inhibitor-induced cytotoxicity of CLL cells (Fig. 6 B ). In proliferative centers, CD40L-expressing T cells represent an important feature of the CLL microenvironment (29). Therefore, we analyzed the effect of SYKII in combination with CD40 ligation. Stimulation with CD40L (100 ng/mL) significantly increased SYKII- mediated cytotoxicity (Fig. 6 C ), suggesting that SYK inhibitors might ensure potential targeting of both, CD40 stimulated, proliferating and resting CLL cells. The signal transducer SYK is a crucial factor for function and survival of B cells. In the absence of SYK, B-cell maturation is blocked at the early pro–B-cell state in murine knockout models (30). Furthermore, inducible depletion of Ig a , the linker between the BCR and its signaling cascade, results in rapid death of mature B cells in mice, revealing the importance of BCR signaling for B-cell survival independent of antigen contact (31). Moreover, SYK formally acts as a proto-oncogene and is suggested to be involved in tumorgenesis of diverse hematologic malignancies including mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), lymphoblastic leukemia, and follicular lymphoma (26, 32–34). Here, we hypothesized that SYK plays a crucial role in CLL by transmitting BCR-mediated signals to the dependent downstream signal transduction pathways. In contrast to a previous report claiming normal SYK expression in CLL compared with normal B cells (35), but lacking quantitative analysis, we show SYK overexpression in CLL compared with healthy B cells. SYK protein expression was enhanced in unmutated compared with mutated CLL, potentially reflecting the increased BCR signaling in unmutated CLL reported by others (6–9). In accordance with its role for tonic BCR signaling, active SYK is crucial for DLBCL cell survival (26). In line with these data, all tested SYK inhibitors resulted in apoptosis induction and reduction of phosphorylation of downstream targets in CLL in this study. SYK inhibitor treatment also affected normal B cells. However, we observed a nearly 2-fold higher expression along with an f 2-fold higher phosphorylation status of SYK in CLL compared with healthy B cells, suggesting that SYK inhibition, although not fully CLL specific, may be feasible in CLL. In fact, the prodrug of R406, R788 (Fostamatinib disodium, FosD), an orally available SYK inhibitor under development for rheumatoid arthritis, showed significant in vitro activity against BCR-dependent non–Hodgkin lymphomas (26) and, very recently, even high clinical activity in patients with relapsed/refractory CLL. CLL/SLL patients showed an overall response rate of 54%, whereas other B-cell malignancies included in the study such as DLBCL (21% overall response rate), follicular lymphoma (10%), and mantle cell lymphoma (11%) showed considerably lower response rates (36), lending direct in vivo evidence to the concept of SYK inhibition in CLL. Our data may contribute to the understanding of these effects. We observed a decrease of CLL cell viability along with an increase of Caspase 3 activation in CLL cells upon treatment with SYKII or R406. Importantly, these events correlated with prognostic factors suggesting that SYK targeting may be most effective in patients with worst prognosis. Recently, a similar ...
Citations
... These processes include the activation of PI3K and AKT (also known as PKB (protein kinase B)), as well as the phosphorylation of other signaling proteins. These findings have been documented in CLL [119] and acute myelogenous leukemia (AML) [120]. The present study found that the use of SYK siRNA resulted in the attenuation of the rate of proliferation and an augmentation in cell death in SCLC cell lines that expressed SYK [118]. ...
Simple Summary
The heterogeneous expression of signaling molecules within the tumor, including kinases, is the major contributor to the acquisition of drug resistance and poor survival observed in small-cell lung cancer (SCLC). The addition of immunotherapy to chemotherapy has only marginally prolonged survival in patients with extensive-stage SCLC (ES-SCLC). Recent clinical trials have combined immunotherapy with the pharmacological inhibitors of kinases often dysregulated in SCLC. However, the regime has not been effective in the long term. Here, we review studies and clinical trials exploring dysregulated kinases in SCLC progression and resistance to chemotherapies and immunotherapies. We also discuss the possibility of repurposing kinase inhibitors against SCLC that have already demonstrated promising results for other types of cancers.
Abstract
SCLC is refractory to conventional therapies; targeted therapies and immunological checkpoint inhibitor (ICI) molecules have prolonged survival only marginally. In addition, ICIs help only a subgroup of SCLC patients. Different types of kinases play pivotal roles in therapeutics-driven cellular functions. Therefore, there is a significant need to understand the roles of kinases in regulating therapeutic responses, acknowledge the existing knowledge gaps, and discuss future directions for improved therapeutics for recalcitrant SCLC. Here, we extensively review the effect of dysregulated kinases in SCLC. We further discuss the pharmacological inhibitors of kinases used in targeted therapies for recalcitrant SCLC. We also describe the role of kinases in the ICI-mediated activation of antitumor immune responses. Finally, we summarize the clinical trials evaluating the potential of kinase inhibitors and ICIs. This review overviews dysregulated kinases in SCLC and summarizes their potential as targeted therapeutic agents. We also discuss their clinical efficacy in enhancing anticancer responses mediated by ICIs.
... Finally, we compared C-13 with R406 (the active metabolite of Fostamatinib R788), a specific, ATP-competitive inhibitor of Syk (Syk IC50 = 41 nM) that has shown efficacy in the treatment of autoimmune diseases [34] and in preclinical leukaemia studies [35,36]. We compared the effects of increasing concentration of the two drugs on the survival of HCT-116, DLD-1 and HT29 CRC cell lines that express low to high levels of Syk, respectively ( Fig 6H). ...
... Syk is also essential for FcεRI-triggered mast cell activation, and in type II and type III hypersensitivity reactions mediated by Fcγ receptors. Fostamatinib (R788), a soluble pro-drug form of the Syk inhibitor R406, has been used as anti-inflammatory therapeutics in phase II clinical trials for the treatment of rheumatoid arthritis (RA) [34], in B-cell lymphocytic leukemia (B-CLL) [35] and in diffuse large B cell lymphoma (DLBCL) patients [46] with a positive response to therapy. A pro-survival role for Syk is also seen in retinoblastoma where the addition of the Syk inhibitor BAY61-3606 to the chemotherapeutic regimen used to treat retinoblastoma in a mouse model significantly improves outcomes [17]. ...
Spleen tyrosine kinase (Syk) expression have been both positively and negatively associated with tumorigenesis. Our goal was to evaluate the contribution of Syk and its two splice variants, full length Syk (L) and short isoform Syk (S), in the tumor biology of colorectal cancer cells (CRC). The analysis of Syk expression in primary human colorectal tumors, as well as the analysis of TCGA database, revealed a high Syk mRNA expression score in colorectal cancer tumors, suggesting a tumor promotor role of Syk in CRC. Our analysis showed that Syk (L) isoform is highly expressed in the majority of the tumor tissues and that it remains expressed in tumors in which global Syk expression is downregulated, suggesting the dependence of tumors to Syk (L) isoform. We also identified a small cluster of tumor tissues, which express a high proportion of Syk (S) isoform. This specific cluster is associated with overexpressed genes related to translation and mitochondria, and down regulated genes implicated in the progression of mitosis. For our functional studies, we used short hairpin RNA tools to target the expression of Syk in CRC cells bearing the activating K-Ras (G13D) mutation. Our results showed that while global Syk knock down increases cell proliferation and cell motility, Syk (L) expression silencing affects the viability and induces the apoptosis of the cells, confirming the dependence of cells on Syk (L) isoform for their survival. Finally, we report the promising potential of compound C-13, an original non-enzymatic inhibitor of Syk isolated in our group. In vitro studies showed that C-13 exerts cytotoxic effects on Syk-positive CRC cells by inhibiting their proliferation and their motility, and by inducing their apoptosis, while Syk-negative cell lines viability was not affected. Moreover, the oral and intraperitoneal administration of C-13 reduced the tumor growth of CRC DLD-1 cells xenografts in Nude mice in vivo .
... Second, curcumin also suppressed the activation of Spleen Tyrosine Kinase (Syk). Syk is a key tyrosine kinase that is constitutively activated in B cell lymphoma and is essential for tumor cells' survival and growth [85,86]. ...
B cells are the only player of humoral immune responses by the production of various types of antibodies. However, B cells are also involved in the pathogenesis of several immune-mediated diseases. Moreover, different types of B cell lymphoma have also been characterized. Selective depletion of B cells by anti-CD20 and other B cell-depleting agents in the clinic can improve a wide range of immune-mediated diseases. B cells' capacity to act as cytokine-producing cells explains how they can control immune cells' activity and contribute to disease pathogenesis. Thus, researchers investigated a safe, low-cost, and effective treatment modality for targeting B cells. In this respect, curcumin, the biologically active ingredient of turmeric, has a wide range of pharmacological activities. Evidence showed that curcumin could affect various immune cells, such as monocytes and macrophages, dendritic cells, and T lymphocytes. However, there are few pieces of evidence about the effects of curcumin on B cells. This study aims to review the available evidence about curcumin's modulatory effects on B cells' proliferation, differentiation, and function in different states. Apart from normal B cells, the modulatory effects of curcumin on B cell lymphoma will also discuss.
... Upregulation of PI3K/Akt/mTOR signaling can also be targeted by selective PI3K isoform inhibitors to overcome ibrutinib resistance either alone or in combination with other standard therapies [46] . Therapies inhibiting other signaling pathways such as inhibitor to MALT1 [47] , IRAK4 [48] , or SYK [49] , as well as inhibitors to chromatin modifiers including histone deacetylase such as panobinostat [50] and EZH2 such as tazemetostat [51] can also overcome ibrutinib resistance. There are clinical trials studying these novel inhibitors, such as a recent phase I/II study, that demonstrated the safety and efficacy of entospletinib (SYK inhibitor) in combination with obinutuzumab in relapsed/refractory (r/r) CLL [52] or an ongoing phase I study of a novel MALT inhibitor JNJ067856633 in r/r CLL and non-Hodgkin lymphoma (NHL). ...
Lymphoma is a diverse disease with a variety of different subtypes, each characterized by unique pathophysiology, tumor microenvironment, and underlying signaling pathways leading to oncogenesis. With our increasing understanding of the molecular biology of lymphoma, there have been a number of novel targeted therapies and immunotherapy approaches that have been developed for the treatment of this complex disease. Despite rapid progress in the field, however, many patients still relapse largely due to the development of drug resistance to these therapies. A better understanding of the mechanisms underlying resistance is needed to develop more novel treatment strategies that circumvent these mechanisms and design better treatment algorithms that personalize therapies to patients and sequence these therapies in the most optimal manner. This review focuses on the recent advances in therapies in lymphoma, including targeted therapies, monoclonal antibodies, antibody-drug conjugates, cellular therapy, bispecific antibodies, and checkpoint inhibitors. We discuss the genetic and cellular principles of drug resistance that span across all the therapies, as well as some of the unique mechanisms of resistance that are specific to these individual classes of therapies and the strategies that have been developed to address these modes of resistance.
... SYK is overexpressed in CLL and its pharmacologic targeting in the neoplastic B-cells blocked activation of multiple prosurvival BCR signalling-dependent kinases (e.g., AKT, PLCγ2, STAT3, ERK1/2), resulting in apoptosis. [2][3][4] Our pre-clinical experiments have shown that targeting SYK with a selective small molecule inhibitor entospletinib (GS-9973) abrogated BCR-signalling and induced apoptosis of CLL cells co-cultured on the BAFF (B-cell activation factor)-expressing stroma. 5 In these conditions, which partially mimic the tumour microenvironment, treatment with entospletinib, but not with ibrutinib or phosphoinotiside-3 kinase inhibitor idelalisib, led to decreased STAT3 activation and downmodulation of the antiapoptotic MCL1 mRNA and protein levels. ...
Spleen tyrosine kinase (SYK) is indispensable in B‐cell receptor signalling. SYK inhibitor entospletinib demonstrated clinical efficacy in patients with chronic lymphocytic leukaemia (CLL). However, pharmacodynamic effects of SYK inhibition in CLL cells and immunomodulatory effects of B‐cell receptor‐signalling inhibitors in patients with CLL are poorly understood. We conducted a phase 2 trial of entospletinib in combination with obinutuzumab, an anti‐CD20 antibody, in 17 patients with relapsed/refractory CLL. Pharmacodynamic analysis demonstrated that treatment with entospletinib led to rapid downmodulation of pSTAT3 and the anti‐apoptotic protein MCL1 in CLL cells. Meanwhile, 6 months of combination therapy was accompanied by a reduction in interferon‐γ secretion in CD4⁺ T‐cells and a reversal of exhausted phenotype, as evidenced by downregulation of PD‐1. Thus, SYK inhibition downmodulates MCL‐1 and partially restores T‐cell immunity in CLL. Trial registration number NCT03010358.
... Certains de ses partenaires tels que Lyn, BTK, PLCγ2, BLNK, sont également dérégulés dans la LLC (Buchner et al., 2009;Wang et al., 2013). L'ensemble de ces surexpressions conduit à une potentialisation de la signalisation du BCR traduit par une augmentation de la survie des cellules B de LLC. ...
... Par exemple, dans la leucémie lymphoïde chronique (LLC), une surexpression de Syk, ainsi que son activation basale plus élevée, sont détectées dans les cellules B pathologiques versus les lymphocytes B normaux. L'inhibition de l'expression de Syk ou bien un traitement par des inhibiteurs pharmacologiques induisent une entrée en apoptose des cellules B leucémiques(Buchner et al., 2009;Chen et al., 2008b;Gobessi et al., 2009). Ces effets ont été également observés dans d'autres hémopathies décrites dans le tableau 2. En réponse à une stimulation antigénique, la survie des cellules B de LLC dépend d'un seuil imposé par les niveaux d'expression du BCR membranaire, de Syk et/ou de Zap70. ...
... Dans ce contexte, la phosphorylation constitutive de Syk sur le résidu tyrosine 352 par les SFKs (Lyn, Lck) conduit à son activation et à une régulation positive de la survie des cellules B leucémiques. De plus, la phosphorylation de résidus tyrosine 525 et 526 est également impliquée dans cette activation constitutive menant à l'augmentation de la phosphorylation des effecteurs qui induisent la prolifération des cellules B de LLC(Buchner et al., 2009;Carsetti et al., 2009;Gobessi et al., 2009). Le rôle promoteur de tumeur de Syk n'est tout de même pas restreint aux cellules hématopoïétiques. ...
La prolifération cellulaire incontrôlée caractérisant les pathologies tumorales implique tout particulièrement des dérégulations de voies de signalisation cellulaire. Les protéines Vav1, Syk et β-caténine comptent parmi les effecteurs importants de signalisation dont l’expression et/ou l’activité sont dérégulées dans les cellules cancéreuses. Ce travail de thèse a porté sur l’étude des liens fonctionnels entre Vav1, Syk et la β-caténine dans une lignée cellulaire de carcinome pulmonaire. Nous avons montré que dans ces cellules Syk interagit avec Vav1 mais que la kinase phosphoryle le facteur d’échange sur des résidus différents de ceux des cellules hématopoïétiques. Syk, comme Vav1, interagit avec la β-caténine ; elle module certaines de ses modifications post-traductionnelles ainsi que l’expression de certains de ses gènes cibles. Les trois protéines sont impliquées dans la régulation de la migration cellulaire via les GTPases Rac1 et Cdc42. Par ailleurs, nous montrons que le gène Syk est un nouveau gène cible de la β-caténine avec l’identification de deux sites de fixation du complexe β-caténine/TCF4 sur son promoteur. En conclusion, nos travaux identifient et caractérisent le trio Vav1, Syk et β-caténine dans des cellules épithéliales comme dans les cellules hématopoïétiques. Nos données permettront d’établir un différentiel des fonctions de ce trio entre les deux types cellulaires pour mieux appréhender leurs rôles dans la tumorigénèse.
... In hematopoietic malignancies, the non-receptor spleen tyrosine kinase (SYK) mainly contributes to cancer cell survival and proliferation [3][4][5] by mediating tonic and chronic signaling through B-cell antigen receptors in different B-cell-derived lymphoma types [6][7][8]. On the other hand, we and others demonstrated that SYK is also expressed in non-hematopoietic cells and that it behaves as a tumor suppressor in mammary epithelial cells [9][10][11][12]. ...
Spleen tyrosine kinase (SYK) can behave as an oncogene or a tumor suppressor, depending on the cell and tissue type. As pharmacological SYK inhibitors are currently evaluated in clinical trials, it is important to gain more information on the molecular mechanisms underpinning these opposite roles. To this aim, we reconstructed and compared its signaling networks using phosphoproteomic data from breast cancer and Burkitt lymphoma cell lines where SYK behaves as a tumor suppressor and promoter. Bioinformatic analyses allowed for unveiling the main differences in signaling pathways, network topology and signal propagation from SYK to its potential effectors. In breast cancer cells, the SYK target-enriched signaling pathways included intercellular adhesion and Hippo signaling components that are often linked to tumor suppression. In Burkitt lymphoma cells, the SYK target-enriched signaling pathways included molecules that could play a role in SYK pro-oncogenic function in B-cell lymphomas. Several protein interactions were profoundly rewired in the breast cancer network compared with the Burkitt lymphoma network. These data demonstrate that proteomic profiling combined with mathematical network modeling allows untangling complex pathway interplays and revealing difficult to discern interactions among the SYK pathways that positively and negatively affect tumor formation and progression.
... 6,34 Importantly, inhibition of SYK causes apoptosis in several types of cancers, such as lung cancer, small cell lung cancer, pancreatic cancer, breast cancer, and B-Bell lymphocytic leukemia, indicating the potential of SYK as an anticancer target. [35][36][37][38][39][40][41] Fostamatinib, 42,43 BAY61-3606, 44 cerdulatinib, 45 MK-8457, 46 and Entospletinib 28 are SYK inhibitors and have been assessed in clinical trials to treat cancer and autoimmune disease. Nevertheless, unacceptable physicochemical properties and poor oral bioavailability were observed in these developed SYK inhibitors. ...
The cytosolic non-receptor protein kinase, spleen tyrosine kinase (SYK), is an attractive drug target in autoimmune, inflammatory disorder, and cancers indications. Here, we employed pharmacophore-based drug screening combined with biochemical assay and molecular dynamics (MD) simulations to identify and characterize inhibitors targeting SYK. The built pharmacophore model, phar-TanI, successfully identified tanshinone (TanI (IC50 = 1.72 μM)) and its analogs (TanIIA (IC50 = 3.2 μM), ST32da (IC50 = 46 μM), and ST32db (IC50 = 51 μM)) which apparently attenuated the activities of SYK in vitro. Additionally, the MD simulations followed by Ligplot analyses revealed that TanI and TanIIA interfered SYK activity through binding deeply into the active site. Besides, TanI and TanIIA mainly interact with residues L377, A400, V433, M448, M450, A451, E452, L453, G454, P455, and L501, which are functional hotspots for structure-based inhibitor optimization against SYK. The structure–activity relationships (SAR) study of the identified SYK inhibitors demonstrated that the pharmacophore model, phar-TanI is reliable and precise in screening inhibitors against SYK. This study disclosed the structure–function relationships of tanshinones from Traditional Chinese Medicine (Danshen), revealing their binding site and mode of action in inhibiting SYK and provides applicability in developing new therapeutic agents.
... Activation of these pathways leads to proliferation, differentiation, cytoskeletal remodeling, cytokine release and survival [2]. In several hematological malignancies, such as CLL [13], diffuse large B-cell lymphoma (DLBCL) [14], mantle cell lymphoma (MCL) [15] and follicular lymphoma (FL) [16], SYK acts as a proto-oncogene and is thus involved in tumorigenesis. Here, tonic BCR signaling leads to constitutively activated SYK [17]. ...
Background:
Impaired B-cell receptor (BCR) function has been associated with the progress of several B-cell malignancies. The spleen tyrosine kinase (SYK) represents a potential therapeutic target in a subset of B-cell neoplasias. In precursor B-acute lymphoblastic leukemia (B-ALL), the pathogenic role and therapeutic potential of SYK is still controversially discussed. We evaluate the application of the SYK inhibitor entospletinib (Ento) in pre- and pro-B-ALL cell lines, characterizing the biologic and molecular effects.
Methods:
SYK expression was characterized in pre-B-ALL (NALM-6) and pro-B-ALL cell lines (SEM and RS4;11). The cell lines were exposed to different Ento concentrations and the cell biological response analyzed by proliferation, metabolic activity, apoptosis induction, cell-cycle distribution and morphology. BCR pathway gene expression and protein modulations were further characterized.
Results:
Ento significantly induced anti-proliferative and pro-apoptotic effects in NALM-6 and SEM, while barely affecting RS4;11. Targeted RNAseq revealed pronounced gene expression modulation only in NALM-6, while Western Blot analyses demonstrated that vital downstream effector proteins, such as pAKT, pERK, pGSK3β, p53 and BCL-6, were affected by Ento exposure in the inhibitor-sensitive cell lines.
Conclusion:
Different acting modes of Ento, independent of pre-BCR dependency, were characterized, unexpected in SEM. Accordingly, SYK classifies as a potential target structure in a subset of pro-B-ALLs.
... SYK is a cytoplasmic TK acting to couple BCR to intracellular signalling pathways in B lymphocytes [118]. SYK is a pro-survival factor, and therefore, its knockdown or inhibition leads to apoptosis in the cell subsets of CLL and ALL of the B cell lineage that express active SYK [119][120][121][122]. The observed effect might be due to the significant downstream reduction of major inflammatory mediators such as tumor necrosis factor alpha (TNFα), interleukins (ILs)-1, 6 and 18 that suggest the role of SYK in mediating the proinflammatory response [116]. ...
Simple Summary
Protein phosphorylation is a key regulatory mechanism that controls a wide variety of cellular responses. This process is catalysed by the members of the protein kinase superfamily that are classified into two main families based on their ability to phosphorylate either tyrosine or serine and threonine residues in their substrates. Massive research efforts have been invested in dissecting the functions of tyrosine kinases, revealing their importance in the initiation and progression of human malignancies. Based on these investigations, numerous tyrosine kinase inhibitors have been included in clinical protocols and proved to be effective in targeted therapies for various haematological malignancies. In this review, we provide insights into the role of tyrosine kinases in leukaemia and discuss their targeting for therapeutic purposes with the currently available inhibitory compounds.
Abstract
Protein kinases constitute a large group of enzymes catalysing protein phosphorylation and controlling multiple signalling events. The human protein kinase superfamily consists of 518 members and represents a complicated system with intricate internal and external interactions. Protein kinases are classified into two main families based on the ability to phosphorylate either tyrosine or serine and threonine residues. Among the 90 tyrosine kinase genes, 58 are receptor types classified into 20 groups and 32 are of the nonreceptor types distributed into 10 groups. Tyrosine kinases execute their biological functions by controlling a variety of cellular responses, such as cell division, metabolism, migration, cell–cell and cell matrix adhesion, cell survival and apoptosis. Over the last 30 years, a major focus of research has been directed towards cancer-associated tyrosine kinases owing to their critical contributions to the development and aggressiveness of human malignancies through the pathological effects on cell behaviour. Leukaemia represents a heterogeneous group of haematological malignancies, characterised by an uncontrolled proliferation of undifferentiated hematopoietic cells or leukaemia blasts, mostly derived from bone marrow. They are usually classified as chronic or acute, depending on the rates of their progression, as well as myeloid or lymphoblastic, according to the type of blood cells involved. Overall, these malignancies are relatively common amongst both children and adults. In malignant haematopoiesis, multiple tyrosine kinases of both receptor and nonreceptor types, including AXL receptor tyrosine kinase (AXL), Discoidin domain receptor 1 (DDR1), Vascular endothelial growth factor receptor (VEGFR), Fibroblast growth factor receptor (FGFR), Mesenchymal–epithelial transition factor (MET), proto-oncogene c-Src (SRC), Spleen tyrosine kinase (SYK) and pro-oncogenic Abelson tyrosine-protein kinase 1 (ABL1) mutants, are implicated in the pathogenesis and drug resistance of practically all types of leukaemia. The role of ABL1 kinase mutants and their therapeutic inhibitors have been extensively analysed in scientific literature, and therefore, in this review, we provide insights into the impact and mechanism of action of other tyrosine kinases involved in the development and progression of human leukaemia and discuss the currently available and emerging treatment options based on targeting these molecules.
