Essential Drug Targets and Pathways in PI-Resistant MM Cells

Background

Proteasome inhibitors (PI) have emerged as a powerful, cell biology-based treatment option for multiple myeloma (MM) and build a central backbone for MM treatment with three proteasome-inhibiting drugs currently approved: bortezomib (BTZ), carfilzomib (CFZ) and ixazomib. However, despite the high anti-MM activity of PI, MM cells adapt to the selective pressure of PI treatment in most cases to date and most MM patients relapse during or after treatment with PI, develop PI-refractory disease and ultimately die. Therefore, understanding and overcoming PI resistance is a key challenge for MM therapy. Our previous in vitro studies on PI-resistant MM suggest that PI-adapted, MM cells show very distinct features of general metabolism and cell biology that differentiate them from PI-sensitive MM, derived from the same cell line. We hypothesize that this highly specialized and adapted nature of PI-resistant MM offers novel areas of vulnerability, that differ from the therapeutic targets in PI-sensitive MM. The aim of our study was to identify essential drug targets and pathways in PI-resistant MM using genome-wide functional screening with the CRISPR/Cas9 system that could serve as novel therapeutic targets in PI-resistant MM.

Methods

We used genome-wide CRISPR/Cas9-based loss-of-function screening with Brunello library in L363-BTZ and RPMI-8226-BTZ cells, adapted to grow in the presence of 90 nM BTZ. The overlapping bortezomib genetic sensitivity candidates were further validated in the set of BTZ-resistant cells (L363-BTZ, RPMI-8226-BTZ, MM1S-BTZ and AMO-BTZ) cells using shRNA silencing or single-gene specific knockout or genetic overexpression using CCK8 viability assay. Subsequent functional analysis of the highest ranking BTZ sensitivity candidates in BTZ-adapted cells included apoptosis and cell cycle analysis, qPCR and western blotting, SILAC, proteasome activity determination using activity-based probes and FRAP analysis.

Results

CRISPR/Cas9-screening identified two candidate genes for BTZ sensitivity, ECPAS (KIAA0368; Ecm29 Proteasome Adaptor and Scaffold protein) and PSME1 (an 11S regulator complex subunit), as consistent screening hits in two independent BTZ-adapted MM cell lines. Both genes are related to proteasome, but do not build the proteasome core particle and do not have a proteolytic activity. Specific knock-down or knock-out of ECPAS sensitized PI-naïve cells to BTZ and CFZ, while significantly more sensitizing BTZ-adapted cells to both PI. Likewise, overexpression of PSMF1, an inhibitor of 11S regulator complex, sensitized BTZ-resistant as well as sensitive cells to BTZ. ECPAS-depleted BTZ-adapted cells showed accumulation of poly-ubiquitinated proteasome substrate proteins, induction of the unfolded protein response, cell cycle arrest and induction of apoptosis, together with changes in protein synthesis after the treatment with 50 nM bortezomib, in contrast to BTZ-adapted control cells. FRAP analysis of cells with GFP-tagged PSMD6 revealed that the intracellular mobility of proteasomes in ECPAS-depleted cells was reduced. Importantly, proteasome activity determined by activity-based probes was not impaired in ECPAS-depleted cells.

Conclusion

In conclusion, BTZ-resistant MM cells uniquely show a high dependency on the proteasome adaptor and scaffold protein ECPAS, which has been shown to be involved in coupling of proteasome in different compartments and promotes proteasome dissociation under oxidative stress. Specifically in PI-resistant MM, ECPAS is important to ensure functional proteasome, is involved in controlling the intracellular mobility of proteasomes, likely to ensure high proteasome turnover. ECPAS therefore represents a novel candidate that may be targeted to specifically re-sensitize PI-resistant MM cells to proteasome inhibitor treatment.