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 1

Lysosomal autophagy inhibition induces significant changes in apoptosis and autophagy proteins.

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Lysosomal autophagy inhibition induces significant changes in apoptosis ...
(A) Trypan blue viability assay of A375P melanoma cells treated with Bafilomycin-A1 (100 nM), DC661 (3 μM), HCQ (10 or 30 μM), hexadecylsulfonyl fluoride (HDSF, 60 μM), pepstatin A (10 μg/mL), E64 (10 μg/mL), or Leu-Leu methyl ester hydrobromide (LLoMe, 20 μM) for 48 hours. (B) LC-MS/MS–based proteome analysis of A375P cells treated with DMSO, DC661 (3 μM), or HCQ (10 or 30 μM) for 24 hours. Heatmap of the top 50 elevated proteins in DC661 verses control. Autophagy, apoptosis (names shown in bold), or other signaling pathway proteins significantly elevated (FDR, <5% and fold change, ≥2) in cells treated with 3 μM DC661, 10 μM HCQ, or 30 μM HCQ compared with those treated with vehicle control. (C) The autophagy cargo receptor proteins that have proapoptotic effects in cancer cells are shown in a Venn diagram. (D–M) A375P cells were treated with nontarget siRNA (siNT) or siRNA against TAX1BP1, BNIP3, ULK1, or ATG7 for 48 hours, followed by treatment with either DMSO or DC661 (3 μM) for 24 hours. (D and G) Immunoblotting of TAX1BP1 or BNIP3 and β-actin in the whole-cell lysates of A375P cells. (E and H) Seventy-two-hour MTT assay with 3 μM DC661 or (F and I) 7-day colony formation assay with 0.3 μM DC661 in A375P cells treated with the indicated siRNA. (J and L) Seventy-two-hour MTT assay with 3 μM DC661 and (K and M) 7-day colony formation assay with 0.3 μM DC661 in A375P cells treated with the indicated siRNA. All viability assays were performed in triplicate. ****P ≤ 0.0001. ANOVA test was used when more than 2 groups were compared. See also Supplemental Figure 1 and Supplemental Figure 2, A–C.