Contributions
Abstract: S870
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 17:00 - 17:15
Location: Room K1
Background
The addition of the anti-CD20 monoclonal antibody (mAb) rituximab to chemotherapy improves responses in almost all chronic lymphocytic leukaemia (CLL) patients, with the exception of those having NOTCH1 mutations. NOTCH1 is a cell surface receptor releasing its intracellular domain (NICD1) after two ligand‑induced cleavage steps performed by metalloproteases and γ-secretase. NICD1 acts as transcription factor and NOTCH1 mutations in CLL lead to longer lasting transcription factor activity.
Aims
To understand the relationship between rituximab and NOTCH1, we studied the effects rituximab treatment has on NOTCH1 signalling.
Methods
Freshly isolated peripheral blood mononuclear cells (PBMCs) from CLL patients attending St. Bartholomew’s Hospital, London were enriched for CD19+ cells and treated with rituximab. Whole protein lysates were obtained after 15, 30 and 60 min of mAb treatment; RNA was isolated after 150 min. NICD1 was semi-quantitatively assessed by western blot. Expression of HES1, the best established NICD1 target gene, and CCL2 was quantified by TaqMan-probe-based quantitative PCR. Rituximab F(ab’)2 fragments and trastuzumab were used as controls.
Results
Peripheral blood CLL cells showed low NOTCH1 signalling activity. Following in-vitro treatment with rituximab, we observed NOTCH1 receptor activation with NICD1 release in a time-dependent manner. In line with this, HES1 gene expression was up-regulated after rituximab treatment. The extent to which HES1 expression was up‑regulated varied in between individual samples. Samples had variable amounts of residual non-tumour PBMCs and the sample with the highest up‑regulation of HES1 expression had the highest amount of residual non-tumour cells. We therefore reasoned that activation of immune effector cells via the Fc‑fragment of rituximab might be involved in activating NOTCH1. Since relative effector cell numbers were low (≤4%), physical contact between CLL cells and activated ligand-expressing effector cells was unlikely to serve as only explanation. Effector cells such as monocytes secrete matrix metalloproteases (MMPs) upon activation. Therefore, we hypothesised that secreted metalloproteases might contribute to cleavage of the NOTCH1 receptor. Particularly MMP9 and MMP8 were found in the supernatant of healthy donor PBMCs treated with rituximab. Using this supernatant, NOTCH1 activation could be evoked in SU‑DHL4 cells, which did not show NOTCH1 activation when adding rituximab to their standard culture medium. Also, the supernatant of trastuzumab treated PBMCs led to NOTCH1 activation in SU-DHL4 cells, whereas using rituximab F(ab’)2 fragments had no impact. MMP9 and MMP8 are mainly secreted by monocytes. In line with this, up-regulation of HES1 expression after rituximab treatment correlated well with up-regulation of CCL2 expression. CCL2 is mainly produced by monocytes and secreted upon their activation.
Conclusion
Our data shows that rituximab treatment increases NOTCH1 signalling. In B-cells, only cell surface bound ADAM10 and ADAM17 were so far associated with first cleavage of NOTCH1. Here we show that proteins secreted by activated immune effector cells contribute to activate NOTCH1. MMP8 and MMP9 are the most likely candidates and active recombinant proteins will be used to prove their involvement. Activation of NOTCH1 in a pro-inflammatory environment occurs independent of the NOTCH1 mutation status, but abnormally long activity of mutant NICD1 probably has a different/stronger impact on the CLL transcriptome than wild-type NICD1.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Microenvironment, Notch, Rituximab
Abstract: S870
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 17:00 - 17:15
Location: Room K1
Background
The addition of the anti-CD20 monoclonal antibody (mAb) rituximab to chemotherapy improves responses in almost all chronic lymphocytic leukaemia (CLL) patients, with the exception of those having NOTCH1 mutations. NOTCH1 is a cell surface receptor releasing its intracellular domain (NICD1) after two ligand‑induced cleavage steps performed by metalloproteases and γ-secretase. NICD1 acts as transcription factor and NOTCH1 mutations in CLL lead to longer lasting transcription factor activity.
Aims
To understand the relationship between rituximab and NOTCH1, we studied the effects rituximab treatment has on NOTCH1 signalling.
Methods
Freshly isolated peripheral blood mononuclear cells (PBMCs) from CLL patients attending St. Bartholomew’s Hospital, London were enriched for CD19+ cells and treated with rituximab. Whole protein lysates were obtained after 15, 30 and 60 min of mAb treatment; RNA was isolated after 150 min. NICD1 was semi-quantitatively assessed by western blot. Expression of HES1, the best established NICD1 target gene, and CCL2 was quantified by TaqMan-probe-based quantitative PCR. Rituximab F(ab’)2 fragments and trastuzumab were used as controls.
Results
Peripheral blood CLL cells showed low NOTCH1 signalling activity. Following in-vitro treatment with rituximab, we observed NOTCH1 receptor activation with NICD1 release in a time-dependent manner. In line with this, HES1 gene expression was up-regulated after rituximab treatment. The extent to which HES1 expression was up‑regulated varied in between individual samples. Samples had variable amounts of residual non-tumour PBMCs and the sample with the highest up‑regulation of HES1 expression had the highest amount of residual non-tumour cells. We therefore reasoned that activation of immune effector cells via the Fc‑fragment of rituximab might be involved in activating NOTCH1. Since relative effector cell numbers were low (≤4%), physical contact between CLL cells and activated ligand-expressing effector cells was unlikely to serve as only explanation. Effector cells such as monocytes secrete matrix metalloproteases (MMPs) upon activation. Therefore, we hypothesised that secreted metalloproteases might contribute to cleavage of the NOTCH1 receptor. Particularly MMP9 and MMP8 were found in the supernatant of healthy donor PBMCs treated with rituximab. Using this supernatant, NOTCH1 activation could be evoked in SU‑DHL4 cells, which did not show NOTCH1 activation when adding rituximab to their standard culture medium. Also, the supernatant of trastuzumab treated PBMCs led to NOTCH1 activation in SU-DHL4 cells, whereas using rituximab F(ab’)2 fragments had no impact. MMP9 and MMP8 are mainly secreted by monocytes. In line with this, up-regulation of HES1 expression after rituximab treatment correlated well with up-regulation of CCL2 expression. CCL2 is mainly produced by monocytes and secreted upon their activation.
Conclusion
Our data shows that rituximab treatment increases NOTCH1 signalling. In B-cells, only cell surface bound ADAM10 and ADAM17 were so far associated with first cleavage of NOTCH1. Here we show that proteins secreted by activated immune effector cells contribute to activate NOTCH1. MMP8 and MMP9 are the most likely candidates and active recombinant proteins will be used to prove their involvement. Activation of NOTCH1 in a pro-inflammatory environment occurs independent of the NOTCH1 mutation status, but abnormally long activity of mutant NICD1 probably has a different/stronger impact on the CLL transcriptome than wild-type NICD1.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Microenvironment, Notch, Rituximab