THE IMPACT OF THE TUMOUR MICROENVIRONMENT ON B-CELL RECEPTOR (BCR) EXPRESSION AND SIGNALLING IN CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL).
(Abstract release date: 05/19/16)
EHA Library. Dobson R. 06/09/16; 132572; E1023
Ms. Rachel Dobson
Contributions
Contributions
Abstract
Abstract: E1023
Type: Eposter Presentation
Background
Chronic lymphocytic leukaemia (CLL) is thought to be driven following antigen engagement of the B cell receptor (BCR). This has been highlighted further by the BCR Kinase inhibitors Ibrutinib and Idelalisib, which have proved instrumental in the treatment of CLL. However, these inhibitors appear to only suppress the disease without being curative. A number of patients have developed resistance to ibrutinib following mutation of the BTK or PLCγ2 gene, whilst other patients are unable to tolerate these drugs due to adverse events or progress whilst on therapy for unknown reasons. B-cell activating factor (BAFF) and interleukin-4 (IL-4) are known to increase CLL cell viability in vitro, and are thought to be present within CLL lymph nodes. Therefore we have investigated the role of BAFF and IL-4 in regulating BCR signalling in CLL cells and promoting resistance to ibrutinib and idelalisib.
Aims
To understand how IL-4 and BAFF regulate BCR pathway inhibition by ibrutinib and idelalisib.
Methods
Experiments were carried out using primary CLL cells. All isolates used contained >85% CD19+CD5+ cells. Protein expression was evaluated by flow cytometry or immunoblotting and signalling capacity was detected by calcium flux analysis in response to soluble αIgM by flow cytometry. Cell viability was assessed using annexin v/propidium iodide staining.
Results
As previously shown CLL cells treated with BAFF (500 ng/ml) or IL-4 (10 ng/ml) only for 48h significantly protected against basal apoptosis. This anti-apoptotic effect was further augmented when cells were treated with BAFF and IL-4 in combination. CLL cells treated in vitro with ibrutinib and idelalisib induced approximately 24.3% and 28.8% apoptosis respectively following treatment for 48h. However, apoptosis induced by the BCR-kinase inhibitors was reversed following pre-treatment with IL-4 or BAFF. CLL cells incubated in vitro with IL-4 for 24h significantly augmented sIgM expression and αIgM induced phosphorylated ERK and calcium flux. These IL-4 induced effects on sIgM expression were more prominent in Unmutated(U)-CLL samples, and could be reversed using the JAK3 inhibitor tofacitinib (CP-690550). Whilst, IL-4 had little effect on sIgD. Interestingly pre-treating CLL cells with IL-4 significantly reversed the ability of ibrutinib (1 µM) and idelalisib (1 µM) to inhibit αIgM-mediated signalling at 24h. In contrast BAFF treatment showed no clear effect on sIgM expression and downstream signalling. However, in a proportion of CLL samples treated simultaneously with BAFF and IL-4, BAFF reduced IL-4 specific increases in sIgM augmentation. Next we pre-treated CLL cells with tofacitinib (10 µM) for 1 hr prior to IL-4 treatment. Tofacitinib significantly reversed the effects observed with IL-4 and restored the ability of ibrutinib and idelalisib to inhibit αIgM induced signalling. This effect was specific to the inhibition of IL-4 since tofacitinib (≤10 µM) did not induce CLL cell death. Finally we showed that BAFF, and IL-4 to a greater extent, resulted in an increase in miRNA155 expression which has previously been shown to be associated with more progressive disease and enhanced BCR signalling.
Conclusion
Our data suggests that IL-4 augments sIgM recovery and down-stream signalling in CLL, which may compromise the effectiveness of BCR kinase inhibitors. However these effects were not observed for BAFF. Furthermore, co-treatment of BCR-kinase inhibitors in combination with tofacitinib may indicate a promising treatment strategy for the treatment of CLL.
Session topic: E-poster
Keyword(s): B cell chronic lymphocytic leukemia, Janus Kinase inhibitor, Microenvironment
Type: Eposter Presentation
Background
Chronic lymphocytic leukaemia (CLL) is thought to be driven following antigen engagement of the B cell receptor (BCR). This has been highlighted further by the BCR Kinase inhibitors Ibrutinib and Idelalisib, which have proved instrumental in the treatment of CLL. However, these inhibitors appear to only suppress the disease without being curative. A number of patients have developed resistance to ibrutinib following mutation of the BTK or PLCγ2 gene, whilst other patients are unable to tolerate these drugs due to adverse events or progress whilst on therapy for unknown reasons. B-cell activating factor (BAFF) and interleukin-4 (IL-4) are known to increase CLL cell viability in vitro, and are thought to be present within CLL lymph nodes. Therefore we have investigated the role of BAFF and IL-4 in regulating BCR signalling in CLL cells and promoting resistance to ibrutinib and idelalisib.
Aims
To understand how IL-4 and BAFF regulate BCR pathway inhibition by ibrutinib and idelalisib.
Methods
Experiments were carried out using primary CLL cells. All isolates used contained >85% CD19+CD5+ cells. Protein expression was evaluated by flow cytometry or immunoblotting and signalling capacity was detected by calcium flux analysis in response to soluble αIgM by flow cytometry. Cell viability was assessed using annexin v/propidium iodide staining.
Results
As previously shown CLL cells treated with BAFF (500 ng/ml) or IL-4 (10 ng/ml) only for 48h significantly protected against basal apoptosis. This anti-apoptotic effect was further augmented when cells were treated with BAFF and IL-4 in combination. CLL cells treated in vitro with ibrutinib and idelalisib induced approximately 24.3% and 28.8% apoptosis respectively following treatment for 48h. However, apoptosis induced by the BCR-kinase inhibitors was reversed following pre-treatment with IL-4 or BAFF. CLL cells incubated in vitro with IL-4 for 24h significantly augmented sIgM expression and αIgM induced phosphorylated ERK and calcium flux. These IL-4 induced effects on sIgM expression were more prominent in Unmutated(U)-CLL samples, and could be reversed using the JAK3 inhibitor tofacitinib (CP-690550). Whilst, IL-4 had little effect on sIgD. Interestingly pre-treating CLL cells with IL-4 significantly reversed the ability of ibrutinib (1 µM) and idelalisib (1 µM) to inhibit αIgM-mediated signalling at 24h. In contrast BAFF treatment showed no clear effect on sIgM expression and downstream signalling. However, in a proportion of CLL samples treated simultaneously with BAFF and IL-4, BAFF reduced IL-4 specific increases in sIgM augmentation. Next we pre-treated CLL cells with tofacitinib (10 µM) for 1 hr prior to IL-4 treatment. Tofacitinib significantly reversed the effects observed with IL-4 and restored the ability of ibrutinib and idelalisib to inhibit αIgM induced signalling. This effect was specific to the inhibition of IL-4 since tofacitinib (≤10 µM) did not induce CLL cell death. Finally we showed that BAFF, and IL-4 to a greater extent, resulted in an increase in miRNA155 expression which has previously been shown to be associated with more progressive disease and enhanced BCR signalling.
Conclusion
Our data suggests that IL-4 augments sIgM recovery and down-stream signalling in CLL, which may compromise the effectiveness of BCR kinase inhibitors. However these effects were not observed for BAFF. Furthermore, co-treatment of BCR-kinase inhibitors in combination with tofacitinib may indicate a promising treatment strategy for the treatment of CLL.
Session topic: E-poster
Keyword(s): B cell chronic lymphocytic leukemia, Janus Kinase inhibitor, Microenvironment
Abstract: E1023
Type: Eposter Presentation
Background
Chronic lymphocytic leukaemia (CLL) is thought to be driven following antigen engagement of the B cell receptor (BCR). This has been highlighted further by the BCR Kinase inhibitors Ibrutinib and Idelalisib, which have proved instrumental in the treatment of CLL. However, these inhibitors appear to only suppress the disease without being curative. A number of patients have developed resistance to ibrutinib following mutation of the BTK or PLCγ2 gene, whilst other patients are unable to tolerate these drugs due to adverse events or progress whilst on therapy for unknown reasons. B-cell activating factor (BAFF) and interleukin-4 (IL-4) are known to increase CLL cell viability in vitro, and are thought to be present within CLL lymph nodes. Therefore we have investigated the role of BAFF and IL-4 in regulating BCR signalling in CLL cells and promoting resistance to ibrutinib and idelalisib.
Aims
To understand how IL-4 and BAFF regulate BCR pathway inhibition by ibrutinib and idelalisib.
Methods
Experiments were carried out using primary CLL cells. All isolates used contained >85% CD19+CD5+ cells. Protein expression was evaluated by flow cytometry or immunoblotting and signalling capacity was detected by calcium flux analysis in response to soluble αIgM by flow cytometry. Cell viability was assessed using annexin v/propidium iodide staining.
Results
As previously shown CLL cells treated with BAFF (500 ng/ml) or IL-4 (10 ng/ml) only for 48h significantly protected against basal apoptosis. This anti-apoptotic effect was further augmented when cells were treated with BAFF and IL-4 in combination. CLL cells treated in vitro with ibrutinib and idelalisib induced approximately 24.3% and 28.8% apoptosis respectively following treatment for 48h. However, apoptosis induced by the BCR-kinase inhibitors was reversed following pre-treatment with IL-4 or BAFF. CLL cells incubated in vitro with IL-4 for 24h significantly augmented sIgM expression and αIgM induced phosphorylated ERK and calcium flux. These IL-4 induced effects on sIgM expression were more prominent in Unmutated(U)-CLL samples, and could be reversed using the JAK3 inhibitor tofacitinib (CP-690550). Whilst, IL-4 had little effect on sIgD. Interestingly pre-treating CLL cells with IL-4 significantly reversed the ability of ibrutinib (1 µM) and idelalisib (1 µM) to inhibit αIgM-mediated signalling at 24h. In contrast BAFF treatment showed no clear effect on sIgM expression and downstream signalling. However, in a proportion of CLL samples treated simultaneously with BAFF and IL-4, BAFF reduced IL-4 specific increases in sIgM augmentation. Next we pre-treated CLL cells with tofacitinib (10 µM) for 1 hr prior to IL-4 treatment. Tofacitinib significantly reversed the effects observed with IL-4 and restored the ability of ibrutinib and idelalisib to inhibit αIgM induced signalling. This effect was specific to the inhibition of IL-4 since tofacitinib (≤10 µM) did not induce CLL cell death. Finally we showed that BAFF, and IL-4 to a greater extent, resulted in an increase in miRNA155 expression which has previously been shown to be associated with more progressive disease and enhanced BCR signalling.
Conclusion
Our data suggests that IL-4 augments sIgM recovery and down-stream signalling in CLL, which may compromise the effectiveness of BCR kinase inhibitors. However these effects were not observed for BAFF. Furthermore, co-treatment of BCR-kinase inhibitors in combination with tofacitinib may indicate a promising treatment strategy for the treatment of CLL.
Session topic: E-poster
Keyword(s): B cell chronic lymphocytic leukemia, Janus Kinase inhibitor, Microenvironment
Type: Eposter Presentation
Background
Chronic lymphocytic leukaemia (CLL) is thought to be driven following antigen engagement of the B cell receptor (BCR). This has been highlighted further by the BCR Kinase inhibitors Ibrutinib and Idelalisib, which have proved instrumental in the treatment of CLL. However, these inhibitors appear to only suppress the disease without being curative. A number of patients have developed resistance to ibrutinib following mutation of the BTK or PLCγ2 gene, whilst other patients are unable to tolerate these drugs due to adverse events or progress whilst on therapy for unknown reasons. B-cell activating factor (BAFF) and interleukin-4 (IL-4) are known to increase CLL cell viability in vitro, and are thought to be present within CLL lymph nodes. Therefore we have investigated the role of BAFF and IL-4 in regulating BCR signalling in CLL cells and promoting resistance to ibrutinib and idelalisib.
Aims
To understand how IL-4 and BAFF regulate BCR pathway inhibition by ibrutinib and idelalisib.
Methods
Experiments were carried out using primary CLL cells. All isolates used contained >85% CD19+CD5+ cells. Protein expression was evaluated by flow cytometry or immunoblotting and signalling capacity was detected by calcium flux analysis in response to soluble αIgM by flow cytometry. Cell viability was assessed using annexin v/propidium iodide staining.
Results
As previously shown CLL cells treated with BAFF (500 ng/ml) or IL-4 (10 ng/ml) only for 48h significantly protected against basal apoptosis. This anti-apoptotic effect was further augmented when cells were treated with BAFF and IL-4 in combination. CLL cells treated in vitro with ibrutinib and idelalisib induced approximately 24.3% and 28.8% apoptosis respectively following treatment for 48h. However, apoptosis induced by the BCR-kinase inhibitors was reversed following pre-treatment with IL-4 or BAFF. CLL cells incubated in vitro with IL-4 for 24h significantly augmented sIgM expression and αIgM induced phosphorylated ERK and calcium flux. These IL-4 induced effects on sIgM expression were more prominent in Unmutated(U)-CLL samples, and could be reversed using the JAK3 inhibitor tofacitinib (CP-690550). Whilst, IL-4 had little effect on sIgD. Interestingly pre-treating CLL cells with IL-4 significantly reversed the ability of ibrutinib (1 µM) and idelalisib (1 µM) to inhibit αIgM-mediated signalling at 24h. In contrast BAFF treatment showed no clear effect on sIgM expression and downstream signalling. However, in a proportion of CLL samples treated simultaneously with BAFF and IL-4, BAFF reduced IL-4 specific increases in sIgM augmentation. Next we pre-treated CLL cells with tofacitinib (10 µM) for 1 hr prior to IL-4 treatment. Tofacitinib significantly reversed the effects observed with IL-4 and restored the ability of ibrutinib and idelalisib to inhibit αIgM induced signalling. This effect was specific to the inhibition of IL-4 since tofacitinib (≤10 µM) did not induce CLL cell death. Finally we showed that BAFF, and IL-4 to a greater extent, resulted in an increase in miRNA155 expression which has previously been shown to be associated with more progressive disease and enhanced BCR signalling.
Conclusion
Our data suggests that IL-4 augments sIgM recovery and down-stream signalling in CLL, which may compromise the effectiveness of BCR kinase inhibitors. However these effects were not observed for BAFF. Furthermore, co-treatment of BCR-kinase inhibitors in combination with tofacitinib may indicate a promising treatment strategy for the treatment of CLL.
Session topic: E-poster
Keyword(s): B cell chronic lymphocytic leukemia, Janus Kinase inhibitor, Microenvironment
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