Postdoctoral Research Fellow Brigham and Women's Hospital/Harvard Medical School Boston, Massachusetts, United States
Introduction: Multiple myeloma (MM) is the second most common hematological malignancy in the world and remains largely incurable. We previously demonstrated that MM cells are dependent on intact proteostasis mechanisms and are specifically sensitive to perturbations of this balance. For instance, proteasome inhibitors (PI) such as bortezomib, carfilzomib and ixazomib are part of the treatment of MM across all stages of disease. However, acquired resistance is inevitable for most patients, leading to shorter remissions and eventual patient demise. New therapeutic strategies to overcome resistance are therefore urgently needed. We recently showed that the aspartic protease DDI2 is necessary for MM cell survival due to its critical role in activating the proteasome stress response (PSR) master-regulator NRF1, leading to de novo proteasome subunit transcription. We hypothesized that targeting DDI2 is a novel therapeutic strategy in MM. 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 compounds. We herein set out to develop MM reporter cell lines to conduct high-throughput repurposing screening of FDA-approved small molecules.
Methods: We generated a reporter construct comprising 8 repeats of 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, the same construct will lead to expression of mCherry under an EF1α promotor. We transduced MM cell lines with varying baseline sensitivities to PI with this construct. We also used AMO1-VR, but DDI2 WT and DDI2 KO, as a model of acquired bortezomib resistance with intact or impaired PSR. To induce PSR, we pulse treated cells with a sublethal dose of carfilzomib as previously described (Chen et al, Blood Adv 2022) 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 pulse treatment with carfilzomib, consistent with our prior data showing peak of proteasome subunit transcription after PI treatment around 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 of de novo proteasome subunit transcription.
Conclusions: Our data showed that 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.
