APPLYING PROBED CAPILLARY ISOELECTRIC FOCUSING (PROBED CIEF) FOR RESPONSE MONITORING OF SIGNAL INHIBITOR THERAPY DIRECTED AGAINST BRUTONS TYROSINE KINASE (BTK) IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
(Abstract release date: 05/19/16)
EHA Library. Burthem J. 06/09/16; 134701; PB1801
Dr. John Burthem
Contributions
Contributions
Abstract
Abstract: PB1801
Type: Publication Only
Background
Signals mediated through the B cell receptor (BCR) of chronic lymphocytic leukemia (CLL) are central to disease pathobiology. Bruton’s Tyrosine Kinase (BTK) is a key regulator of BCR signalling. Small molecule inhibitors of BTK (BTKi) have therefore emerged as effective therapeutic agents. BTKi treatment is particularly successful in CLL; however responses may not be complete or durable. It is therefore important that we have tools to monitor disease response and direct therapy. Probed capillary isoelectric focussing (probed cIEF) uses fine capillaries that contain an ampholyte pH-gradient to separate protein within cell-lysates according to their charge. Many post-translational modifications induce significant changes to charge, and modified proteins are readily separated. Antibodies that recognise specific signal-molecules are then used to probe the samples and to generate profiles that demonstrate the presence and relative abundance of different charge-forms of the target molecule. These charge-forms reflect structural alteration, lipid modification or different phosphorylation states, modifications that reflect differential activation of the signalling molecule. The technique offers the capability to detect and evaluate the full range of changes to BTK, and therefore can be used to support clinical monitoring of drug effectiveness.
Aims
To evaluate the use of probed cIEF in signal response monitoring for patients treated with BTK inhibitors.
Methods
CLL cells were cultured in-vitro in the presence or absence of BCR activation (anti-IgM cross-linkage) with and without BTKi (ibrutinib). Cells lysates were separated according to charge using an ampholyte pH-gradient (nested 5-8). To assess BTK response, separated protein was probed with total BTK antibody to generate profiles of the presence and relative abundance (area under curve for peak volumes (Compass software)) of different charge-forms of the molecule. Ex-vivo cells of CLL pre and post in-vivo BTK signal inhibitor therapy were analysed to compare with in-vitro profiles.
Results
Phosphoprotein traces were reliably obtained yielding reproducible dose-response profiles. A significant complexity of charge-forms was identified for BTK, with a range of peaks differing substantially between resting and activated cell states. Ibrutinib greatly changed the profile of BTK, both through new peak formation and peak shifts that were not attributable to recognised effects of the drug against standard regulatory motifs. The peak changes we observe are therefore complex, they combine the expected prevention of BTK activation, but also new and unexpected changes not simply a return to the “resting state profile” of the molecule. We suggest these form a molecular signature representing a biomarker of drug response. Studies of inhibitor action in clinically treated and clinically responsive CLL cells mirrored that of our profile of BTKi treated cells in-vitro indicating our profile to represent biochemical modification of BTK signalling after clinically relevant therapy.
Conclusion
Probed cIEF offers the capability to monitor complex signal-interactions, and to identify important signalling changes in a simple, objective and reproducible manner. Future work will aim to precisely identify the biochemical changes that underlie the specific peaks of the BTK profile that may represent clinically useful biomarkers of treatment response.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Proteomics, Signaling
Type: Publication Only
Background
Signals mediated through the B cell receptor (BCR) of chronic lymphocytic leukemia (CLL) are central to disease pathobiology. Bruton’s Tyrosine Kinase (BTK) is a key regulator of BCR signalling. Small molecule inhibitors of BTK (BTKi) have therefore emerged as effective therapeutic agents. BTKi treatment is particularly successful in CLL; however responses may not be complete or durable. It is therefore important that we have tools to monitor disease response and direct therapy. Probed capillary isoelectric focussing (probed cIEF) uses fine capillaries that contain an ampholyte pH-gradient to separate protein within cell-lysates according to their charge. Many post-translational modifications induce significant changes to charge, and modified proteins are readily separated. Antibodies that recognise specific signal-molecules are then used to probe the samples and to generate profiles that demonstrate the presence and relative abundance of different charge-forms of the target molecule. These charge-forms reflect structural alteration, lipid modification or different phosphorylation states, modifications that reflect differential activation of the signalling molecule. The technique offers the capability to detect and evaluate the full range of changes to BTK, and therefore can be used to support clinical monitoring of drug effectiveness.
Aims
To evaluate the use of probed cIEF in signal response monitoring for patients treated with BTK inhibitors.
Methods
CLL cells were cultured in-vitro in the presence or absence of BCR activation (anti-IgM cross-linkage) with and without BTKi (ibrutinib). Cells lysates were separated according to charge using an ampholyte pH-gradient (nested 5-8). To assess BTK response, separated protein was probed with total BTK antibody to generate profiles of the presence and relative abundance (area under curve for peak volumes (Compass software)) of different charge-forms of the molecule. Ex-vivo cells of CLL pre and post in-vivo BTK signal inhibitor therapy were analysed to compare with in-vitro profiles.
Results
Phosphoprotein traces were reliably obtained yielding reproducible dose-response profiles. A significant complexity of charge-forms was identified for BTK, with a range of peaks differing substantially between resting and activated cell states. Ibrutinib greatly changed the profile of BTK, both through new peak formation and peak shifts that were not attributable to recognised effects of the drug against standard regulatory motifs. The peak changes we observe are therefore complex, they combine the expected prevention of BTK activation, but also new and unexpected changes not simply a return to the “resting state profile” of the molecule. We suggest these form a molecular signature representing a biomarker of drug response. Studies of inhibitor action in clinically treated and clinically responsive CLL cells mirrored that of our profile of BTKi treated cells in-vitro indicating our profile to represent biochemical modification of BTK signalling after clinically relevant therapy.
Conclusion
Probed cIEF offers the capability to monitor complex signal-interactions, and to identify important signalling changes in a simple, objective and reproducible manner. Future work will aim to precisely identify the biochemical changes that underlie the specific peaks of the BTK profile that may represent clinically useful biomarkers of treatment response.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Proteomics, Signaling
Abstract: PB1801
Type: Publication Only
Background
Signals mediated through the B cell receptor (BCR) of chronic lymphocytic leukemia (CLL) are central to disease pathobiology. Bruton’s Tyrosine Kinase (BTK) is a key regulator of BCR signalling. Small molecule inhibitors of BTK (BTKi) have therefore emerged as effective therapeutic agents. BTKi treatment is particularly successful in CLL; however responses may not be complete or durable. It is therefore important that we have tools to monitor disease response and direct therapy. Probed capillary isoelectric focussing (probed cIEF) uses fine capillaries that contain an ampholyte pH-gradient to separate protein within cell-lysates according to their charge. Many post-translational modifications induce significant changes to charge, and modified proteins are readily separated. Antibodies that recognise specific signal-molecules are then used to probe the samples and to generate profiles that demonstrate the presence and relative abundance of different charge-forms of the target molecule. These charge-forms reflect structural alteration, lipid modification or different phosphorylation states, modifications that reflect differential activation of the signalling molecule. The technique offers the capability to detect and evaluate the full range of changes to BTK, and therefore can be used to support clinical monitoring of drug effectiveness.
Aims
To evaluate the use of probed cIEF in signal response monitoring for patients treated with BTK inhibitors.
Methods
CLL cells were cultured in-vitro in the presence or absence of BCR activation (anti-IgM cross-linkage) with and without BTKi (ibrutinib). Cells lysates were separated according to charge using an ampholyte pH-gradient (nested 5-8). To assess BTK response, separated protein was probed with total BTK antibody to generate profiles of the presence and relative abundance (area under curve for peak volumes (Compass software)) of different charge-forms of the molecule. Ex-vivo cells of CLL pre and post in-vivo BTK signal inhibitor therapy were analysed to compare with in-vitro profiles.
Results
Phosphoprotein traces were reliably obtained yielding reproducible dose-response profiles. A significant complexity of charge-forms was identified for BTK, with a range of peaks differing substantially between resting and activated cell states. Ibrutinib greatly changed the profile of BTK, both through new peak formation and peak shifts that were not attributable to recognised effects of the drug against standard regulatory motifs. The peak changes we observe are therefore complex, they combine the expected prevention of BTK activation, but also new and unexpected changes not simply a return to the “resting state profile” of the molecule. We suggest these form a molecular signature representing a biomarker of drug response. Studies of inhibitor action in clinically treated and clinically responsive CLL cells mirrored that of our profile of BTKi treated cells in-vitro indicating our profile to represent biochemical modification of BTK signalling after clinically relevant therapy.
Conclusion
Probed cIEF offers the capability to monitor complex signal-interactions, and to identify important signalling changes in a simple, objective and reproducible manner. Future work will aim to precisely identify the biochemical changes that underlie the specific peaks of the BTK profile that may represent clinically useful biomarkers of treatment response.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Proteomics, Signaling
Type: Publication Only
Background
Signals mediated through the B cell receptor (BCR) of chronic lymphocytic leukemia (CLL) are central to disease pathobiology. Bruton’s Tyrosine Kinase (BTK) is a key regulator of BCR signalling. Small molecule inhibitors of BTK (BTKi) have therefore emerged as effective therapeutic agents. BTKi treatment is particularly successful in CLL; however responses may not be complete or durable. It is therefore important that we have tools to monitor disease response and direct therapy. Probed capillary isoelectric focussing (probed cIEF) uses fine capillaries that contain an ampholyte pH-gradient to separate protein within cell-lysates according to their charge. Many post-translational modifications induce significant changes to charge, and modified proteins are readily separated. Antibodies that recognise specific signal-molecules are then used to probe the samples and to generate profiles that demonstrate the presence and relative abundance of different charge-forms of the target molecule. These charge-forms reflect structural alteration, lipid modification or different phosphorylation states, modifications that reflect differential activation of the signalling molecule. The technique offers the capability to detect and evaluate the full range of changes to BTK, and therefore can be used to support clinical monitoring of drug effectiveness.
Aims
To evaluate the use of probed cIEF in signal response monitoring for patients treated with BTK inhibitors.
Methods
CLL cells were cultured in-vitro in the presence or absence of BCR activation (anti-IgM cross-linkage) with and without BTKi (ibrutinib). Cells lysates were separated according to charge using an ampholyte pH-gradient (nested 5-8). To assess BTK response, separated protein was probed with total BTK antibody to generate profiles of the presence and relative abundance (area under curve for peak volumes (Compass software)) of different charge-forms of the molecule. Ex-vivo cells of CLL pre and post in-vivo BTK signal inhibitor therapy were analysed to compare with in-vitro profiles.
Results
Phosphoprotein traces were reliably obtained yielding reproducible dose-response profiles. A significant complexity of charge-forms was identified for BTK, with a range of peaks differing substantially between resting and activated cell states. Ibrutinib greatly changed the profile of BTK, both through new peak formation and peak shifts that were not attributable to recognised effects of the drug against standard regulatory motifs. The peak changes we observe are therefore complex, they combine the expected prevention of BTK activation, but also new and unexpected changes not simply a return to the “resting state profile” of the molecule. We suggest these form a molecular signature representing a biomarker of drug response. Studies of inhibitor action in clinically treated and clinically responsive CLL cells mirrored that of our profile of BTKi treated cells in-vitro indicating our profile to represent biochemical modification of BTK signalling after clinically relevant therapy.
Conclusion
Probed cIEF offers the capability to monitor complex signal-interactions, and to identify important signalling changes in a simple, objective and reproducible manner. Future work will aim to precisely identify the biochemical changes that underlie the specific peaks of the BTK profile that may represent clinically useful biomarkers of treatment response.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Proteomics, Signaling
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