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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 BCR-induced LYN and JNK activation and EGR-1 upregulation. (A) Patients’ cells (UPN9) were pretreated with dasatinib (100nM) or SP600125 (10 μM) for 1 h and stimulated for 5 min or 15 min with soluble anti-IgM (10 μg/ml). Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. (B) The same experiment was done with PP2 (10 μM) on UPN 9 and UPN 13 under the same conditions of BCR stimulation for 10 min. Lines 1 and 2 have to be compared to evidence the effect of PP2 on the constitutive level of phosphorylation for Lyn. Similarly lines 3 and 4 reflect this effect upon BCR stimulation. (C) BCR-induced phospho-JNK (p54 and p46) was analyzed under treatment with dasatinib (100nM) or SP600125 (10 μM) used herein as a positive control of phospho-JNK inhibition. (D) Impact of dasatinib on BCR-induced EGR-1 expression. MCL cells were pretreated with various concentrations of dasatinib as indicated and stimulated with immobilized anti-IgM. EGR-1 mRNA and protein were analyzed by qRT-PCR at 1 h of stimulation (left panel) and western-blot at 3 h of stimulation (right panel). Relative mRNA expression was calculated compared with unstimulated cells.
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Figure 5: PP2 and dasatinib inhibit BCR-induced LYN and JNK activation and EGR-1 upregulation. (A) Patients’ cells (UPN9) were pretreated with dasatinib (100nM) or SP600125 (10 μM) for 1 h and stimulated for 5 min or 15 min with soluble anti-IgM (10 μg/ml). Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. (B) The same experiment was done with PP2 (10 μM) on UPN 9 and UPN 13 under the same conditions of BCR stimulation for 10 min. Lines 1 and 2 have to be compared to evidence the effect of PP2 on the constitutive level of phosphorylation for Lyn. Similarly lines 3 and 4 reflect this effect upon BCR stimulation. (C) BCR-induced phospho-JNK (p54 and p46) was analyzed under treatment with dasatinib (100nM) or SP600125 (10 μM) used herein as a positive control of phospho-JNK inhibition. (D) Impact of dasatinib on BCR-induced EGR-1 expression. MCL cells were pretreated with various concentrations of dasatinib as indicated and stimulated with immobilized anti-IgM. EGR-1 mRNA and protein were analyzed by qRT-PCR at 1 h of stimulation (left panel) and western-blot at 3 h of stimulation (right panel). Relative mRNA expression was calculated compared with unstimulated cells.

Mentions: Since PP2 and dasatinib efficiently blocked activation of BCR-associated LYN in MCL cells, we next evaluated the impact of these compounds on JNK phosphorylation, EGR-1 expression and on cell survival upon BCR engagement. As shown in Figure 5A, a strong increase of phospho-Tyr397 LYN was observed in response to BCR ligation (lane 1 compared to lane 4) and treatment with dasatinib (100nM) completely blocked this effect while SP600125 that affect JNK did not. Similarly, PP2 decreased BCR-induced phospho-Tyr397 LYN in primary MCL cells (Figure 5B). Dasatinib also reduced BCR-induced phospho-JNK p46 (Figure 5C, lane 1 compared to lane 3 and lane 4 compared to lane 6), positioning JNK as a downstream target of LYN in response to BCR engagement. We next evaluated the impact of dasatinib on basal and BCR-induced level of EGR-1 as a target of JNK. As shown in Figure 5D (left panels), dasatinib (100nM) decreased basal expression of EGR1 mRNA and totally abrogated its upregulation in response to BCR ligation (UPN3, UPN13). Dasatinib also slightly decreased basal level of EGR1 protein and blocked its BCR-induced upregulation (Figure 5D, right panels and Additional file 4: Figure S3). Finally, we evaluated the impact of PP2 and dasatinib treatment on BCR-induced cell survival. Increasing concentrations of dasatinib abrogated the BCR-induced survival response in a dose-dependent manner and significantly suppressed this survival signal in all UPN cases tested (from 34 ±6% to 54±7% apoptotic cells for untreated and dasatinib-treated BCR-activated cells, respectively; n=6; p<0.001) (Figure 6A). Similarly, PP2 treatment also reduced or abolished BCR-induced cell survival (Figure 6B). Overall, these results highlight the importance of LYN, JNK and EGR1 as intermediates of BCR signaling in mediating survival signals in MCL cells and point out to the efficiency of dasatinib in suppressing cell survival signal emanating from the BCR.


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 BCR-induced LYN and JNK activation and EGR-1 upregulation. (A) Patients’ cells (UPN9) were pretreated with dasatinib (100nM) or SP600125 (10 μM) for 1 h and stimulated for 5 min or 15 min with soluble anti-IgM (10 μg/ml). Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. (B) The same experiment was done with PP2 (10 μM) on UPN 9 and UPN 13 under the same conditions of BCR stimulation for 10 min. Lines 1 and 2 have to be compared to evidence the effect of PP2 on the constitutive level of phosphorylation for Lyn. Similarly lines 3 and 4 reflect this effect upon BCR stimulation. (C) BCR-induced phospho-JNK (p54 and p46) was analyzed under treatment with dasatinib (100nM) or SP600125 (10 μM) used herein as a positive control of phospho-JNK inhibition. (D) Impact of dasatinib on BCR-induced EGR-1 expression. MCL cells were pretreated with various concentrations of dasatinib as indicated and stimulated with immobilized anti-IgM. EGR-1 mRNA and protein were analyzed by qRT-PCR at 1 h of stimulation (left panel) and western-blot at 3 h of stimulation (right panel). Relative mRNA expression was calculated compared with unstimulated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585857&req=5

Figure 5: PP2 and dasatinib inhibit BCR-induced LYN and JNK activation and EGR-1 upregulation. (A) Patients’ cells (UPN9) were pretreated with dasatinib (100nM) or SP600125 (10 μM) for 1 h and stimulated for 5 min or 15 min with soluble anti-IgM (10 μg/ml). Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. (B) The same experiment was done with PP2 (10 μM) on UPN 9 and UPN 13 under the same conditions of BCR stimulation for 10 min. Lines 1 and 2 have to be compared to evidence the effect of PP2 on the constitutive level of phosphorylation for Lyn. Similarly lines 3 and 4 reflect this effect upon BCR stimulation. (C) BCR-induced phospho-JNK (p54 and p46) was analyzed under treatment with dasatinib (100nM) or SP600125 (10 μM) used herein as a positive control of phospho-JNK inhibition. (D) Impact of dasatinib on BCR-induced EGR-1 expression. MCL cells were pretreated with various concentrations of dasatinib as indicated and stimulated with immobilized anti-IgM. EGR-1 mRNA and protein were analyzed by qRT-PCR at 1 h of stimulation (left panel) and western-blot at 3 h of stimulation (right panel). Relative mRNA expression was calculated compared with unstimulated cells.
Mentions: Since PP2 and dasatinib efficiently blocked activation of BCR-associated LYN in MCL cells, we next evaluated the impact of these compounds on JNK phosphorylation, EGR-1 expression and on cell survival upon BCR engagement. As shown in Figure 5A, a strong increase of phospho-Tyr397 LYN was observed in response to BCR ligation (lane 1 compared to lane 4) and treatment with dasatinib (100nM) completely blocked this effect while SP600125 that affect JNK did not. Similarly, PP2 decreased BCR-induced phospho-Tyr397 LYN in primary MCL cells (Figure 5B). Dasatinib also reduced BCR-induced phospho-JNK p46 (Figure 5C, lane 1 compared to lane 3 and lane 4 compared to lane 6), positioning JNK as a downstream target of LYN in response to BCR engagement. We next evaluated the impact of dasatinib on basal and BCR-induced level of EGR-1 as a target of JNK. As shown in Figure 5D (left panels), dasatinib (100nM) decreased basal expression of EGR1 mRNA and totally abrogated its upregulation in response to BCR ligation (UPN3, UPN13). Dasatinib also slightly decreased basal level of EGR1 protein and blocked its BCR-induced upregulation (Figure 5D, right panels and Additional file 4: Figure S3). Finally, we evaluated the impact of PP2 and dasatinib treatment on BCR-induced cell survival. Increasing concentrations of dasatinib abrogated the BCR-induced survival response in a dose-dependent manner and significantly suppressed this survival signal in all UPN cases tested (from 34 ±6% to 54±7% apoptotic cells for untreated and dasatinib-treated BCR-activated cells, respectively; n=6; p<0.001) (Figure 6A). Similarly, PP2 treatment also reduced or abolished BCR-induced cell survival (Figure 6B). Overall, these results highlight the importance of LYN, JNK and EGR1 as intermediates of BCR signaling in mediating survival signals in MCL cells and point out to the efficiency of dasatinib in suppressing cell survival signal emanating from the BCR.

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