Abstract A126: Characterization of proteasome stress response reporter mammalian cell lines for the discovery of DDI2 inhibitors in multiple myeloma

Abstract

Abstract Background The goal of this study was to develop and characterize proteasome stress response (PSR) reporter cell lines to aid in the identification of inhibitors of the aspartic protease DDI2. We recently showed that multiple myeloma (MM) is dependent on DDI2 for survival due to its critical role in activating PSR master-regulator NRF1. Cleaved NRF1 induces de novo proteasome subunit transcription in face of proteasome insufficiency, thus maintaining proteostasis and mediating resistance to proteasome inhibitors (PI), a mainstay of MM therapy. We hypothesize that targeting DDI2 represent an unexplored, attractive therapeutic strategy in MM, able to overcome acquired and innate resistant to PI, a major clinical hurdle in the care of MM patients. We further hypothesize that due to intrinsic proteostasis vulnerability in MM, DDI2 inhibitors will have a favorable therapeutic index in the clinics. However, specific DDI2 inhibitors are not available and mammalian reporter cell lines of PSR activation do not exist, limiting our capabilities to successfully screen for new drugs. Methods We generated a reporter construct comprising 8 repeats of NRF1 DNA binding site, antioxidant response elements (ARE), upstream of a minimal promoter followed by destabilized GFP. To monitor for successful transfection, copy number integration and global changes in transcription, we cloned mCherry under an EF1α promotor within the same vector. We used this lentiviral construct to transduce MM cell lines characterized by distinct sensitivity to PIs, including AMO1-VR, as a model of acquired bortezomib resistance, and DDI2 KO AMO1-VR to assess for DDI2-independent PSR suppression. To induce PSR, we pulse treated cells with a sublethal dose of carfilzomib and monitored changes in the ratio of GFP/mCherry intensity over time via flow cytometry and immunofluorescence. Results We found that the reporter cell lines showed a peak of GFP/mCherry intensity 8-12 hours after carfilzomib treatment, consistent with our prior data showing peak of proteasome subunit transcription at 10 hours. Importantly, the GFP/mCherry ratio returned to baseline after approximately 24 hours from treatment, in the absence of significant cell apoptosis. DDI2 KO AMO1-VR showed significantly blunted peak of GFP/mCherry intensity, consistent with reduced capability to induce proteasome subunit transcription. We hypothesize that redundant transcription factors, such as NRF2 are responsible for the blunted PSR observed, consistent with increased expression of NRF2 in DDI2 KO cells. Importantly, treatment with sublethal doses of genetoxic drug doxorubicin did not induce significant activation of reporter construct.   Conclusions We have successfully developed the first mammalian reporter cell line of PSR with preliminary data showing adequate delta and kinetics to assist in pharmacological screening of molecules blocking the PSR and inducing apoptosis in MM. This powerful tool will inform screening and subsequent validation of newly found/synthesized DDI2 inhibitors, currently under development. Citation Format: Cameron VanCleave, Tianzeng Chen, Matthew Ho, Maria Moscvin, Peter G Czarnecki, Giada Bianchi. Characterization of proteasome stress response reporter mammalian cell lines for the discovery of DDI2 inhibitors in multiple myeloma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A126.