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 6

NAC reverses LLP in an MFSD12-dependent manner.

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NAC reverses LLP in an MFSD12-dependent manner. (A–C) A375P-galectin-3-G...
(A–C) A375P-galectin-3-GFP cells or A375P cells were treated with MFSD12 siRNA or nontarget siRNA (siNT) for 48 hours, followed by treatment with DMSO, 3 μM DC661, or 10 mM NAC for 24 hours, 6 hours, or 72 hours. (A) Quantification of galectin-3 puncta in A375P-galectin-3-GFP cells after 24 hours. Galectin-3–positive puncta are shown with white arrows. (B) Fluorescence images of A375P cells stained with FOAM-LPO (1 μM, 5 min) to detect LLP after 6 hours. (C) Trypan blue cell viability in A375P cells after 72 hours of treatment with DC661, NAC, or both. (D) Schematic of lysosomal pull down using Lyso-IP. (E) Relative quantification of metabolites in whole-cell lysates (WCL), lysosomal IP unbound fractions (UB), and lysosomal IP bound samples (Lyso IP) with NAC or vehicle treatment after 24 hours. Total peak area accounts for metabolite abundance in the entire sample. Quantifications are depicted as mean ± SD from 3 biological replicates per condition. Scale bar: 20 μm. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001. ANOVA test was used when more than 2 groups were compared.