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Biological rational for sequential targeting of Bruton tyrosine kinase and Bcl-2 to overcome CD40-induced ABT-199 resistance in mantle cell lymphoma.

Chiron D, Dousset C, Brosseau C, Touzeau C, Maïga S, Moreau P, Pellat-Deceunynck C, Le Gouill S, Amiot M - Oncotarget (2015)

Bottom Line: We show here for two patients undergoing ibrutinib therapy that mobilized MCL cells are highly sensitive to ABT-199.These results provide evidence that in situ ABT-199 resistance can be overcome when MCL cells escape from the lymph nodes.Altogether, our data support the clinical application of ABT-199 therapy both as a single agent and in sequential combination with BTK inhibitors.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR892 - CNRS, UMR 6299, Université de Nantes, France.

ABSTRACT
The aggressive biological behavior of mantle cell lymphoma (MCL) and its short response to current treatment highlight a great need for better rational therapy. Herein, we investigate the ability of ABT-199, the Bcl-2-selective BH3 mimetic, to kill MCL cells. Among MCL cell lines tested (n = 8), only three were sensitive (LD50 < 200 nM). In contrast, all primary MCL samples tested (n = 11) were highly sensitive to ABT-199 (LD50 < 10 nM). Mcl-1 and Bcl-xL both confer resistance to ABT-199-specific killing and BCL2/(BCLXL+MCL1) mRNA ratio is a strong predictor of sensitivity. By mimicking the microenvironment through CD40 stimulation, we show that ABT-199 sensitivity is impaired through activation of NF-kB pathway and Bcl-x(L) up-regulation. We further demonstrate that resistance is rapidly lost when MCL cells detach from CD40L-expressing fibroblasts. It has been reported that ibrutinib induces lymphocytosis in vivo holding off malignant cells from their protective microenvironment. We show here for two patients undergoing ibrutinib therapy that mobilized MCL cells are highly sensitive to ABT-199. These results provide evidence that in situ ABT-199 resistance can be overcome when MCL cells escape from the lymph nodes. Altogether, our data support the clinical application of ABT-199 therapy both as a single agent and in sequential combination with BTK inhibitors.

No MeSH data available.


Related in: MedlinePlus

Influence of Bcl-2 family anti-apoptotic proteins on ABT-199 sensitivity in MCL cells(A) Analysis of the relative expression of BCLXL, MCL1 and BCL2 mRNA by RT-qPCR in MCL cell lines (n = 8) and primary MCL cells (n = 8). The relative expression was normalized to JeKo-1 cell line. (B) The BCL2/(MCL1+BCLXL) mRNA ratio correlates with ABT-199 sensitivity in MCL cells. Cells with a LD50 < 200 nM were defined as sensitive whereas cells with a LD50 > 1000 nM were defined as resistant. The cut-off value (0.67) was determined as the mean of BCL2/(MCL1+BCLXL) ratio of resistant cells + (standard deviation) x 2 (True positive rate: 100%) (C) Both Mcl-1 and Bcl-xL confer primary resistance to ABT-199. Z138 and JeKo-1 cell lines were transfected with Si Control (Ct), Mcl-1 or Bcl-xL. Following transfection, cells were treated with ABT-199 for 24 hours and cell death was quantified by Apo2.7 staining. p-value was determined using the paired Student's t test: *p < .05; **p < .01. (D) The protein levels of Mcl-1 and Bcl-xL were determined by immunoblotting.
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Figure 1: Influence of Bcl-2 family anti-apoptotic proteins on ABT-199 sensitivity in MCL cells(A) Analysis of the relative expression of BCLXL, MCL1 and BCL2 mRNA by RT-qPCR in MCL cell lines (n = 8) and primary MCL cells (n = 8). The relative expression was normalized to JeKo-1 cell line. (B) The BCL2/(MCL1+BCLXL) mRNA ratio correlates with ABT-199 sensitivity in MCL cells. Cells with a LD50 < 200 nM were defined as sensitive whereas cells with a LD50 > 1000 nM were defined as resistant. The cut-off value (0.67) was determined as the mean of BCL2/(MCL1+BCLXL) ratio of resistant cells + (standard deviation) x 2 (True positive rate: 100%) (C) Both Mcl-1 and Bcl-xL confer primary resistance to ABT-199. Z138 and JeKo-1 cell lines were transfected with Si Control (Ct), Mcl-1 or Bcl-xL. Following transfection, cells were treated with ABT-199 for 24 hours and cell death was quantified by Apo2.7 staining. p-value was determined using the paired Student's t test: *p < .05; **p < .01. (D) The protein levels of Mcl-1 and Bcl-xL were determined by immunoblotting.

Mentions: (A) Cell lines were cultured with increasing doses of ABT-199 for 48 hours to determine the median lethal dose (LD50: 15-10000 nM). (B) MCL cells from peripheral blood were obtained after gradient density centrifugation on Ficoll Hypaque. MCL cells were cultured with 10 nM of ABT-199 for 24 hours. Diag: diagnosis, Rel: relapse, ND: data not determined. The relative expression of BCL2, MCL1 and BCLXL mRNA was defined on purified CD19+ cells as described in the Methods section and BCL2/(MCL1+BCLXL) mRNA ratio is indicated. Analysis of BCLXL, MCL1 and BCL2 relative expression in primary MCL cells and cell lines are shown in Figure 1A. Correlation between BCL2/(MCL1+BCLXL) ratio and ABT-199 sensitivity is shown in Figure 1B.


Biological rational for sequential targeting of Bruton tyrosine kinase and Bcl-2 to overcome CD40-induced ABT-199 resistance in mantle cell lymphoma.

Chiron D, Dousset C, Brosseau C, Touzeau C, Maïga S, Moreau P, Pellat-Deceunynck C, Le Gouill S, Amiot M - Oncotarget (2015)

Influence of Bcl-2 family anti-apoptotic proteins on ABT-199 sensitivity in MCL cells(A) Analysis of the relative expression of BCLXL, MCL1 and BCL2 mRNA by RT-qPCR in MCL cell lines (n = 8) and primary MCL cells (n = 8). The relative expression was normalized to JeKo-1 cell line. (B) The BCL2/(MCL1+BCLXL) mRNA ratio correlates with ABT-199 sensitivity in MCL cells. Cells with a LD50 < 200 nM were defined as sensitive whereas cells with a LD50 > 1000 nM were defined as resistant. The cut-off value (0.67) was determined as the mean of BCL2/(MCL1+BCLXL) ratio of resistant cells + (standard deviation) x 2 (True positive rate: 100%) (C) Both Mcl-1 and Bcl-xL confer primary resistance to ABT-199. Z138 and JeKo-1 cell lines were transfected with Si Control (Ct), Mcl-1 or Bcl-xL. Following transfection, cells were treated with ABT-199 for 24 hours and cell death was quantified by Apo2.7 staining. p-value was determined using the paired Student's t test: *p < .05; **p < .01. (D) The protein levels of Mcl-1 and Bcl-xL were determined by immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496181&req=5

Figure 1: Influence of Bcl-2 family anti-apoptotic proteins on ABT-199 sensitivity in MCL cells(A) Analysis of the relative expression of BCLXL, MCL1 and BCL2 mRNA by RT-qPCR in MCL cell lines (n = 8) and primary MCL cells (n = 8). The relative expression was normalized to JeKo-1 cell line. (B) The BCL2/(MCL1+BCLXL) mRNA ratio correlates with ABT-199 sensitivity in MCL cells. Cells with a LD50 < 200 nM were defined as sensitive whereas cells with a LD50 > 1000 nM were defined as resistant. The cut-off value (0.67) was determined as the mean of BCL2/(MCL1+BCLXL) ratio of resistant cells + (standard deviation) x 2 (True positive rate: 100%) (C) Both Mcl-1 and Bcl-xL confer primary resistance to ABT-199. Z138 and JeKo-1 cell lines were transfected with Si Control (Ct), Mcl-1 or Bcl-xL. Following transfection, cells were treated with ABT-199 for 24 hours and cell death was quantified by Apo2.7 staining. p-value was determined using the paired Student's t test: *p < .05; **p < .01. (D) The protein levels of Mcl-1 and Bcl-xL were determined by immunoblotting.
Mentions: (A) Cell lines were cultured with increasing doses of ABT-199 for 48 hours to determine the median lethal dose (LD50: 15-10000 nM). (B) MCL cells from peripheral blood were obtained after gradient density centrifugation on Ficoll Hypaque. MCL cells were cultured with 10 nM of ABT-199 for 24 hours. Diag: diagnosis, Rel: relapse, ND: data not determined. The relative expression of BCL2, MCL1 and BCLXL mRNA was defined on purified CD19+ cells as described in the Methods section and BCL2/(MCL1+BCLXL) mRNA ratio is indicated. Analysis of BCLXL, MCL1 and BCL2 relative expression in primary MCL cells and cell lines are shown in Figure 1A. Correlation between BCL2/(MCL1+BCLXL) ratio and ABT-199 sensitivity is shown in Figure 1B.

Bottom Line: We show here for two patients undergoing ibrutinib therapy that mobilized MCL cells are highly sensitive to ABT-199.These results provide evidence that in situ ABT-199 resistance can be overcome when MCL cells escape from the lymph nodes.Altogether, our data support the clinical application of ABT-199 therapy both as a single agent and in sequential combination with BTK inhibitors.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR892 - CNRS, UMR 6299, Université de Nantes, France.

ABSTRACT
The aggressive biological behavior of mantle cell lymphoma (MCL) and its short response to current treatment highlight a great need for better rational therapy. Herein, we investigate the ability of ABT-199, the Bcl-2-selective BH3 mimetic, to kill MCL cells. Among MCL cell lines tested (n = 8), only three were sensitive (LD50 < 200 nM). In contrast, all primary MCL samples tested (n = 11) were highly sensitive to ABT-199 (LD50 < 10 nM). Mcl-1 and Bcl-xL both confer resistance to ABT-199-specific killing and BCL2/(BCLXL+MCL1) mRNA ratio is a strong predictor of sensitivity. By mimicking the microenvironment through CD40 stimulation, we show that ABT-199 sensitivity is impaired through activation of NF-kB pathway and Bcl-x(L) up-regulation. We further demonstrate that resistance is rapidly lost when MCL cells detach from CD40L-expressing fibroblasts. It has been reported that ibrutinib induces lymphocytosis in vivo holding off malignant cells from their protective microenvironment. We show here for two patients undergoing ibrutinib therapy that mobilized MCL cells are highly sensitive to ABT-199. These results provide evidence that in situ ABT-199 resistance can be overcome when MCL cells escape from the lymph nodes. Altogether, our data support the clinical application of ABT-199 therapy both as a single agent and in sequential combination with BTK inhibitors.

No MeSH data available.


Related in: MedlinePlus