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Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma.

Boukhiar MA, Roger C, Tran J, Gressin R, Martin A, Ajchenbaum-Cymbalista F, Varin-Blank N, Ledoux D, Baran-Marszak F - Exp Hematol Oncol (2013)

Bottom Line: Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1.Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival.Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM, UMR U978, Adaptateur de Signalisation en Hématologie, F-93000, Bobigny, France. fanny.baran-marszak@avc.aphp.fr.

ABSTRACT

Background: We previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL.

Methods: Primary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry.

Results: We showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed.

Conclusions: This study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis.

No MeSH data available.


Related in: MedlinePlus

PP2 and dasatinib inhibit constitutive phosphorylation of LYN and induce apoptosis of primary MCL cells. (A) Constitutive phosphorylation profiles of LYN in MCL patients’ samples. Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. The blots were stripped and re-probed for total LYN. (B) Total proteins from HBL-2 cells were immunoprecipitated with an anti LYN antibody (Ig-LYN) or with an irrelevant IgG control and immunobloted (IB) with either an anti-phosphotyrosine antibody (P-Tyr) or an anti-LYN antibody. (C) Primary MCL cells (UPN3, UPN14) were treated with variable concentrations of PP2 (2 to 20 μM) or dasatinib (1 to 200nM) for 2 h. Phospho-Tyr397 LYN and LYN total were analyzed by western-blot. (D) Primary MCL cells were treated with various concentrations of PP2 (UPN3, top panel) or 10 μM of PP2 (UPN 1–3-7–8-9–14, middle panel) for 24 h and apoptosis was measured by flow cytometry after gating on CD19+ cells. All measurements were done in duplicate and the mean is provided. Results are also shown as median ± quartile (box) ± SE (bars) bottom panel). Differences between groups were determined using the paired Student t test. (E) Primary MCL cells were treated with dasatinib for 24 h with various concentrations (top panel) or with 100nM (middle panel). Apoptosis was measured as described above. Results are also shown as median ± quartile (box) ± SE (bars) bottom panel).
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Figure 4: PP2 and dasatinib inhibit constitutive phosphorylation of LYN and induce apoptosis of primary MCL cells. (A) Constitutive phosphorylation profiles of LYN in MCL patients’ samples. Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. The blots were stripped and re-probed for total LYN. (B) Total proteins from HBL-2 cells were immunoprecipitated with an anti LYN antibody (Ig-LYN) or with an irrelevant IgG control and immunobloted (IB) with either an anti-phosphotyrosine antibody (P-Tyr) or an anti-LYN antibody. (C) Primary MCL cells (UPN3, UPN14) were treated with variable concentrations of PP2 (2 to 20 μM) or dasatinib (1 to 200nM) for 2 h. Phospho-Tyr397 LYN and LYN total were analyzed by western-blot. (D) Primary MCL cells were treated with various concentrations of PP2 (UPN3, top panel) or 10 μM of PP2 (UPN 1–3-7–8-9–14, middle panel) for 24 h and apoptosis was measured by flow cytometry after gating on CD19+ cells. All measurements were done in duplicate and the mean is provided. Results are also shown as median ± quartile (box) ± SE (bars) bottom panel). Differences between groups were determined using the paired Student t test. (E) Primary MCL cells were treated with dasatinib for 24 h with various concentrations (top panel) or with 100nM (middle panel). Apoptosis was measured as described above. Results are also shown as median ± quartile (box) ± SE (bars) bottom panel).

Mentions: The BCR signal is initially transmitted by LYN kinase leading to activation of various signaling pathways including JNK. We therefore evaluated the activation status of LYN in MCL cells and its involvement in cell survival. Using an anti-phospho-SFK recognizing the catalytic site of several Src kinases among which the Tyr397 of LYN, we detected in 9 out of 10 UPN cases tested (UPN 1,3,5,7,8,9,10,13,14) such a specific signal to variable extents of constitutive phosphorylation forming a 53–56 kDa doublet (Figure 4A and Additional file 3: Figure S2). We confirmed that this doublet corresponded to phospho-LYN by an immunoprecipitation assay using an anti-LYN antibody (Figure 4B). Considering the constitutive activation of LYN in MCL cells, we next evaluated the impact of PP2, a synthetic pyrazolopyrimidine selective inhibitor of SFK, and dasatinib (BMS-354825), an oral multi kinase inhibitor which also inhibits the trans-autophosphorylation of the active Tyr397 residue of LYN [30]. Treatment of primary cells with PP2 or dasatinib led to a dose-dependent decrease of Tyr397 LYN phosphorylation and complete inhibition was achieved up to 10 μM and 100nM for PP2 and dasatinib respectively (Figure 4C). Inhibition of phospho-Tyr397 LYN by PP2 was associated with a significant and dose-dependent increase of apoptosis rate (from 49±4% to 67±3% apoptotic cells for untreated and 24 h treated (10 μM) cells respectively; p=0.006; n=6) (Figure 4D). Treatment with dasatinib for 24 h also led to a significant and dose-dependent increase of apoptosis (from 46±5% to 64±5% apoptotic cells for untreated and treated (100nM) cells, respectively; p=0.0001; n=7) (Figure 4E). Remarkably, dasatinib had little apoptosis effect on phospho-Tyr397 LYN-negative cells (UPN4) at a concentration up to 200nM (Figure 4E, top panel). Altogether, these results indicate that MCL cells display a constitutive phosphorylation of BCR-associated LYN and that treatment with dasatinib or PP2 suppressed LYN activation and increased spontaneous apoptosis.


Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma.

Boukhiar MA, Roger C, Tran J, Gressin R, Martin A, Ajchenbaum-Cymbalista F, Varin-Blank N, Ledoux D, Baran-Marszak F - Exp Hematol Oncol (2013)

PP2 and dasatinib inhibit constitutive phosphorylation of LYN and induce apoptosis of primary MCL cells. (A) Constitutive phosphorylation profiles of LYN in MCL patients’ samples. Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. The blots were stripped and re-probed for total LYN. (B) Total proteins from HBL-2 cells were immunoprecipitated with an anti LYN antibody (Ig-LYN) or with an irrelevant IgG control and immunobloted (IB) with either an anti-phosphotyrosine antibody (P-Tyr) or an anti-LYN antibody. (C) Primary MCL cells (UPN3, UPN14) were treated with variable concentrations of PP2 (2 to 20 μM) or dasatinib (1 to 200nM) for 2 h. Phospho-Tyr397 LYN and LYN total were analyzed by western-blot. (D) Primary MCL cells were treated with various concentrations of PP2 (UPN3, top panel) or 10 μM of PP2 (UPN 1–3-7–8-9–14, middle panel) for 24 h and apoptosis was measured by flow cytometry after gating on CD19+ cells. All measurements were done in duplicate and the mean is provided. Results are also shown as median ± quartile (box) ± SE (bars) bottom panel). Differences between groups were determined using the paired Student t test. (E) Primary MCL cells were treated with dasatinib for 24 h with various concentrations (top panel) or with 100nM (middle panel). Apoptosis was measured as described above. Results are also shown as median ± quartile (box) ± SE (bars) bottom panel).
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Related In: Results  -  Collection

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Figure 4: PP2 and dasatinib inhibit constitutive phosphorylation of LYN and induce apoptosis of primary MCL cells. (A) Constitutive phosphorylation profiles of LYN in MCL patients’ samples. Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. The blots were stripped and re-probed for total LYN. (B) Total proteins from HBL-2 cells were immunoprecipitated with an anti LYN antibody (Ig-LYN) or with an irrelevant IgG control and immunobloted (IB) with either an anti-phosphotyrosine antibody (P-Tyr) or an anti-LYN antibody. (C) Primary MCL cells (UPN3, UPN14) were treated with variable concentrations of PP2 (2 to 20 μM) or dasatinib (1 to 200nM) for 2 h. Phospho-Tyr397 LYN and LYN total were analyzed by western-blot. (D) Primary MCL cells were treated with various concentrations of PP2 (UPN3, top panel) or 10 μM of PP2 (UPN 1–3-7–8-9–14, middle panel) for 24 h and apoptosis was measured by flow cytometry after gating on CD19+ cells. All measurements were done in duplicate and the mean is provided. Results are also shown as median ± quartile (box) ± SE (bars) bottom panel). Differences between groups were determined using the paired Student t test. (E) Primary MCL cells were treated with dasatinib for 24 h with various concentrations (top panel) or with 100nM (middle panel). Apoptosis was measured as described above. Results are also shown as median ± quartile (box) ± SE (bars) bottom panel).
Mentions: The BCR signal is initially transmitted by LYN kinase leading to activation of various signaling pathways including JNK. We therefore evaluated the activation status of LYN in MCL cells and its involvement in cell survival. Using an anti-phospho-SFK recognizing the catalytic site of several Src kinases among which the Tyr397 of LYN, we detected in 9 out of 10 UPN cases tested (UPN 1,3,5,7,8,9,10,13,14) such a specific signal to variable extents of constitutive phosphorylation forming a 53–56 kDa doublet (Figure 4A and Additional file 3: Figure S2). We confirmed that this doublet corresponded to phospho-LYN by an immunoprecipitation assay using an anti-LYN antibody (Figure 4B). Considering the constitutive activation of LYN in MCL cells, we next evaluated the impact of PP2, a synthetic pyrazolopyrimidine selective inhibitor of SFK, and dasatinib (BMS-354825), an oral multi kinase inhibitor which also inhibits the trans-autophosphorylation of the active Tyr397 residue of LYN [30]. Treatment of primary cells with PP2 or dasatinib led to a dose-dependent decrease of Tyr397 LYN phosphorylation and complete inhibition was achieved up to 10 μM and 100nM for PP2 and dasatinib respectively (Figure 4C). Inhibition of phospho-Tyr397 LYN by PP2 was associated with a significant and dose-dependent increase of apoptosis rate (from 49±4% to 67±3% apoptotic cells for untreated and 24 h treated (10 μM) cells respectively; p=0.006; n=6) (Figure 4D). Treatment with dasatinib for 24 h also led to a significant and dose-dependent increase of apoptosis (from 46±5% to 64±5% apoptotic cells for untreated and treated (100nM) cells, respectively; p=0.0001; n=7) (Figure 4E). Remarkably, dasatinib had little apoptosis effect on phospho-Tyr397 LYN-negative cells (UPN4) at a concentration up to 200nM (Figure 4E, top panel). Altogether, these results indicate that MCL cells display a constitutive phosphorylation of BCR-associated LYN and that treatment with dasatinib or PP2 suppressed LYN activation and increased spontaneous apoptosis.

Bottom Line: Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1.Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival.Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM, UMR U978, Adaptateur de Signalisation en Hématologie, F-93000, Bobigny, France. fanny.baran-marszak@avc.aphp.fr.

ABSTRACT

Background: We previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL.

Methods: Primary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry.

Results: We showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed.

Conclusions: This study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis.

No MeSH data available.


Related in: MedlinePlus