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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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 7

N-acetyl cysteine prevents DC661-induced calreticulin surface expression.

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N-acetyl cysteine prevents DC661-induced calreticulin surface expression...
(A–D) Flow cytometry for calreticulin (CALR) and propidium iodide (PI), with quantification of 2–3 independent experiments. (A) Murine MC38 cells were treated with 3 μM DC661, 10 mM N-acetyl cysteine (NAC), or both for 24 hours. (B) MC38 cells were treated with siPpt11 or siNT for 48 hours in the presence or absence of 10 mM NAC for 24 hours. (C) Murine B16F10 cells were treated with 3 μM DC661, 10 mM NAC, or both for 24 hours. (D) MC38 cells were treated with cell death inhibitors (40 μM Z-VAD-FMK, 50 μM Nec-1, 2 μM Liprox-1) with and without 3 μM DC661 for 24 hours. (E) Immunoblots of MHC class I, PSMB9, PSMB8, and β-actin in the lysates of murine B16F10 and MC38 cells treated with indicated concentrations of DC661 for 6 and 24 hours. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. ANOVA test was used when more than 2 groups were compared.