CHARACTERIZATION OF A PERK KINASE INHIBITOR WITH ANTI-MYELOMA ACTIVITY
(Abstract release date: 05/19/16)
EHA Library. Kastritis E. 06/09/16; 132790; E1241
Dr. Efstathios Kastritis
Contributions
Contributions
Abstract
Abstract: E1241
Type: Eposter Presentation
Background
Due to the immunoglobulin production, multiple myeloma (MM) plasma cells are dependent on the unfolded protein response process (UPR), which controls protein production and ensures its proper translation and folding. A study by Michallet et al (2011) showed that knockdown of one of the three well-known arms of the UPR, PERK (protein kinase R (PKR)-like ER kinase) in MM cells resulted in autophagic cell death. This outcome indicated the important role of PERK activation for the metabolic shift of plasma cell to myeloma cell but also its ability to impede the apoptotic effect.
Aims
In this study we used a small ATP competitive molecular inhibitor, GSK2606414, that acts by targeting PERK enzyme activity in its inactive DFG conformation at the ATP-binding region, while displaying ≥385 fold selectivity over c-kit, Aurora B, BRK and many other kinases.
Methods
mRNA expression of PERK, CHOP and ATF4 in MM patients and cell lines was determined by real-time PCR. To deterine the effects of GSK2606414 on MM cell proliferation and apoptosis WST1 proliferation assay Annexin-PI staining was used respectively. To determine the effects of GSK2606414 in key genes of the UPR, UPR RT2 profiler PCR array was used.
Results
We initially screened 25 CD138+ MM patients and 6 human myeloma cell lines (HMCLs) for PERK mRNA expression. Our results showed that PERK mRNA is highly expressed in almost all patients (5-10 fold higher than the mean PERK expression of HMCLs).To test the effect of GSK2606414 on the proliferation of MM cells, 4 HMCLs were treated with different doses of GSK2606414 at two time points (24 and 48 hours). Treatment of cells with 3-30μM GSK2606414 resulted in a dose-dependent inhibition of cell proliferation in all HMCLs ranging for 20-95% reduction of proliferative activity, thus, indicating the dependency of these cells on PERK activity.Treatment with GSK2606414 at 20μM in H929 and L363 HMCLs for 24 hours resulted 25 % and 15 % increase in apoptotic cells by Annexin-PI staining respectively compared to the untreated cells. However, the most important finding was a significant synergistic effect of GSK2606414 with bortezomib in these cells. Specifically when H929 and L363 cells were treated with 5nM bortezomib in combination with 20μM GSK2606414, synergistic effect was seen where apoptotic cells reached 99% and 77% respectively, compared to bortezomib-treated cells (87% and 42% respectively).To determine the gene target effects of GSK2606414, ATF4 and CHOP mRNA expression levels were determined in H929 cell line after 24 hour of treatment. Treatment with GSK2606414 alone did not alter the expression levels of CHOP but reduced more than 50% the expression levels of ATF4. When combined with bortezomib CHOP and ATF4 levels were reduced 20% and 60% respectively while treatment with bortezomib alone increased the levels of CHOP and ATF4 by 50-100%. H929 and L363, were pre-treated with GSK2606414 and then subjected to ER stress conditions by treatment with tunicamycin (TM). In the presence of exogeneous UPR inducers, such as TM, GSK2656157 had a significant effect on the expression of the above genes at much lower IC50 range (≤100 nM) suggesting the in a more active UPR (sustained ER stress) the effects are more pronounced. Thus, the combination of GSK2606414 with bortezomib may inhibit one of the mechanisms of MM cells to escape ER stress induced by bortezomib. To determine the effects of the GSK2606414 on myeloma cells, a UPR RT2 Profiler PCR array containing 84 key genes for the UPR pathway, was used in order to screen for differentially expressed genes between treated and non-treated cells. Results of this array will be presented at the meeting.
Conclusion
In conclusion, given the on-target pharmacological effects of PERK inhibitor on MM, development of PERK inhibitors may offer a therapeutic advantage that would affect MM pathogenesis and treatment.
Session topic: E-poster
Keyword(s): Bortezomib, Inhibitor, Multiple myeloma, Proliferation
Type: Eposter Presentation
Background
Due to the immunoglobulin production, multiple myeloma (MM) plasma cells are dependent on the unfolded protein response process (UPR), which controls protein production and ensures its proper translation and folding. A study by Michallet et al (2011) showed that knockdown of one of the three well-known arms of the UPR, PERK (protein kinase R (PKR)-like ER kinase) in MM cells resulted in autophagic cell death. This outcome indicated the important role of PERK activation for the metabolic shift of plasma cell to myeloma cell but also its ability to impede the apoptotic effect.
Aims
In this study we used a small ATP competitive molecular inhibitor, GSK2606414, that acts by targeting PERK enzyme activity in its inactive DFG conformation at the ATP-binding region, while displaying ≥385 fold selectivity over c-kit, Aurora B, BRK and many other kinases.
Methods
mRNA expression of PERK, CHOP and ATF4 in MM patients and cell lines was determined by real-time PCR. To deterine the effects of GSK2606414 on MM cell proliferation and apoptosis WST1 proliferation assay Annexin-PI staining was used respectively. To determine the effects of GSK2606414 in key genes of the UPR, UPR RT2 profiler PCR array was used.
Results
We initially screened 25 CD138+ MM patients and 6 human myeloma cell lines (HMCLs) for PERK mRNA expression. Our results showed that PERK mRNA is highly expressed in almost all patients (5-10 fold higher than the mean PERK expression of HMCLs).To test the effect of GSK2606414 on the proliferation of MM cells, 4 HMCLs were treated with different doses of GSK2606414 at two time points (24 and 48 hours). Treatment of cells with 3-30μM GSK2606414 resulted in a dose-dependent inhibition of cell proliferation in all HMCLs ranging for 20-95% reduction of proliferative activity, thus, indicating the dependency of these cells on PERK activity.Treatment with GSK2606414 at 20μM in H929 and L363 HMCLs for 24 hours resulted 25 % and 15 % increase in apoptotic cells by Annexin-PI staining respectively compared to the untreated cells. However, the most important finding was a significant synergistic effect of GSK2606414 with bortezomib in these cells. Specifically when H929 and L363 cells were treated with 5nM bortezomib in combination with 20μM GSK2606414, synergistic effect was seen where apoptotic cells reached 99% and 77% respectively, compared to bortezomib-treated cells (87% and 42% respectively).To determine the gene target effects of GSK2606414, ATF4 and CHOP mRNA expression levels were determined in H929 cell line after 24 hour of treatment. Treatment with GSK2606414 alone did not alter the expression levels of CHOP but reduced more than 50% the expression levels of ATF4. When combined with bortezomib CHOP and ATF4 levels were reduced 20% and 60% respectively while treatment with bortezomib alone increased the levels of CHOP and ATF4 by 50-100%. H929 and L363, were pre-treated with GSK2606414 and then subjected to ER stress conditions by treatment with tunicamycin (TM). In the presence of exogeneous UPR inducers, such as TM, GSK2656157 had a significant effect on the expression of the above genes at much lower IC50 range (≤100 nM) suggesting the in a more active UPR (sustained ER stress) the effects are more pronounced. Thus, the combination of GSK2606414 with bortezomib may inhibit one of the mechanisms of MM cells to escape ER stress induced by bortezomib. To determine the effects of the GSK2606414 on myeloma cells, a UPR RT2 Profiler PCR array containing 84 key genes for the UPR pathway, was used in order to screen for differentially expressed genes between treated and non-treated cells. Results of this array will be presented at the meeting.
Conclusion
In conclusion, given the on-target pharmacological effects of PERK inhibitor on MM, development of PERK inhibitors may offer a therapeutic advantage that would affect MM pathogenesis and treatment.
Session topic: E-poster
Keyword(s): Bortezomib, Inhibitor, Multiple myeloma, Proliferation
Abstract: E1241
Type: Eposter Presentation
Background
Due to the immunoglobulin production, multiple myeloma (MM) plasma cells are dependent on the unfolded protein response process (UPR), which controls protein production and ensures its proper translation and folding. A study by Michallet et al (2011) showed that knockdown of one of the three well-known arms of the UPR, PERK (protein kinase R (PKR)-like ER kinase) in MM cells resulted in autophagic cell death. This outcome indicated the important role of PERK activation for the metabolic shift of plasma cell to myeloma cell but also its ability to impede the apoptotic effect.
Aims
In this study we used a small ATP competitive molecular inhibitor, GSK2606414, that acts by targeting PERK enzyme activity in its inactive DFG conformation at the ATP-binding region, while displaying ≥385 fold selectivity over c-kit, Aurora B, BRK and many other kinases.
Methods
mRNA expression of PERK, CHOP and ATF4 in MM patients and cell lines was determined by real-time PCR. To deterine the effects of GSK2606414 on MM cell proliferation and apoptosis WST1 proliferation assay Annexin-PI staining was used respectively. To determine the effects of GSK2606414 in key genes of the UPR, UPR RT2 profiler PCR array was used.
Results
We initially screened 25 CD138+ MM patients and 6 human myeloma cell lines (HMCLs) for PERK mRNA expression. Our results showed that PERK mRNA is highly expressed in almost all patients (5-10 fold higher than the mean PERK expression of HMCLs).To test the effect of GSK2606414 on the proliferation of MM cells, 4 HMCLs were treated with different doses of GSK2606414 at two time points (24 and 48 hours). Treatment of cells with 3-30μM GSK2606414 resulted in a dose-dependent inhibition of cell proliferation in all HMCLs ranging for 20-95% reduction of proliferative activity, thus, indicating the dependency of these cells on PERK activity.Treatment with GSK2606414 at 20μM in H929 and L363 HMCLs for 24 hours resulted 25 % and 15 % increase in apoptotic cells by Annexin-PI staining respectively compared to the untreated cells. However, the most important finding was a significant synergistic effect of GSK2606414 with bortezomib in these cells. Specifically when H929 and L363 cells were treated with 5nM bortezomib in combination with 20μM GSK2606414, synergistic effect was seen where apoptotic cells reached 99% and 77% respectively, compared to bortezomib-treated cells (87% and 42% respectively).To determine the gene target effects of GSK2606414, ATF4 and CHOP mRNA expression levels were determined in H929 cell line after 24 hour of treatment. Treatment with GSK2606414 alone did not alter the expression levels of CHOP but reduced more than 50% the expression levels of ATF4. When combined with bortezomib CHOP and ATF4 levels were reduced 20% and 60% respectively while treatment with bortezomib alone increased the levels of CHOP and ATF4 by 50-100%. H929 and L363, were pre-treated with GSK2606414 and then subjected to ER stress conditions by treatment with tunicamycin (TM). In the presence of exogeneous UPR inducers, such as TM, GSK2656157 had a significant effect on the expression of the above genes at much lower IC50 range (≤100 nM) suggesting the in a more active UPR (sustained ER stress) the effects are more pronounced. Thus, the combination of GSK2606414 with bortezomib may inhibit one of the mechanisms of MM cells to escape ER stress induced by bortezomib. To determine the effects of the GSK2606414 on myeloma cells, a UPR RT2 Profiler PCR array containing 84 key genes for the UPR pathway, was used in order to screen for differentially expressed genes between treated and non-treated cells. Results of this array will be presented at the meeting.
Conclusion
In conclusion, given the on-target pharmacological effects of PERK inhibitor on MM, development of PERK inhibitors may offer a therapeutic advantage that would affect MM pathogenesis and treatment.
Session topic: E-poster
Keyword(s): Bortezomib, Inhibitor, Multiple myeloma, Proliferation
Type: Eposter Presentation
Background
Due to the immunoglobulin production, multiple myeloma (MM) plasma cells are dependent on the unfolded protein response process (UPR), which controls protein production and ensures its proper translation and folding. A study by Michallet et al (2011) showed that knockdown of one of the three well-known arms of the UPR, PERK (protein kinase R (PKR)-like ER kinase) in MM cells resulted in autophagic cell death. This outcome indicated the important role of PERK activation for the metabolic shift of plasma cell to myeloma cell but also its ability to impede the apoptotic effect.
Aims
In this study we used a small ATP competitive molecular inhibitor, GSK2606414, that acts by targeting PERK enzyme activity in its inactive DFG conformation at the ATP-binding region, while displaying ≥385 fold selectivity over c-kit, Aurora B, BRK and many other kinases.
Methods
mRNA expression of PERK, CHOP and ATF4 in MM patients and cell lines was determined by real-time PCR. To deterine the effects of GSK2606414 on MM cell proliferation and apoptosis WST1 proliferation assay Annexin-PI staining was used respectively. To determine the effects of GSK2606414 in key genes of the UPR, UPR RT2 profiler PCR array was used.
Results
We initially screened 25 CD138+ MM patients and 6 human myeloma cell lines (HMCLs) for PERK mRNA expression. Our results showed that PERK mRNA is highly expressed in almost all patients (5-10 fold higher than the mean PERK expression of HMCLs).To test the effect of GSK2606414 on the proliferation of MM cells, 4 HMCLs were treated with different doses of GSK2606414 at two time points (24 and 48 hours). Treatment of cells with 3-30μM GSK2606414 resulted in a dose-dependent inhibition of cell proliferation in all HMCLs ranging for 20-95% reduction of proliferative activity, thus, indicating the dependency of these cells on PERK activity.Treatment with GSK2606414 at 20μM in H929 and L363 HMCLs for 24 hours resulted 25 % and 15 % increase in apoptotic cells by Annexin-PI staining respectively compared to the untreated cells. However, the most important finding was a significant synergistic effect of GSK2606414 with bortezomib in these cells. Specifically when H929 and L363 cells were treated with 5nM bortezomib in combination with 20μM GSK2606414, synergistic effect was seen where apoptotic cells reached 99% and 77% respectively, compared to bortezomib-treated cells (87% and 42% respectively).To determine the gene target effects of GSK2606414, ATF4 and CHOP mRNA expression levels were determined in H929 cell line after 24 hour of treatment. Treatment with GSK2606414 alone did not alter the expression levels of CHOP but reduced more than 50% the expression levels of ATF4. When combined with bortezomib CHOP and ATF4 levels were reduced 20% and 60% respectively while treatment with bortezomib alone increased the levels of CHOP and ATF4 by 50-100%. H929 and L363, were pre-treated with GSK2606414 and then subjected to ER stress conditions by treatment with tunicamycin (TM). In the presence of exogeneous UPR inducers, such as TM, GSK2656157 had a significant effect on the expression of the above genes at much lower IC50 range (≤100 nM) suggesting the in a more active UPR (sustained ER stress) the effects are more pronounced. Thus, the combination of GSK2606414 with bortezomib may inhibit one of the mechanisms of MM cells to escape ER stress induced by bortezomib. To determine the effects of the GSK2606414 on myeloma cells, a UPR RT2 Profiler PCR array containing 84 key genes for the UPR pathway, was used in order to screen for differentially expressed genes between treated and non-treated cells. Results of this array will be presented at the meeting.
Conclusion
In conclusion, given the on-target pharmacological effects of PERK inhibitor on MM, development of PERK inhibitors may offer a therapeutic advantage that would affect MM pathogenesis and treatment.
Session topic: E-poster
Keyword(s): Bortezomib, Inhibitor, Multiple myeloma, Proliferation
{{ help_message }}
{{filter}}