Lysosomal lipid peroxidation regulates tumor immunity
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
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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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 4

Cathepsin inhibition or calcium chelation does not prevent DC661-induced cell death.

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Cathepsin inhibition or calcium chelation does not prevent DC661-induced...
(A) A375P-galectin-3-EGFP cells were given nontarget siRNA (siNT) or PPT1 siRNA (siPPT1) for 48 hours, followed by treatment with DMSO, 60 μM HDSF, or 3 μM DC661 for 6 hours. (B) Cathepsin-L enzyme activity (red) and quantification in A375P cells treated with 3 μM DC661, 10 μg/mL cysteine protease inhibitor E64, or the combination of both for 6 hours. (C) Immunoblots for indicated proteins in A375P cell lysates treated with pepstatin A (PepA, 10 μg/mL), 10 μg/mL E64, and PepA+E64 with or without 3 μM DC661 for 24 hours. (D) Seventy-two-hour MTT assay plot with increasing concentrations of DC661 (0.01 to 10 μM), with and without PepA, E64, and PepA+E64 in A375P cells. (E) Seven-day colony formation assay in A375P cells treated with 10 μg/mL PepA, 10 μg/mL E64, and PepA+E64 with or without 0.3 μM DC661. (F and G) A375P or A375P-galectin-3-EGFP cells were treated with 3 μM DC661, 1 μM calcium chelator BAPTA-AM, or both for 24 hours. (F) Fluorescence images of A375P cells stained with Fluo-4, AM, to detect calcium release. (G) A375P-galectin-3-EGFP cells showing galectin-3 puncta (white arrows) and quantification after treatment with DC661, BAPTA-AM, or both. (H) Seventy-two-hour MTT assay plot with increasing concentrations of DC661 (0.01 to 10 μM), with and without indicated concentrations of BAPTA-AM in A375P cells. Scale bar: 20 μm. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. ANOVA test was used when more than 2 groups were compared. All viability experiments were done in triplicate.