Contributions
Abstract: PB1630
Type: Publication Only
Session title: Myeloma and other monoclonal gammopathies - Biology & Translational Research
Background
The development of proteasome inhibitors (PI) – Bortezomib (BTZ), Carfilzomib (CFZ), Ixazomib (IXA) - for the treatment of multiple myeloma (MM) has dramatically increased treatment responses and improved patient outcomes. However, the acquisition of resistance is a major limitation in PI therapy. Many mechanisms have been proposed for PI resistance, including genetic mutations in proteasome subunits, alterations in drug efflux transporters and antioxidant response pathways, among others.
Aims
In this study, we investigate the molecular mechanisms of PI resistance and the existence of cross-resistance between the three PI. Additionally, we explore potential strategies to overcome PI resistance.
Methods
Three PI-resistant MM cell lines were established by chronic exposure of PI-sensitive cells (NCI-H929) to non-lethal doses of BTZ (H929-BTZ cells), CFZ (H929-CFZ cells) and IXA (H929-IXA cells). The validation of the PI resistant models, the evaluation of the potential cross-resistance between PIs and the efficacy of the immunomodulatory drugs (IMiDs), lenalidomide (LEN) and pomalidomide (POM), were assessed using the fluorometric microculture cytotoxicity assay (FMCA). Expression and activity of ATP Binding Cassette (ABC) family efflux transporters (P-glycoprotein; multidrug resistance protein 1 (MRP1) and MRP2; breast cancer resistance protein (BCRP)) were analysed by flow cytometry. Gene expression analysis (Nuclear Factor, Erythroid 2 Like 2 (NFE2L2; Kelch Like ECH Associated Protein 1 (KEAP1), Nuclear Factor Kappa B Subunit 1 (NFKB1); ABC Subfamily B Member 1 (ABCB1); ABC Subfamily G Member 2 (ABCG2); ABC Subfamily C Member 1 (ABCC1) and ABCC2) was performed by qPCR assay. A p-value of 0,05 was used as cut-off for significance.
Results
The confirmation of the resistance of the three PI-resistant MM cell lines was shown by the increase of the half maximal inhibitory concentration (IC50) by 4.7 folds (H929-BTZ IC50 2.8nM), 6.7 folds (H929-CFZ IC50 2.0nM) and 10.5 folds (H929-IXA IC50 20nM) compared to the IC50 value of sensitive cells (IC50 BTZ 0.6nM, IC50 CFZ 0.3nM and IC50 IXA 1.9Nm, respectively). BTZ-resistant cells displayed cross-resistance to CFZ; however, cells resistant to these two PI did not display cross-resistance to IXA (H929-BTZ IC50 IXA 0.5nM; H929-CFZ IC50 IXA 1.9nM). H929-CFZ showed an increased expression of ABCB1 and ABCG2 genes. There were not statistically significant differences in the expression levels of the other evaluated genes (NFE2L2, KEAP1, NFKB1, ABCC1, ABCC2) in PI-resistant cell lines compared to PI-sensitive cell lines. Expression levels of protein efflux transporters were also similar between all cell lines; however, the activity of MRP1/2 in all PI-resistant cell lines was significantly higher than in sensitive cells. The association of IMiDs with PI was effective to overcome PI resistance.
Conclusion
Our results suggest that PI resistance may be mediated by an increased activity of MRP1/2. The absence of cross-resistance between IXA and the other two PI suggest that IXA can be effective even in the presence of resistance to BTZ and/or to CFZ. The association of IMiDs with PI appears effective in overcoming PI resistance. Additional studies are required for a better understanding of the mechanisms of PI-resistance in MM, in order to improve novel therapeutic strategies for refractory and relapsed patients.
Keyword(s): Multiple myeloma, Proteasome inhibitor, Resistance
Abstract: PB1630
Type: Publication Only
Session title: Myeloma and other monoclonal gammopathies - Biology & Translational Research
Background
The development of proteasome inhibitors (PI) – Bortezomib (BTZ), Carfilzomib (CFZ), Ixazomib (IXA) - for the treatment of multiple myeloma (MM) has dramatically increased treatment responses and improved patient outcomes. However, the acquisition of resistance is a major limitation in PI therapy. Many mechanisms have been proposed for PI resistance, including genetic mutations in proteasome subunits, alterations in drug efflux transporters and antioxidant response pathways, among others.
Aims
In this study, we investigate the molecular mechanisms of PI resistance and the existence of cross-resistance between the three PI. Additionally, we explore potential strategies to overcome PI resistance.
Methods
Three PI-resistant MM cell lines were established by chronic exposure of PI-sensitive cells (NCI-H929) to non-lethal doses of BTZ (H929-BTZ cells), CFZ (H929-CFZ cells) and IXA (H929-IXA cells). The validation of the PI resistant models, the evaluation of the potential cross-resistance between PIs and the efficacy of the immunomodulatory drugs (IMiDs), lenalidomide (LEN) and pomalidomide (POM), were assessed using the fluorometric microculture cytotoxicity assay (FMCA). Expression and activity of ATP Binding Cassette (ABC) family efflux transporters (P-glycoprotein; multidrug resistance protein 1 (MRP1) and MRP2; breast cancer resistance protein (BCRP)) were analysed by flow cytometry. Gene expression analysis (Nuclear Factor, Erythroid 2 Like 2 (NFE2L2; Kelch Like ECH Associated Protein 1 (KEAP1), Nuclear Factor Kappa B Subunit 1 (NFKB1); ABC Subfamily B Member 1 (ABCB1); ABC Subfamily G Member 2 (ABCG2); ABC Subfamily C Member 1 (ABCC1) and ABCC2) was performed by qPCR assay. A p-value of 0,05 was used as cut-off for significance.
Results
The confirmation of the resistance of the three PI-resistant MM cell lines was shown by the increase of the half maximal inhibitory concentration (IC50) by 4.7 folds (H929-BTZ IC50 2.8nM), 6.7 folds (H929-CFZ IC50 2.0nM) and 10.5 folds (H929-IXA IC50 20nM) compared to the IC50 value of sensitive cells (IC50 BTZ 0.6nM, IC50 CFZ 0.3nM and IC50 IXA 1.9Nm, respectively). BTZ-resistant cells displayed cross-resistance to CFZ; however, cells resistant to these two PI did not display cross-resistance to IXA (H929-BTZ IC50 IXA 0.5nM; H929-CFZ IC50 IXA 1.9nM). H929-CFZ showed an increased expression of ABCB1 and ABCG2 genes. There were not statistically significant differences in the expression levels of the other evaluated genes (NFE2L2, KEAP1, NFKB1, ABCC1, ABCC2) in PI-resistant cell lines compared to PI-sensitive cell lines. Expression levels of protein efflux transporters were also similar between all cell lines; however, the activity of MRP1/2 in all PI-resistant cell lines was significantly higher than in sensitive cells. The association of IMiDs with PI was effective to overcome PI resistance.
Conclusion
Our results suggest that PI resistance may be mediated by an increased activity of MRP1/2. The absence of cross-resistance between IXA and the other two PI suggest that IXA can be effective even in the presence of resistance to BTZ and/or to CFZ. The association of IMiDs with PI appears effective in overcoming PI resistance. Additional studies are required for a better understanding of the mechanisms of PI-resistance in MM, in order to improve novel therapeutic strategies for refractory and relapsed patients.
Keyword(s): Multiple myeloma, Proteasome inhibitor, Resistance