RESISTANCE TO PROTEASOME INHIBITORS IS MEDIATED BY AN OVER-ACTIVATION OF THE UPR AND THE DNA DAMAGE RESPONSE IN A PRECLINICAL MODEL OF MM
(Abstract release date: 05/19/16)
EHA Library. M Ocio E. 06/09/16; 132783; E1234
Mrs. Enrique M Ocio
Contributions
Contributions
Abstract
Abstract: E1234
Type: Eposter Presentation
Background
MM is still considered incurable partly due to the development of resistance.
Aims
We have developed a cellular model of resistance to proteasome inhibitors to investigate the underlying mechanisms.
Methods
Cells sensitive to proteasome inhibitors (MM1S, MM1R, RPMI-8226) were continuously exposed to increasing concentrations of bortezomib until development of resistance. Viability was analyzed by MTT. Activity of different subunits of the proteasome was analyzed with Proteasome-Glo™ Assay Kit and presence of unfolded proteins with ProteoStat® Aggresome Detection Kit. Western-Blot, flow cytometry and qRT-PCR were also used.
Results
The development of acquired resistance to bortezomib in three cell lines was assessed by MTT assay with IC50 (nM) of 2.5 vs >100; 6.7 vs 78.5 and 21.2 vs >100 for the sensitive vs the resistant counterpart in MM1S, MM1R and RPMI at 24 hours. Interestingly, cells were also resistant to other proteasome inhibitors such as carfilzomib, oprozomib or ixazomib, indicating a class-effect resistance. In absence of bortezomib treatment, resistant cells presented a lower activity of the different subunits of the proteasome compared to the sensitive counterpart by 52% for caspase-like (C-L); 54% for trypsin-like (T-L) and 42% for chemotrypsin-like (CT-L) activities. This was even lower in case of MM1R (26%, 25% and 12%) and RPMI-8226 (21%, 40% and 23%) resistant cells. This inhibition led to an accumulation of unfolded proteins in the resistant MM1S cells as compared with their sensitive counterpart (96% vs 45%). Moreover, the concentration of bortezomib required to inhibit the proteasome was around 20-fold higher in the resistant than in the sensitive MM1S cells (x23; x21 and x27 for the C-L, T-L and CT-L activities). Finally, and most importantly, even with doses of bortezomib achieving a complete inhibition of the three subunits, resistant cells were viable, indicating they were not dependent on the proteasome and had developed alternative mechanisms of survival. In this context, resistant cells significantly upregulated the pro-survival chaperone BIP/GRP78, a critical sensor of the ER stress. This upregulation activated the three main arms of the unfolded protein response (UPR): increased levels of p-IRE1, ATF-6 and activation of PERK as observed through phosphorylation of its downstream substrate EIFα. A sequelae of the activation of the UPR is the transcriptional activation of chaperones, and, accordingly, there was increased expression of HSP-40 and, particularly, HSP-70 (but not HSP-90) in resistant cells. The ER stress also induced a DNA damage response with an increase in pH2Ax in resistant cells and a subsequent upregulation of p53 protein levels. Moreover, the increase in p-IRE-1 induced the nuclear translocation of NFkB, where it could exert its pro-survival and transcriptional function. In fact, we also observed an increase in the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP, some of them known substrates of NFkB. Interestingly, these resistant cells had an increased sensitivity to Pifithrin, a Hsp-70 and p53 inhibitor (6 hours Pifithrin treatment induced 69% vs 26% apoptosis in resistant vs sensitive cells).
Conclusion
We have demonstrated some of the mechanisms associated with resistance to proteasome inhibitors: activation of the UPR (with subsequent upregulation of the chaperone system), DNA damage response and NFkB signaling with increased levels of antiapoptotic Bcl-2 family members. This work was supported by ISCIII-FIS PI15/00067 and /02156 and GRS 1175/A/15.
Session topic: E-poster
Keyword(s): Proteasome inhibitor, Resistance
Type: Eposter Presentation
Background
MM is still considered incurable partly due to the development of resistance.
Aims
We have developed a cellular model of resistance to proteasome inhibitors to investigate the underlying mechanisms.
Methods
Cells sensitive to proteasome inhibitors (MM1S, MM1R, RPMI-8226) were continuously exposed to increasing concentrations of bortezomib until development of resistance. Viability was analyzed by MTT. Activity of different subunits of the proteasome was analyzed with Proteasome-Glo™ Assay Kit and presence of unfolded proteins with ProteoStat® Aggresome Detection Kit. Western-Blot, flow cytometry and qRT-PCR were also used.
Results
The development of acquired resistance to bortezomib in three cell lines was assessed by MTT assay with IC50 (nM) of 2.5 vs >100; 6.7 vs 78.5 and 21.2 vs >100 for the sensitive vs the resistant counterpart in MM1S, MM1R and RPMI at 24 hours. Interestingly, cells were also resistant to other proteasome inhibitors such as carfilzomib, oprozomib or ixazomib, indicating a class-effect resistance. In absence of bortezomib treatment, resistant cells presented a lower activity of the different subunits of the proteasome compared to the sensitive counterpart by 52% for caspase-like (C-L); 54% for trypsin-like (T-L) and 42% for chemotrypsin-like (CT-L) activities. This was even lower in case of MM1R (26%, 25% and 12%) and RPMI-8226 (21%, 40% and 23%) resistant cells. This inhibition led to an accumulation of unfolded proteins in the resistant MM1S cells as compared with their sensitive counterpart (96% vs 45%). Moreover, the concentration of bortezomib required to inhibit the proteasome was around 20-fold higher in the resistant than in the sensitive MM1S cells (x23; x21 and x27 for the C-L, T-L and CT-L activities). Finally, and most importantly, even with doses of bortezomib achieving a complete inhibition of the three subunits, resistant cells were viable, indicating they were not dependent on the proteasome and had developed alternative mechanisms of survival. In this context, resistant cells significantly upregulated the pro-survival chaperone BIP/GRP78, a critical sensor of the ER stress. This upregulation activated the three main arms of the unfolded protein response (UPR): increased levels of p-IRE1, ATF-6 and activation of PERK as observed through phosphorylation of its downstream substrate EIFα. A sequelae of the activation of the UPR is the transcriptional activation of chaperones, and, accordingly, there was increased expression of HSP-40 and, particularly, HSP-70 (but not HSP-90) in resistant cells. The ER stress also induced a DNA damage response with an increase in pH2Ax in resistant cells and a subsequent upregulation of p53 protein levels. Moreover, the increase in p-IRE-1 induced the nuclear translocation of NFkB, where it could exert its pro-survival and transcriptional function. In fact, we also observed an increase in the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP, some of them known substrates of NFkB. Interestingly, these resistant cells had an increased sensitivity to Pifithrin, a Hsp-70 and p53 inhibitor (6 hours Pifithrin treatment induced 69% vs 26% apoptosis in resistant vs sensitive cells).
Conclusion
We have demonstrated some of the mechanisms associated with resistance to proteasome inhibitors: activation of the UPR (with subsequent upregulation of the chaperone system), DNA damage response and NFkB signaling with increased levels of antiapoptotic Bcl-2 family members. This work was supported by ISCIII-FIS PI15/00067 and /02156 and GRS 1175/A/15.
Session topic: E-poster
Keyword(s): Proteasome inhibitor, Resistance
Abstract: E1234
Type: Eposter Presentation
Background
MM is still considered incurable partly due to the development of resistance.
Aims
We have developed a cellular model of resistance to proteasome inhibitors to investigate the underlying mechanisms.
Methods
Cells sensitive to proteasome inhibitors (MM1S, MM1R, RPMI-8226) were continuously exposed to increasing concentrations of bortezomib until development of resistance. Viability was analyzed by MTT. Activity of different subunits of the proteasome was analyzed with Proteasome-Glo™ Assay Kit and presence of unfolded proteins with ProteoStat® Aggresome Detection Kit. Western-Blot, flow cytometry and qRT-PCR were also used.
Results
The development of acquired resistance to bortezomib in three cell lines was assessed by MTT assay with IC50 (nM) of 2.5 vs >100; 6.7 vs 78.5 and 21.2 vs >100 for the sensitive vs the resistant counterpart in MM1S, MM1R and RPMI at 24 hours. Interestingly, cells were also resistant to other proteasome inhibitors such as carfilzomib, oprozomib or ixazomib, indicating a class-effect resistance. In absence of bortezomib treatment, resistant cells presented a lower activity of the different subunits of the proteasome compared to the sensitive counterpart by 52% for caspase-like (C-L); 54% for trypsin-like (T-L) and 42% for chemotrypsin-like (CT-L) activities. This was even lower in case of MM1R (26%, 25% and 12%) and RPMI-8226 (21%, 40% and 23%) resistant cells. This inhibition led to an accumulation of unfolded proteins in the resistant MM1S cells as compared with their sensitive counterpart (96% vs 45%). Moreover, the concentration of bortezomib required to inhibit the proteasome was around 20-fold higher in the resistant than in the sensitive MM1S cells (x23; x21 and x27 for the C-L, T-L and CT-L activities). Finally, and most importantly, even with doses of bortezomib achieving a complete inhibition of the three subunits, resistant cells were viable, indicating they were not dependent on the proteasome and had developed alternative mechanisms of survival. In this context, resistant cells significantly upregulated the pro-survival chaperone BIP/GRP78, a critical sensor of the ER stress. This upregulation activated the three main arms of the unfolded protein response (UPR): increased levels of p-IRE1, ATF-6 and activation of PERK as observed through phosphorylation of its downstream substrate EIFα. A sequelae of the activation of the UPR is the transcriptional activation of chaperones, and, accordingly, there was increased expression of HSP-40 and, particularly, HSP-70 (but not HSP-90) in resistant cells. The ER stress also induced a DNA damage response with an increase in pH2Ax in resistant cells and a subsequent upregulation of p53 protein levels. Moreover, the increase in p-IRE-1 induced the nuclear translocation of NFkB, where it could exert its pro-survival and transcriptional function. In fact, we also observed an increase in the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP, some of them known substrates of NFkB. Interestingly, these resistant cells had an increased sensitivity to Pifithrin, a Hsp-70 and p53 inhibitor (6 hours Pifithrin treatment induced 69% vs 26% apoptosis in resistant vs sensitive cells).
Conclusion
We have demonstrated some of the mechanisms associated with resistance to proteasome inhibitors: activation of the UPR (with subsequent upregulation of the chaperone system), DNA damage response and NFkB signaling with increased levels of antiapoptotic Bcl-2 family members. This work was supported by ISCIII-FIS PI15/00067 and /02156 and GRS 1175/A/15.
Session topic: E-poster
Keyword(s): Proteasome inhibitor, Resistance
Type: Eposter Presentation
Background
MM is still considered incurable partly due to the development of resistance.
Aims
We have developed a cellular model of resistance to proteasome inhibitors to investigate the underlying mechanisms.
Methods
Cells sensitive to proteasome inhibitors (MM1S, MM1R, RPMI-8226) were continuously exposed to increasing concentrations of bortezomib until development of resistance. Viability was analyzed by MTT. Activity of different subunits of the proteasome was analyzed with Proteasome-Glo™ Assay Kit and presence of unfolded proteins with ProteoStat® Aggresome Detection Kit. Western-Blot, flow cytometry and qRT-PCR were also used.
Results
The development of acquired resistance to bortezomib in three cell lines was assessed by MTT assay with IC50 (nM) of 2.5 vs >100; 6.7 vs 78.5 and 21.2 vs >100 for the sensitive vs the resistant counterpart in MM1S, MM1R and RPMI at 24 hours. Interestingly, cells were also resistant to other proteasome inhibitors such as carfilzomib, oprozomib or ixazomib, indicating a class-effect resistance. In absence of bortezomib treatment, resistant cells presented a lower activity of the different subunits of the proteasome compared to the sensitive counterpart by 52% for caspase-like (C-L); 54% for trypsin-like (T-L) and 42% for chemotrypsin-like (CT-L) activities. This was even lower in case of MM1R (26%, 25% and 12%) and RPMI-8226 (21%, 40% and 23%) resistant cells. This inhibition led to an accumulation of unfolded proteins in the resistant MM1S cells as compared with their sensitive counterpart (96% vs 45%). Moreover, the concentration of bortezomib required to inhibit the proteasome was around 20-fold higher in the resistant than in the sensitive MM1S cells (x23; x21 and x27 for the C-L, T-L and CT-L activities). Finally, and most importantly, even with doses of bortezomib achieving a complete inhibition of the three subunits, resistant cells were viable, indicating they were not dependent on the proteasome and had developed alternative mechanisms of survival. In this context, resistant cells significantly upregulated the pro-survival chaperone BIP/GRP78, a critical sensor of the ER stress. This upregulation activated the three main arms of the unfolded protein response (UPR): increased levels of p-IRE1, ATF-6 and activation of PERK as observed through phosphorylation of its downstream substrate EIFα. A sequelae of the activation of the UPR is the transcriptional activation of chaperones, and, accordingly, there was increased expression of HSP-40 and, particularly, HSP-70 (but not HSP-90) in resistant cells. The ER stress also induced a DNA damage response with an increase in pH2Ax in resistant cells and a subsequent upregulation of p53 protein levels. Moreover, the increase in p-IRE-1 induced the nuclear translocation of NFkB, where it could exert its pro-survival and transcriptional function. In fact, we also observed an increase in the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP, some of them known substrates of NFkB. Interestingly, these resistant cells had an increased sensitivity to Pifithrin, a Hsp-70 and p53 inhibitor (6 hours Pifithrin treatment induced 69% vs 26% apoptosis in resistant vs sensitive cells).
Conclusion
We have demonstrated some of the mechanisms associated with resistance to proteasome inhibitors: activation of the UPR (with subsequent upregulation of the chaperone system), DNA damage response and NFkB signaling with increased levels of antiapoptotic Bcl-2 family members. This work was supported by ISCIII-FIS PI15/00067 and /02156 and GRS 1175/A/15.
Session topic: E-poster
Keyword(s): Proteasome inhibitor, Resistance
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