Abstract 2974: Bortezomib resistant multiple myeloma shows a specific dependency on Ecm29 in vitro and in vivo
Authors: Andrej Besse, Lenka Besse, Sara C. Stolze, Marianne Kraus, Ondrej Slaby, Hirofumi Nakagami, Christoph Driessen
Journal: Cancer Research (2025)

Abstract Background: Resistance to proteasome inhibitors (PI) bortezomib (BTZ) is a major obstacle to the successful treatment of multiple myeloma (MM). Thus, the identification of a specific target in PI-resistant MM is an unmet medical need. The highly specialized and adapted nature of PI-resistant MM may offer novel areas of vulnerability. Using genome-wide CRISPR/Cas9-based functional screening in BTZ-adapted cell lines in vitro we identified the proteasome adaptor and scaffold protein Ecm29 as an essential survival factor for BTZ-adapted cells under BTZ treatment. We aimed to characterize its function in myeloma in vitro and in vivo. Methods: Genome-wide CRISPR/Cas9 screening was used in BTZ-resistant cells to identify overlapping BTZ sensitivity candidates, which were further validated in a set of four BTZ-resistant cells. Viability was determined by CCK8 viability assay. Cell cycle, apoptosis induction, and aggresome formation after BTZ treatment were determined by flow cytometry. NGS, LC-MS/MS, and SILAC were used to determine major changes in Ecm29 knock-down (KD) or knock-out (KO) cells. RT-PCR and westernblot were used to determine unfolded protein response. Two in vivo orthotopic mouse models were used to determine the effect of Ecm29 on BTZ resistance. Results: Ecm29 was identified as a consistent candidate for BTZ sensitivity in two independent screens with BTZ-adapted MM cell lines exposed to BTZ. Validation with specific Ecm29 KD or KO sensitized BTZ-adapted cells to BTZ, in contrast to BTZ-naïve parental cells. Pull-down of proteins bound to Ecm29 in BTZ-adapted cells, in contrast to BTZ-naïve cells, showed significant changes, related to Ecm29 and proteasome localization towards the cytoskeleton and vacuolar compartment. Following, Ecm29 silencing induced changes in the cytoskeleton and vesicular trafficking, decreased protein synthesis and diminished the accumulation of poly-ubiquitinated protein. BTZ treatment caused accumulation of poly-ubiquitinated proteasome substrate proteins, induction of the unfolded protein response, induction of chaperones and aggresome formation, G2-phase cell cycle arrest, and apoptosis in Ecm29-silenced cells, in contrast to BTZ-adapted control cells. Ecm29 depletion significantly reduced the growth of two independent BTZ-adapted cells in vivo in murine bone marrow, following BTZ treatment. Conclusion: The proteasome adaptor and scaffold protein Ecm29 controls proteasome trafficking and localization. It is of central importance in BTZ-adapted MM cells undergoing BTZ treatment due to dependance on different proteasome localization. Ecm29 may therefore represent a novel therapeutic target to specifically re-sensitize PI-resistant MM cells to PI treatment. Support: Supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Andrej Besse, Lenka Besse, Sara C. Stolze, Marianne Kraus, Ondrej Slaby, Hirofumi Nakagami, Christoph Driessen. Bortezomib resistant multiple myeloma shows a specific dependency on Ecm29 in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2974.