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 2

DC661-induced apoptosis and necroptosis.

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DC661-induced apoptosis and necroptosis. (A) Immunoblots of cleaved casp...
(A) Immunoblots of cleaved caspase-3 (Cl. C-3), caspase-7 (Cl. C-7), caspase-9 (Cl. C-9), PARP and β-actin from A375P cell lysates treated with indicated concentrations of DC661 for 24 hours. Staurosporine (ST; 20 ng/mL) was used as a positive control for apoptosis. (B) Immunoblots of lysates from A375P cells treated with 3 μM DC661, 80 μM pan-caspase inhibitor Z-VAD-FMK, or both for 24 hours. (C) Seventy-two-hour MTT assay plot with increasing concentrations of DC661 (0.01 to 10 μM), with and without Z-VAD-FMK 80 μM. (D) Seven-day colony formation assay in A375P cells treated with 0.3 μM DC661, 8 μM Z-VAD-FMK, or their combinations. (E) Trypan blue viability assay with and without 3 μM DC661 for 24 hours in FL5.12 and IL-3–dependent Bax−/−Bak−/− (BB-DKO) primary bone marrow cells. (F) Immunoblots of RIP1, MLKL, their phosphorylated forms, and β-actin in the lysates of A375P cells treated with DC661 for 24 hours. Necroptosis conventional TSZ (TNF-α, Smac mimetic [SM-164], and Z-VAD-FMK) treatment conditions used included the following: C, pretreatment with Z-VAD-FMK (25 μM, 1 hour), followed by SM-164 (2 μM, 1 hour) and TNF-α (20 ng/mL, 22 hours); D, pretreatment with Z-VAD-FMK (80 μM, 1 hour), followed by SM-164 (100 nM, 1 hour) and TNF-α (20 ng/mL, 22 hours). (G) Immunoblots of necroptosis proteins in lysates of A375P cells treated with necroptosis inhibitors necrostatin-1s (Nec-1s, 50 μM) and necrosulfonamide (NS, 2.5 μM) with DC661 1 μM for 24 hours. (H) Seventy-two-hour MTT assay plot with DC661 (0.01 to 10 μM), with and without necrostatin-1 (Nec-1, 50 μM), 50 μM Nec-1s, and 2.5 μM NS in A375P cells. All viability assays were performed in triplicate. ****P ≤ 0.0001; ns, nonsignificant. Two-tailed unpaired t test between 2 groups (C). ANOVA test was used when more than 2 groups were compared (E and H). See also Supplemental Figure 2, D–G.