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Ferroptosis Induction in Multiple Myeloma Cells Triggers an Epigenomic Stress Response Associated with DNA Repair and Cellular Senescence

ABSTRACT: Acquired and primary therapy resistance remain a major hurdle in clinical treatment of multiple myeloma (MM). Despite the availability of broad spectrum anti-cancer drugs, most MM patients eventually relapse and become refractory to current treatments. Therefore, we explored whether therapy-resistant MM cells are sensitive to ferroptosis induction, an iron-catalyzed mode of cell death associated with increased lipid peroxidation. In this study, we exposed glucocorticoid-resistant MM1R and glucocorticoid-sensitive MM1S cells to ferroptosis inducers RSL3 and evaluated their transcriptional changes via RNA sequencing. Compared to untreated controls, ferroptotic RSL3-treated cells displayed a significant upregulation of genes involved in inflammation, kinase signaling, cellular stress, and cell death pathways. Pre-treatment with ferroptosis inhibitor Fer-1 partly reverted these RSL3-induced transcriptional changes and revealed that especially genes involved in metal binding, nuclear receptor signaling, chromatin remodeling, and gene transcription regulation are pivotal for ferroptosis induction. Overall, these findings suggest that ferroptosis effectively targets MM1 cells and that RSL3-mediated ferroptosis triggers similar oxidative stress and cell death pathways in both MM1R and MM1S cells, irrespective of their glucocorticoid-sensitivity status.

ORGANISM(S): Homo sapiens

PROVIDER: GSE182638 | GEO | 2021/11/08

REPOSITORIES: GEO

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Project description:Ferroptosis is a form of regulated cell death characterized by iron-dependent overaccumulation of lipid peroxides. It can be prevented by cellular mechanisms such as GPX4-mediated elimination of the lipid peroxides at the cost of glutathione (GSH). Since originally being discovered as a property of RAS-mutant cancer cells, ferroptosis has been shown to be regulated by several important oncogenes and tumor suppressors. In particular, LIFR, the receptor of the pleiotropic cytokine LIF, has recently been shown to be required for ferroptosis, as loss of Lifr in mouse liver tumor confers the cells resistance to ferroptosis. In this study, however, we found that the LIFR-targeting drugs, EC330 and EC359, are potent inducers of ferroptosis, thus reflecting an interesting “gain-of-function” effect of EC330/EC359 on LIFR-associated ferroptosis. Treatment of various types of cells with EC330/EC359 causes a rapid nonapoptotic cell death with characteristic morphology of ferroptosis, which can be inhibited by iron chelator (DFO) and lipid ROS scavenger (Fer-1), but not pan-caspase inhibitor (z-VAD-fmk). Consistent with previous observations, the efficiency of EC330/EC359 appears to be correlated with the expression levels of LIF and LIFR. RNA-seq analysis showed that the EC330/EC359 treatment leads to (i) a gene expression profile highly resembling that of RSL3, the GPX4-targeting ferroptosis inducer, and (ii) a specific decrease of expression of a few gene encoding membrane-located proteins, especially those located on the mitochondria (e.g., TOMM6 and COX14), implying defects in cellular/mitochondrial membrane functions. Indeed, transmission electron microscopy imaging of the EC330/EC359-treated cells shows shrunken mitochondria and loss of mitochondrial cristae. EC330/EC359 also decreases the activity of GPX4 in the cells to the level comparable with RSL3; meanwhile, the intracellular level of GSH is also decreased, and thus GPX4 inactivation and GSH depletion may jointly disable the cells to eliminate the toxic lipid peroxides. Lastly, although the STAT3 and AKT signaling pathways downstream of LIFR are inhibited by EC330/EC359, inhibition of these pathways per se can only induce non-ferroptotic cell death, suggesting that the EC330/EC359-induced ferroptosis is independent of these downstream pathways; the LIFR-NFkB-LCN2 axis discovered recently in mouse liver tumor cells might not explain the EC330/EC359-induced ferroptosis either, because LCN2 is not detectable in the herein used cell lines. Taken together, these results reveal an unexpected role of the LIFR-targeting drugs EC330/EC359 as potent inducer of ferroptosis and, in combination with previous studies, suggest that there could be either unknown mechanism for the LIFR-associated ferroptosis or unknown target for EC330/EC359 to effect the ferroptosis induction.

Project description:Ferroptosis is a form of regulated cell death driven by lipid peroxidation of polyunsaturated fatty acids (PUFAs). Endogenous PUFAs are nonconjugated PUFAs and their peroxidation proceeds via the hydrogen-atom transfer (HAT) mechanism. We previously reported that lipids with conjugated double bonds undergo lipid peroxidation mostly via a different mechanism, peroxyl radical addition (PRA), and were much more readily oxidizable than nonconjugated ones. In this study, we aim to elucidate the effects of various unsaturated lipids in sensitizing ferroptosis. We found that while some peroxidation-reactive lipids, such as 7-dehydrocholesterol, vitamins D3 and A, and coenzyme Q10, suppress ferroptosis, both nonconjugated and conjugated PUFAs enhanced cell death induced by RSL3, a ferroptosis inducer. Importantly, we showed that conjugated linolenic acid (CLA 18:3) could act as a ferroptosis inducer by itself and conjugated linoleic acid (CLA 18:2) was more potent in sensitizing cells to RSL3-induced cell death than any nonconjugated PUFAs. We next sought to elucidate the mechanism underlying the different ferroptosis-inducing potency of conjugated and nonconjugated PUFAs. Lipidomics revealed that conjugated and nonconjugated PUFAs are incorporated into distinct cellular lipid species. Furthermore, the different peroxidation mechanisms predict the formation of higher levels of reactive electrophilic aldehydes from conjugated PUFAs than nonconjugated PUFAs, which was confirmed by aldehyde-trapping and mass spectrometry. RNA sequencing revealed that protein processing in the endoplasmic reticulum and proteasome are among the most significantly upregulated pathways in cells treated with CLA 18:3, suggesting increased ER stress and activation of unfolded protein response. Significantly, using click chemistry, we observed increased protein adduction by oxidized lipids in cells treated with an alkynylated CLA 18:2 probe. These results suggest that protein damage by lipid electrophiles is a key step in ferroptosis.