Bortezomib Resistant Multiple Myeloma Shows a Specific Dependency on Ecpas in Vitro and In Vivo

Background Resistance to proteasome inhibitors bortezomib (BTZ) and carfilzomib (CFZ) is a major obstacle to the successful treatment of multiple myeloma (MM). Thus, understanding and overcoming proteasome inhibitor resistance is a key challenge for MM therapy and identification of a specific target in proteasome inhibitor-resistant MM is an unmet need. Previous in vitro studies on proteasome inhibitor-resistant MM cells suggest that proteasome inhibitor-adapted MM cells show very distinct biological features that differentiate them from the sensitive MM, derived from the same cell line. We hypothesize that this highly specialized and adapted nature of proteasome inhibizor-resistant MM offers novel areas of vulnerability, that differ from the therapeutic targets in proteasome inhibitor-sensitive MM. Using genome-wide CRISPR/Cas9-based functional screening in several BTZ-adapted cells in vitro we identified the proteasome adaptor and scaffold protein Ecm29 (ECPAS) as a specific survival dependency of the BTZ-adapted cells under BTZ treatment. We aimed to characterize its function in myeloma in vitro and in vivo.

Methods Genome-wide CRISPR/Cas9-based loss-of-function screening with Brunello library was used in two BTZ-resistant cells to identify overlapping BTZ sensitivity candidates, which were further validated in the set of four BTZ-resistant cells (L363-BTZ, RPMI-8226-BTZ, MM1S-BTZ and AMO-BTZ). Viability after shRNA silencing or single-gene specific knockout of ECPAS was determined by CCK8 viability assay. Cell cycle, apoptosis induction and aggresome formation after BTZ treatment of ECPAS-depleted cell was determined by flow cytometry. NGS sequencing, LC/MS-MS and SILAC was used to determine major changes in ECPAS-depleted cells in standard culture condition or upon BTZ treatment. Two in vivo mouse models, based on orthotopic injection of AMO-BTZ cells into murine femur and systemic injection of MM1S-BTZ cells into murine tail-vein (cells with or without ECPAS), were used to determine the effect of ECPAS on BTZ resistance in vivo. Monitoring of tumor progression in vivo was done by Luciferin-based imaging for a period of 4 weeks of treatment with subcutaneous BTZ twice weekly.

Results ECPAS was identified as a consistent screening hit and sensitivity candidate in two independent BTZ-adapted MM cell lines exposed to BTZ. Specific knock-down or knock-out of ECPAS sensitized PI-naïve cells to BTZ and CFZ, while significantly more sensitized the BTZ-adapted cells to both proteasome inhibitors. In the untreated state, ECPAS-depleted cells showed changes in the expression of genes related to cytoskeleton and vesicular trafficking, decreased protein synthesis and lower accumulation of polyubiquitinated protein. ECPAS-depleted BTZ-adapted MM cells showed more significant accumulation of poly-ubiquitinated proteasome substrate proteins, induction of the unfolded protein response, induction of chaperons and aggresome formation together with cell cycle arrest and induction of apoptosis after the treatment with BTZ, in contrast to BTZ-adapted control cells. Importantly, the activity of proteasome β-subunits and their inhibition by BTZ was not impaired in ECPAS-depleted cells. Analysis of the growth of two BTZ-adapted cells in vivo in the murine bone marrow showed that ECPAS depletion significantly reduced growth of BTZ-adapted cells under BTZ treatment in both in vivo models.

Conclusion In conclusion, under BTZ treatment, BTZ-resistant MM cells show a unique high in vitro and in vivo dependency on the proteasome adaptor and scaffold protein ECPAS, which couples the proteasomes in different compartments and controls stability of 26S proteasome under different conditions. ECPAS deficiency in BTZ-resistant cells significantly stimulates the functional effect of BTZ on BTZ-adapted MM cells. Thus, ECPAS represents a novel therapeutic candidate that may be used to specifically re-sensitize proteasome inhibitor-resistant MM cells to proteasome inhibitor treatment.

No relevant conflicts of interest to declare.