Lysosomal lipid peroxidation regulates tumor immunity
Monika Bhardwaj, … , David W. Speicher, Ravi K. Amaravadi
Monika Bhardwaj, … , David W. Speicher, Ravi K. Amaravadi
Published February 16, 2023
Citation Information: J Clin Invest. 2023;133(8):e164596. https://doi.org/10.1172/JCI164596.
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Research Article Oncology

Lysosomal lipid peroxidation regulates tumor immunity

Abstract

Lysosomal inhibition elicited by palmitoyl-protein thioesterase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism for this is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC) but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12 was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naive T cells and enhanced T cell–mediated toxicity. Mice vaccinated with DC661-treated cells engendered adaptive immunity and tumor rejection in “immune hot” tumors but not in “immune cold” tumors. These findings demonstrate that LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

Authors

Monika Bhardwaj, Jennifer J. Lee, Amanda M. Versace, Sandra L. Harper, Aaron R. Goldman, Mary Ann S. Crissey, Vaibhav Jain, Mahendra Pal Singh, Megane Vernon, Andrew E. Aplin, Seokwoo Lee, Masao Morita, Jeffrey D. Winkler, Qin Liu, David W. Speicher, Ravi K. Amaravadi

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Figure 3

DC661-induced ferroptosis and pyroptosis.

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DC661-induced ferroptosis and pyroptosis. (A) qRT-PCR showed the fold ch...
(A) qRT-PCR showed the fold change increase in the transcriptional expression of PTGS2, CHAC, and CARS in A375P cells treated with 3 μM DC661 for 24 hours. (B) A375P cells treated for 24 hours with DC661 (3 μM), liproxstatin-1 (Liprox-1, 2 μM), or ferrostatin-1 (Ferro-1, 10 μM). Lipid peroxidation measured by C-11 BODIPY using flow cytometry. Erastin was used as positive control (see Supplemental Figure 3A). (C–E) Trypan blue cell viability assay in A375P cells treated with 3 μM DC661, with and without ferroptosis inhibitors (C) ferrostatin-1 (Ferro-1, 10 μM), (D) liproxstatin-1 (Liprox-1, 2 μM), and (E) iron chelator deferoxamine (DFO, 5 μM). (F) Western blots were probed for pyroptosis and autophagy proteins in the whole-cell lysates and HMGB1 release in cell supernatant of human WM35 empty vector (EV) and gasdermin-E–KO (KO1 and KO2) cells treated with DC661 1 μM for 48 hours. (G) Bar graph showing average DC661 IC50 values ± SEM of MTT assays in both 10% and 1% FBS conditions in mouse YUMM1.7 WT, EV, and gasdermin-E–KO (Gsdme-KO) cells from 3 independent experiments. Statistical analysis for I was applied on ΔCT values. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Two-tailed unpaired t test between 2 groups (A). ANOVA test was used when more than 2 groups were compared (B–E and G). See also Supplemental Figures 3–5. All viability assays were performed in triplicate.