BAX ACTIVATION IS A POTENTIAL BIOMARKER OF RESPONSE TO FILANESIB (ARRY-520) ALONE AND IN COMBINATION WITH POMALIDOMIDE AND DEXAMETHASONE
(Abstract release date: 05/19/16)
EHA Library. M Ocio E. 06/09/16; 132788; E1239
Mrs. Enrique M Ocio
Contributions
Contributions
Abstract
Abstract: E1239
Type: Eposter Presentation
Background
Pomalidomide in combination with dexamethasone (PD) has demonstrated activity in refractory MM patients, but novel combinations are needed to improve its efficacy. In this context, the kinesin spindle protein (KSP) inhibitor, filanesib (Arry-520), has demonstrated clinical anti-myeloma effect and previous data from our group and others have demonstrated the preclinical synergy of pomalidomide with dexamethasone and filanesib (PDF) what set the stage for the recently activated clinical trial POMDEFIL being run by the Spanish MM group investigating this combination in relapsed MM patients.
Aims
In this abstract, we gained insights into the mechanism of the combination and aimed at identifying potential biomarkers that could predict response.
Methods
MM cells were transiently transfected with non-targeting control short interfering RNA (NT-SiRNA), Bax siRNA ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. In vitro action was evaluated in MM cells by Annexin V staining using flow cytometry. The mechanism of action was analyzed by Western Blot, immunohistochemistry and immunofluorescence techniques.
Results
All 11 MM cell lines tested were sensitive to filanesib and sensitivity to this agent correlated with basal levels of Bax and Bax/Mcl-1 protein ratio. Subcellular fractionation of MM1S cells indicated that filanesib triggered translocation of Bax from the cytoplasm to the mitochondria after 24 and 48 hours, and its subsequent cleavage into the very potent proapoptotic 18 kDa fragment. A simultaneous decrease in the levels of Mcl-1 was observed. Knock-down of Bax in MM1S by using small interfering RNA decreased the sensitivity of these cells to filanesib, as treatment with 10 nM for 24 hours induced apoptosis in only 26% of the siRNA-Bax cells as compared with 50% in the non-targeted cells (as compared with 58% vs 61% for bortezomib, used as a control for this experiment).Next, we evaluated changes of Bax protein levels in comparison with others in MM1S cells after treatment with vehicle, F, PD and PDF treatments. The levels of the proapoptotic protein, Bax, were significantly increased in both cytosolic and mitochondrial fractions with PDF treatment, as assessed by Western blotting after 12 or 48 hours of treatment. Of note, this event preceded the initiation of the apoptosis process as evaluated by cleavage of PARP and annexin V staining. Moreover, total cellular antiapoptotic protein Mcl-1 levels declined with the combination, although less than with F treatment and no significant changes occurred in the mitochondrial and cytosolic compartments by western blot.These results were confirmed in vivo in a model of subcutaneous plasmacytoma in tumors with big plasmacytomas, that after two days of treatment with vehicle, PD, F or PDF were sacrificed and tumors were fixed, paraffin-embedded and stained with anti-Bax and counterstained with haematoxilin for analyses. In vivo treatment with PDF also induced a clear increase in Bax that was mainly present in cells with aberrant monopolar spindles (a hallmark of the KSP inhibition induced by filanesib) and with an increase of apoptosis evaluated by TUNEL (p<0.001).
Conclusion
Bax protein is specific involved in the response to filanesib in monotherapy and plays a significant role in the synergy of its combination with pomalidomide and dexamethasone, being one of the earlier events observed. Therefore, Bax protein could be a potential biomarker of response to filanesib and also this synergistic combination.This work was funded in part by the company Array BioPharma.
Session topic: E-poster
Keyword(s): Apoptosis, Bax, Imids, SiRNA
Type: Eposter Presentation
Background
Pomalidomide in combination with dexamethasone (PD) has demonstrated activity in refractory MM patients, but novel combinations are needed to improve its efficacy. In this context, the kinesin spindle protein (KSP) inhibitor, filanesib (Arry-520), has demonstrated clinical anti-myeloma effect and previous data from our group and others have demonstrated the preclinical synergy of pomalidomide with dexamethasone and filanesib (PDF) what set the stage for the recently activated clinical trial POMDEFIL being run by the Spanish MM group investigating this combination in relapsed MM patients.
Aims
In this abstract, we gained insights into the mechanism of the combination and aimed at identifying potential biomarkers that could predict response.
Methods
MM cells were transiently transfected with non-targeting control short interfering RNA (NT-SiRNA), Bax siRNA ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. In vitro action was evaluated in MM cells by Annexin V staining using flow cytometry. The mechanism of action was analyzed by Western Blot, immunohistochemistry and immunofluorescence techniques.
Results
All 11 MM cell lines tested were sensitive to filanesib and sensitivity to this agent correlated with basal levels of Bax and Bax/Mcl-1 protein ratio. Subcellular fractionation of MM1S cells indicated that filanesib triggered translocation of Bax from the cytoplasm to the mitochondria after 24 and 48 hours, and its subsequent cleavage into the very potent proapoptotic 18 kDa fragment. A simultaneous decrease in the levels of Mcl-1 was observed. Knock-down of Bax in MM1S by using small interfering RNA decreased the sensitivity of these cells to filanesib, as treatment with 10 nM for 24 hours induced apoptosis in only 26% of the siRNA-Bax cells as compared with 50% in the non-targeted cells (as compared with 58% vs 61% for bortezomib, used as a control for this experiment).Next, we evaluated changes of Bax protein levels in comparison with others in MM1S cells after treatment with vehicle, F, PD and PDF treatments. The levels of the proapoptotic protein, Bax, were significantly increased in both cytosolic and mitochondrial fractions with PDF treatment, as assessed by Western blotting after 12 or 48 hours of treatment. Of note, this event preceded the initiation of the apoptosis process as evaluated by cleavage of PARP and annexin V staining. Moreover, total cellular antiapoptotic protein Mcl-1 levels declined with the combination, although less than with F treatment and no significant changes occurred in the mitochondrial and cytosolic compartments by western blot.These results were confirmed in vivo in a model of subcutaneous plasmacytoma in tumors with big plasmacytomas, that after two days of treatment with vehicle, PD, F or PDF were sacrificed and tumors were fixed, paraffin-embedded and stained with anti-Bax and counterstained with haematoxilin for analyses. In vivo treatment with PDF also induced a clear increase in Bax that was mainly present in cells with aberrant monopolar spindles (a hallmark of the KSP inhibition induced by filanesib) and with an increase of apoptosis evaluated by TUNEL (p<0.001).
Conclusion
Bax protein is specific involved in the response to filanesib in monotherapy and plays a significant role in the synergy of its combination with pomalidomide and dexamethasone, being one of the earlier events observed. Therefore, Bax protein could be a potential biomarker of response to filanesib and also this synergistic combination.This work was funded in part by the company Array BioPharma.
Session topic: E-poster
Keyword(s): Apoptosis, Bax, Imids, SiRNA
Abstract: E1239
Type: Eposter Presentation
Background
Pomalidomide in combination with dexamethasone (PD) has demonstrated activity in refractory MM patients, but novel combinations are needed to improve its efficacy. In this context, the kinesin spindle protein (KSP) inhibitor, filanesib (Arry-520), has demonstrated clinical anti-myeloma effect and previous data from our group and others have demonstrated the preclinical synergy of pomalidomide with dexamethasone and filanesib (PDF) what set the stage for the recently activated clinical trial POMDEFIL being run by the Spanish MM group investigating this combination in relapsed MM patients.
Aims
In this abstract, we gained insights into the mechanism of the combination and aimed at identifying potential biomarkers that could predict response.
Methods
MM cells were transiently transfected with non-targeting control short interfering RNA (NT-SiRNA), Bax siRNA ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. In vitro action was evaluated in MM cells by Annexin V staining using flow cytometry. The mechanism of action was analyzed by Western Blot, immunohistochemistry and immunofluorescence techniques.
Results
All 11 MM cell lines tested were sensitive to filanesib and sensitivity to this agent correlated with basal levels of Bax and Bax/Mcl-1 protein ratio. Subcellular fractionation of MM1S cells indicated that filanesib triggered translocation of Bax from the cytoplasm to the mitochondria after 24 and 48 hours, and its subsequent cleavage into the very potent proapoptotic 18 kDa fragment. A simultaneous decrease in the levels of Mcl-1 was observed. Knock-down of Bax in MM1S by using small interfering RNA decreased the sensitivity of these cells to filanesib, as treatment with 10 nM for 24 hours induced apoptosis in only 26% of the siRNA-Bax cells as compared with 50% in the non-targeted cells (as compared with 58% vs 61% for bortezomib, used as a control for this experiment).Next, we evaluated changes of Bax protein levels in comparison with others in MM1S cells after treatment with vehicle, F, PD and PDF treatments. The levels of the proapoptotic protein, Bax, were significantly increased in both cytosolic and mitochondrial fractions with PDF treatment, as assessed by Western blotting after 12 or 48 hours of treatment. Of note, this event preceded the initiation of the apoptosis process as evaluated by cleavage of PARP and annexin V staining. Moreover, total cellular antiapoptotic protein Mcl-1 levels declined with the combination, although less than with F treatment and no significant changes occurred in the mitochondrial and cytosolic compartments by western blot.These results were confirmed in vivo in a model of subcutaneous plasmacytoma in tumors with big plasmacytomas, that after two days of treatment with vehicle, PD, F or PDF were sacrificed and tumors were fixed, paraffin-embedded and stained with anti-Bax and counterstained with haematoxilin for analyses. In vivo treatment with PDF also induced a clear increase in Bax that was mainly present in cells with aberrant monopolar spindles (a hallmark of the KSP inhibition induced by filanesib) and with an increase of apoptosis evaluated by TUNEL (p<0.001).
Conclusion
Bax protein is specific involved in the response to filanesib in monotherapy and plays a significant role in the synergy of its combination with pomalidomide and dexamethasone, being one of the earlier events observed. Therefore, Bax protein could be a potential biomarker of response to filanesib and also this synergistic combination.This work was funded in part by the company Array BioPharma.
Session topic: E-poster
Keyword(s): Apoptosis, Bax, Imids, SiRNA
Type: Eposter Presentation
Background
Pomalidomide in combination with dexamethasone (PD) has demonstrated activity in refractory MM patients, but novel combinations are needed to improve its efficacy. In this context, the kinesin spindle protein (KSP) inhibitor, filanesib (Arry-520), has demonstrated clinical anti-myeloma effect and previous data from our group and others have demonstrated the preclinical synergy of pomalidomide with dexamethasone and filanesib (PDF) what set the stage for the recently activated clinical trial POMDEFIL being run by the Spanish MM group investigating this combination in relapsed MM patients.
Aims
In this abstract, we gained insights into the mechanism of the combination and aimed at identifying potential biomarkers that could predict response.
Methods
MM cells were transiently transfected with non-targeting control short interfering RNA (NT-SiRNA), Bax siRNA ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. In vitro action was evaluated in MM cells by Annexin V staining using flow cytometry. The mechanism of action was analyzed by Western Blot, immunohistochemistry and immunofluorescence techniques.
Results
All 11 MM cell lines tested were sensitive to filanesib and sensitivity to this agent correlated with basal levels of Bax and Bax/Mcl-1 protein ratio. Subcellular fractionation of MM1S cells indicated that filanesib triggered translocation of Bax from the cytoplasm to the mitochondria after 24 and 48 hours, and its subsequent cleavage into the very potent proapoptotic 18 kDa fragment. A simultaneous decrease in the levels of Mcl-1 was observed. Knock-down of Bax in MM1S by using small interfering RNA decreased the sensitivity of these cells to filanesib, as treatment with 10 nM for 24 hours induced apoptosis in only 26% of the siRNA-Bax cells as compared with 50% in the non-targeted cells (as compared with 58% vs 61% for bortezomib, used as a control for this experiment).Next, we evaluated changes of Bax protein levels in comparison with others in MM1S cells after treatment with vehicle, F, PD and PDF treatments. The levels of the proapoptotic protein, Bax, were significantly increased in both cytosolic and mitochondrial fractions with PDF treatment, as assessed by Western blotting after 12 or 48 hours of treatment. Of note, this event preceded the initiation of the apoptosis process as evaluated by cleavage of PARP and annexin V staining. Moreover, total cellular antiapoptotic protein Mcl-1 levels declined with the combination, although less than with F treatment and no significant changes occurred in the mitochondrial and cytosolic compartments by western blot.These results were confirmed in vivo in a model of subcutaneous plasmacytoma in tumors with big plasmacytomas, that after two days of treatment with vehicle, PD, F or PDF were sacrificed and tumors were fixed, paraffin-embedded and stained with anti-Bax and counterstained with haematoxilin for analyses. In vivo treatment with PDF also induced a clear increase in Bax that was mainly present in cells with aberrant monopolar spindles (a hallmark of the KSP inhibition induced by filanesib) and with an increase of apoptosis evaluated by TUNEL (p<0.001).
Conclusion
Bax protein is specific involved in the response to filanesib in monotherapy and plays a significant role in the synergy of its combination with pomalidomide and dexamethasone, being one of the earlier events observed. Therefore, Bax protein could be a potential biomarker of response to filanesib and also this synergistic combination.This work was funded in part by the company Array BioPharma.
Session topic: E-poster
Keyword(s): Apoptosis, Bax, Imids, SiRNA
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