INFLUENCE OF HISTONE DEACETYLASE INHIBITORS (HDACIS) ON THE EXPRESSION OF ADHESION MOLECULES AND MODERN TARGET STRUCTURES IN MULTIPLE MYELOMA (MM)
(Abstract release date: 05/19/16)
EHA Library. Müller S. 06/09/16; 132796; E1247
Mr. Stefan Müller
Contributions
Contributions
Abstract
Abstract: E1247
Type: Eposter Presentation
Background
During the last decades the vital role of the bone marrow (BM) niche was increasingly explored to elucidate the progression and drug resistance in multiple myeloma (MM), this leading to advances of treatment strategies to modulate the interaction between malignant plasma cells (PCs) and the microenvironment. Small molecules (IMiDs, plerixafor, NOX-A12) and biologicals (elotuzumab, daratumumab, indatuximab) have been developed and are in pre- and clinical use.
Aims
We assessed the influence of histone deacetylase inhibitors (HDACis) on the BM niche. For this purpose various HDACis were assessed alone and in combination with antimyeloma agents in a novel 3-dimensional (3D) co-culture, to predict most rational combination use targeting both PCs and BM niche.
Methods
To best mimic the microenvironment, whilst ensuring easy handling, we have established an agarose matrix-based 3D coculture model, suitable for high-throughput screening. The cells are seeded in conical microwells and their proliferation, viability and apoptosis are assessed. These properties are compared both in presence or absence of stroma and to 2D cultures. Cells are incubated with HDACis, their synergisms with established antimyeloma drugs and stroma effects upon the treatment can be visualized via flow-cytometry and viability assays. The expression profile of relevant surface molecules (CXCR4, CD38, SLAM-F7, CD138) in primary MM cells and MM cell lines (MMCLs) are determined using fluorescent labeled antibodies and flow-cytometry.
Results
Inside the microcavities of the agarose matrices, PCs indeed formed 3D cell clusters and showed a similar cell proliferation rate compared to 2D cultures. Cell growth and expression pattern of surface proteins were influenced by the presence of BM stroma cells (BMSCs), leading to a decrease in CXCR4 but persistence of CD138 expression. Our current results suggest evident differences concerning growth and treatment response in our 3D versus 2D culture systems, which we currently further validate using HDACi as a novel substance class in MM within this easy to use culture system. Therein, the novel selective HDAC-6 inhibitor JS28 was selected from more than 20 hydroxamic acid derivates due to trypsin-dependent in vitro HDAC-inhibition assay results, with an IC50 of 59nM and a both 200-fold selectivity for HDAC 6 and HDAC 8 as compared to HDAC 1, respectively. The effect on target could be demonstrated in 2 different cell lines (HeLa and HL30) via western blot, quantifying a higher amount of acetylated tubulin, which is mainly degraded via HDAC 6 and consequently an evident marker for HDAC 6 activity. The phenotypic HDACi effect was determined with assessment in 2 MMCLs U266 and RPMI 8226: treatment of U266 and RPMI cells were performed with use of panobinostat or JS28 for 48 hours, either alone or combined with bortezomib, to test synergism. 2, 4 and 6nM bortezomib were combined with same ratios of both HDACi panobinostat with 2, 4 and 6nM or JS28 with 20, 40 and 60µM. The calculated combination indices (CI) in both MMCLs were <1 (=synergistic) within a range of approximately EC40 to EC55 for both combined panobinostat/bortezomib and JS28/bortezomib treatment schedules.
Conclusion
Our current data underline the importance of accurate 3D co-culture models to mimic in vivo proliferation and drug resistance and to predict later clinical treatment success more effectively. So far, there is insufficient data that conclusively unravel the complex interactions between HDACis, their different subtype selectivity and the rapidly increasing number of treatment options in MM. In this context, our ongoing investigations will focus on cytokine secretion patterns, expression of adhesion molecules, their targeting and their alteration by HDACis.
Session topic: E-poster
Keyword(s): Bone marrow stroma, HDAC inhibitor, Multiple myeloma
Type: Eposter Presentation
Background
During the last decades the vital role of the bone marrow (BM) niche was increasingly explored to elucidate the progression and drug resistance in multiple myeloma (MM), this leading to advances of treatment strategies to modulate the interaction between malignant plasma cells (PCs) and the microenvironment. Small molecules (IMiDs, plerixafor, NOX-A12) and biologicals (elotuzumab, daratumumab, indatuximab) have been developed and are in pre- and clinical use.
Aims
We assessed the influence of histone deacetylase inhibitors (HDACis) on the BM niche. For this purpose various HDACis were assessed alone and in combination with antimyeloma agents in a novel 3-dimensional (3D) co-culture, to predict most rational combination use targeting both PCs and BM niche.
Methods
To best mimic the microenvironment, whilst ensuring easy handling, we have established an agarose matrix-based 3D coculture model, suitable for high-throughput screening. The cells are seeded in conical microwells and their proliferation, viability and apoptosis are assessed. These properties are compared both in presence or absence of stroma and to 2D cultures. Cells are incubated with HDACis, their synergisms with established antimyeloma drugs and stroma effects upon the treatment can be visualized via flow-cytometry and viability assays. The expression profile of relevant surface molecules (CXCR4, CD38, SLAM-F7, CD138) in primary MM cells and MM cell lines (MMCLs) are determined using fluorescent labeled antibodies and flow-cytometry.
Results
Inside the microcavities of the agarose matrices, PCs indeed formed 3D cell clusters and showed a similar cell proliferation rate compared to 2D cultures. Cell growth and expression pattern of surface proteins were influenced by the presence of BM stroma cells (BMSCs), leading to a decrease in CXCR4 but persistence of CD138 expression. Our current results suggest evident differences concerning growth and treatment response in our 3D versus 2D culture systems, which we currently further validate using HDACi as a novel substance class in MM within this easy to use culture system. Therein, the novel selective HDAC-6 inhibitor JS28 was selected from more than 20 hydroxamic acid derivates due to trypsin-dependent in vitro HDAC-inhibition assay results, with an IC50 of 59nM and a both 200-fold selectivity for HDAC 6 and HDAC 8 as compared to HDAC 1, respectively. The effect on target could be demonstrated in 2 different cell lines (HeLa and HL30) via western blot, quantifying a higher amount of acetylated tubulin, which is mainly degraded via HDAC 6 and consequently an evident marker for HDAC 6 activity. The phenotypic HDACi effect was determined with assessment in 2 MMCLs U266 and RPMI 8226: treatment of U266 and RPMI cells were performed with use of panobinostat or JS28 for 48 hours, either alone or combined with bortezomib, to test synergism. 2, 4 and 6nM bortezomib were combined with same ratios of both HDACi panobinostat with 2, 4 and 6nM or JS28 with 20, 40 and 60µM. The calculated combination indices (CI) in both MMCLs were <1 (=synergistic) within a range of approximately EC40 to EC55 for both combined panobinostat/bortezomib and JS28/bortezomib treatment schedules.
Conclusion
Our current data underline the importance of accurate 3D co-culture models to mimic in vivo proliferation and drug resistance and to predict later clinical treatment success more effectively. So far, there is insufficient data that conclusively unravel the complex interactions between HDACis, their different subtype selectivity and the rapidly increasing number of treatment options in MM. In this context, our ongoing investigations will focus on cytokine secretion patterns, expression of adhesion molecules, their targeting and their alteration by HDACis.
Session topic: E-poster
Keyword(s): Bone marrow stroma, HDAC inhibitor, Multiple myeloma
Abstract: E1247
Type: Eposter Presentation
Background
During the last decades the vital role of the bone marrow (BM) niche was increasingly explored to elucidate the progression and drug resistance in multiple myeloma (MM), this leading to advances of treatment strategies to modulate the interaction between malignant plasma cells (PCs) and the microenvironment. Small molecules (IMiDs, plerixafor, NOX-A12) and biologicals (elotuzumab, daratumumab, indatuximab) have been developed and are in pre- and clinical use.
Aims
We assessed the influence of histone deacetylase inhibitors (HDACis) on the BM niche. For this purpose various HDACis were assessed alone and in combination with antimyeloma agents in a novel 3-dimensional (3D) co-culture, to predict most rational combination use targeting both PCs and BM niche.
Methods
To best mimic the microenvironment, whilst ensuring easy handling, we have established an agarose matrix-based 3D coculture model, suitable for high-throughput screening. The cells are seeded in conical microwells and their proliferation, viability and apoptosis are assessed. These properties are compared both in presence or absence of stroma and to 2D cultures. Cells are incubated with HDACis, their synergisms with established antimyeloma drugs and stroma effects upon the treatment can be visualized via flow-cytometry and viability assays. The expression profile of relevant surface molecules (CXCR4, CD38, SLAM-F7, CD138) in primary MM cells and MM cell lines (MMCLs) are determined using fluorescent labeled antibodies and flow-cytometry.
Results
Inside the microcavities of the agarose matrices, PCs indeed formed 3D cell clusters and showed a similar cell proliferation rate compared to 2D cultures. Cell growth and expression pattern of surface proteins were influenced by the presence of BM stroma cells (BMSCs), leading to a decrease in CXCR4 but persistence of CD138 expression. Our current results suggest evident differences concerning growth and treatment response in our 3D versus 2D culture systems, which we currently further validate using HDACi as a novel substance class in MM within this easy to use culture system. Therein, the novel selective HDAC-6 inhibitor JS28 was selected from more than 20 hydroxamic acid derivates due to trypsin-dependent in vitro HDAC-inhibition assay results, with an IC50 of 59nM and a both 200-fold selectivity for HDAC 6 and HDAC 8 as compared to HDAC 1, respectively. The effect on target could be demonstrated in 2 different cell lines (HeLa and HL30) via western blot, quantifying a higher amount of acetylated tubulin, which is mainly degraded via HDAC 6 and consequently an evident marker for HDAC 6 activity. The phenotypic HDACi effect was determined with assessment in 2 MMCLs U266 and RPMI 8226: treatment of U266 and RPMI cells were performed with use of panobinostat or JS28 for 48 hours, either alone or combined with bortezomib, to test synergism. 2, 4 and 6nM bortezomib were combined with same ratios of both HDACi panobinostat with 2, 4 and 6nM or JS28 with 20, 40 and 60µM. The calculated combination indices (CI) in both MMCLs were <1 (=synergistic) within a range of approximately EC40 to EC55 for both combined panobinostat/bortezomib and JS28/bortezomib treatment schedules.
Conclusion
Our current data underline the importance of accurate 3D co-culture models to mimic in vivo proliferation and drug resistance and to predict later clinical treatment success more effectively. So far, there is insufficient data that conclusively unravel the complex interactions between HDACis, their different subtype selectivity and the rapidly increasing number of treatment options in MM. In this context, our ongoing investigations will focus on cytokine secretion patterns, expression of adhesion molecules, their targeting and their alteration by HDACis.
Session topic: E-poster
Keyword(s): Bone marrow stroma, HDAC inhibitor, Multiple myeloma
Type: Eposter Presentation
Background
During the last decades the vital role of the bone marrow (BM) niche was increasingly explored to elucidate the progression and drug resistance in multiple myeloma (MM), this leading to advances of treatment strategies to modulate the interaction between malignant plasma cells (PCs) and the microenvironment. Small molecules (IMiDs, plerixafor, NOX-A12) and biologicals (elotuzumab, daratumumab, indatuximab) have been developed and are in pre- and clinical use.
Aims
We assessed the influence of histone deacetylase inhibitors (HDACis) on the BM niche. For this purpose various HDACis were assessed alone and in combination with antimyeloma agents in a novel 3-dimensional (3D) co-culture, to predict most rational combination use targeting both PCs and BM niche.
Methods
To best mimic the microenvironment, whilst ensuring easy handling, we have established an agarose matrix-based 3D coculture model, suitable for high-throughput screening. The cells are seeded in conical microwells and their proliferation, viability and apoptosis are assessed. These properties are compared both in presence or absence of stroma and to 2D cultures. Cells are incubated with HDACis, their synergisms with established antimyeloma drugs and stroma effects upon the treatment can be visualized via flow-cytometry and viability assays. The expression profile of relevant surface molecules (CXCR4, CD38, SLAM-F7, CD138) in primary MM cells and MM cell lines (MMCLs) are determined using fluorescent labeled antibodies and flow-cytometry.
Results
Inside the microcavities of the agarose matrices, PCs indeed formed 3D cell clusters and showed a similar cell proliferation rate compared to 2D cultures. Cell growth and expression pattern of surface proteins were influenced by the presence of BM stroma cells (BMSCs), leading to a decrease in CXCR4 but persistence of CD138 expression. Our current results suggest evident differences concerning growth and treatment response in our 3D versus 2D culture systems, which we currently further validate using HDACi as a novel substance class in MM within this easy to use culture system. Therein, the novel selective HDAC-6 inhibitor JS28 was selected from more than 20 hydroxamic acid derivates due to trypsin-dependent in vitro HDAC-inhibition assay results, with an IC50 of 59nM and a both 200-fold selectivity for HDAC 6 and HDAC 8 as compared to HDAC 1, respectively. The effect on target could be demonstrated in 2 different cell lines (HeLa and HL30) via western blot, quantifying a higher amount of acetylated tubulin, which is mainly degraded via HDAC 6 and consequently an evident marker for HDAC 6 activity. The phenotypic HDACi effect was determined with assessment in 2 MMCLs U266 and RPMI 8226: treatment of U266 and RPMI cells were performed with use of panobinostat or JS28 for 48 hours, either alone or combined with bortezomib, to test synergism. 2, 4 and 6nM bortezomib were combined with same ratios of both HDACi panobinostat with 2, 4 and 6nM or JS28 with 20, 40 and 60µM. The calculated combination indices (CI) in both MMCLs were <1 (=synergistic) within a range of approximately EC40 to EC55 for both combined panobinostat/bortezomib and JS28/bortezomib treatment schedules.
Conclusion
Our current data underline the importance of accurate 3D co-culture models to mimic in vivo proliferation and drug resistance and to predict later clinical treatment success more effectively. So far, there is insufficient data that conclusively unravel the complex interactions between HDACis, their different subtype selectivity and the rapidly increasing number of treatment options in MM. In this context, our ongoing investigations will focus on cytokine secretion patterns, expression of adhesion molecules, their targeting and their alteration by HDACis.
Session topic: E-poster
Keyword(s): Bone marrow stroma, HDAC inhibitor, Multiple myeloma
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