Samenvatting
The discovery of agents like bortezomib, has significantly improved multiple myeloma (MM) patients outcome. Despite advances in treatment, MM is still incurable due to the development of resistance. To cure this disease we need to optimize current treatment strategies and identify new therapeutical targets.
Inhibitor of apoptosis (IAP) proteins block apoptosis induced by a variety of apoptotic triggers. Alterations in these proteins are frequently found in different human cancers and are associated with drug resistance, disease progression and poor prognosis. IAPs contain baculovirus IAP repeat (BIR) motifs to bind and inhibit caspases resulting in blocking of apoptosis. IAPs can also inhibit apoptosis by the ability of cIAP1, cIAP2 and X-linked IAP to regulate activation of nuclear factor-kB (NF-kB).
NF-kB signalling is constitutively activated in MM and promotes cell survival and proliferation. Two distinct NF-alphaB activation pathways have been defined, the canonical and the noncanonical. Genetic lesions in the noncanonical pathway leading to uncontrolled NF-alphaB activation are appearing in approximately 20% of MM patients. Loss of functional TRAF3 is the most commonly occurring gene deleted/mutated in MM. In normal cells, TRAF3 binds directly to NIK and recruits it to a complex with Ub-ligase activity consisting of TRAF2, cIAP1 and/or cIAP2 leading to NIK degradation. Deletion of TRAF3 results in NIK accumulation and activation of noncanonical NF-kB.
The aim of this study was to investigate the role of cIAP2 in the response of MM cells to bortezomib. We focused on TRAF3 mutated human MM cell lines to exclude effects of cIAP2 expression on the noncanonical pathway. TRAF-3 mutated MM cell lines LP-1 and ANBL-6 were stably transduced with a lentiviral vector containing a cIAP2-eGFP cassette. Expression of cIAP2 was evaluated by quantitative RT-PCR and western blotting and compared to cells transduced with a lentiviral vector containing an empty-eGFP cassette (control). cIAP2 expression was significantly increased both on mRNA and protein level. Cells were exposed to different concentrations of bortezomib; those overexpressing cIAP2 were less sensitive to bortezomib compared to control. MG132, another proteasome inhibitor, was also tested and gave similar results.
Next we examined the effect of cIAP2 on induction of apoptosis when MM cells were treated with bortezomib. In cells overexpressing cIAP2, apoptotic cells were decreased compared to control cells. We continued to examine the effect of cIAP2 on caspase activation. We found that cells overexpressing cIAP2 had less activation of caspase-3, -8 and -9 compared to control cells when treated with bortezomib, confirming our results on induction of apoptosis.
To investigate if cIAP2 is a potential target for MM, we combined the IAP inhibitor AT-406 with bortezomib. Western blot analysis showed that AT-406 (1 and 10 µM) downregulated the expression of cIAP2 protein after 4 hours. We pre-treated the LP-1 cell line for 4h with 1 and 10 µM AT-406 followed by bortezomib treatment for 24h. When we combine bortezomib with AT-406 we see a significant reduction in viability compared to single treatment of bortezomib.
We can conclude that overexpression of cIAP2 in LP-1 and ANBL-6 MM cells protects the cells from bortezomib treatment. Furthermore, inhibition of IAPs results in a better sensitivity to bortezomib. Our study provides the rationale for using the combination of bortezomib and the IAP-inhibitor AT-406, to improve the anti-myeloma effect.
Inhibitor of apoptosis (IAP) proteins block apoptosis induced by a variety of apoptotic triggers. Alterations in these proteins are frequently found in different human cancers and are associated with drug resistance, disease progression and poor prognosis. IAPs contain baculovirus IAP repeat (BIR) motifs to bind and inhibit caspases resulting in blocking of apoptosis. IAPs can also inhibit apoptosis by the ability of cIAP1, cIAP2 and X-linked IAP to regulate activation of nuclear factor-kB (NF-kB).
NF-kB signalling is constitutively activated in MM and promotes cell survival and proliferation. Two distinct NF-alphaB activation pathways have been defined, the canonical and the noncanonical. Genetic lesions in the noncanonical pathway leading to uncontrolled NF-alphaB activation are appearing in approximately 20% of MM patients. Loss of functional TRAF3 is the most commonly occurring gene deleted/mutated in MM. In normal cells, TRAF3 binds directly to NIK and recruits it to a complex with Ub-ligase activity consisting of TRAF2, cIAP1 and/or cIAP2 leading to NIK degradation. Deletion of TRAF3 results in NIK accumulation and activation of noncanonical NF-kB.
The aim of this study was to investigate the role of cIAP2 in the response of MM cells to bortezomib. We focused on TRAF3 mutated human MM cell lines to exclude effects of cIAP2 expression on the noncanonical pathway. TRAF-3 mutated MM cell lines LP-1 and ANBL-6 were stably transduced with a lentiviral vector containing a cIAP2-eGFP cassette. Expression of cIAP2 was evaluated by quantitative RT-PCR and western blotting and compared to cells transduced with a lentiviral vector containing an empty-eGFP cassette (control). cIAP2 expression was significantly increased both on mRNA and protein level. Cells were exposed to different concentrations of bortezomib; those overexpressing cIAP2 were less sensitive to bortezomib compared to control. MG132, another proteasome inhibitor, was also tested and gave similar results.
Next we examined the effect of cIAP2 on induction of apoptosis when MM cells were treated with bortezomib. In cells overexpressing cIAP2, apoptotic cells were decreased compared to control cells. We continued to examine the effect of cIAP2 on caspase activation. We found that cells overexpressing cIAP2 had less activation of caspase-3, -8 and -9 compared to control cells when treated with bortezomib, confirming our results on induction of apoptosis.
To investigate if cIAP2 is a potential target for MM, we combined the IAP inhibitor AT-406 with bortezomib. Western blot analysis showed that AT-406 (1 and 10 µM) downregulated the expression of cIAP2 protein after 4 hours. We pre-treated the LP-1 cell line for 4h with 1 and 10 µM AT-406 followed by bortezomib treatment for 24h. When we combine bortezomib with AT-406 we see a significant reduction in viability compared to single treatment of bortezomib.
We can conclude that overexpression of cIAP2 in LP-1 and ANBL-6 MM cells protects the cells from bortezomib treatment. Furthermore, inhibition of IAPs results in a better sensitivity to bortezomib. Our study provides the rationale for using the combination of bortezomib and the IAP-inhibitor AT-406, to improve the anti-myeloma effect.