Depletion of oxysterol-binding proteins by OSW-1 triggers RIP1/RIP3-independent necroptosis and sensitization to cancer immunotherapy - PubMed (original) (raw)

. 2025 Nov;32(11):2038-2052.

doi: 10.1038/s41418-025-01521-8. Epub 2025 May 6.

Xinyan Lu 1 2 3 4, Dongshi Chen 1 2 3 4, Xiangping Song 3 4, Kaylee Ermine 3 4, Suisui Hao 1 2 3 4, Anupma Jha 3 4, Yixian Huang 4 5, Ying Kang 6, Haibo Qiu 6, Heinz-Josef Lenz 1 2, Song Li 4 5, Zhendong Jin 6, Jian Yu 1 2 3 4, Lin Zhang 7 8 9 10

Affiliations

Depletion of oxysterol-binding proteins by OSW-1 triggers RIP1/RIP3-independent necroptosis and sensitization to cancer immunotherapy

Xinyan Lu et al. Cell Death Differ. 2025 Nov.

Abstract

Oxysterol-binding proteins (OSBPs), lipid transfer proteins functioning at intracellular membrane contact sites, are recently found to be dysregulated in cancer and promote cancer cell survival. However, their role as potential targets in cancer therapy remains largely unexplored. In this study, we found OSW-1, a natural compound and OSBP inhibitor, potently and selectively kills colon cancer cells by activating a previously unknown necroptosis pathway that is independent of receptor-interacting protein 1 (RIP1) and RIP3. OSW-1 stabilizes p53 and degrades OSBPs to promote endoplasmic reticulum (ER) stress and glycogen synthase kinase 3β (GSK3β)/Tip60-mediated p53 acetylation at Lysine 120, which selectively induces its target PUMA. PUMA-mediated mitochondrial calcium influx activates calcium/calmodulin-dependent protein kinase IIδ (CamKIIδ) to promote mixed lineage kinase domain-like (MLKL) phosphorylation and necroptotic cell death. Furthermore, OSW-1-induced necroptosis is highly immunogenic and sensitizes syngeneic colorectal tumors to anti-PD-1 immunotherapy. Together, our results identified a novel RIP1/RIP3-independent necroptosis pathway underlying the extremely potent anticancer activity of OSW-1, which can be harnessed to develop new anticancer therapies by selectively stimulating antitumor immunity.

© 2025. The Author(s).

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Conflict of interest statement

Competing interests: ZJ is the founder and a shareholder of InnoBioPharma, LLC, a spin-off from the University of Iowa, focusing on the development of novel payloads for ADCs from natural products like OSW-1. InnoBioPharma sponsored the project, “Development of novel anticancer agents based on natural products” at the University of Iowa. Ethical approval: This study was reviewed and approved by the ethics committees of the University of Pittsburgh and University of Southern California. All methods including laboratory animal experiments were performed in accordance with the relevant guidelines and regulations of the University of Pittsburgh and University of Southern California.

Figures

Fig. 1

Fig. 1. OSW-1 induces RIP1/RIP3-independent necroptosis in CRC cells.

A MTS analysis of indicated CRC cell lines and NCM356 normal colonic epithelial cells treated with OSW-1 at indicated concentrations for 48 h. B Crystal violet staining of RIP3- HCT116 and RKO cells and NCM356 cells treated with OSW-1 (0.5 nM) for 24 h. C HCT116 cells treated with OSW-1 as in (B) were analyzed by Annexin V/PI staining followed by flow cytometry. Cells treated with cisplatin (50 μM) were used as a positive control for apoptosis. D HCT116 cells were treated for 24 h with OSW-1 (0.5 nM) alone or in combination with the RIP1 inhibitor Necrostatin-1 (Nec-1; 20 μM), the MLKL inhibitor Necrosulfonamide (NSA; 2 μM), and/or the pan-caspase inhibitor z-VAD-fmk (z-VAD; 10 μM). Upper, ATP levels in treated cells; lower, western blotting of total and phosphorylated MLKL (p-MLKL; S358) in whole cell lysates (WCL) and HMGB1 in 20-μl cell culture medium (M). E Transmission electron microscopy (TEM) of HCT116 cells with or without OSW-1 treatment as in (B). White arrowheads indicate plasma membrane, and black arrowheads denote mitochondria. Scale bars: 2 μm. F HCT116 cells transfected with control (Ctr) or RIP1 siRNA were treated with OSW-1 as in (B). Necroptosis was analyzed by measuring ATP levels (upper), Western blotting of indicated proteins (middle), and crystal violet staining (lower). G LoVo cells transfected with Ctr or RIP3 siRNA were treated with OSW-1 and analyzed for necroptosis as in (F). H, I WT and _MLKL_-KO H HCT116 and I RKO cells were treated with OSW-1 and analyzed for necroptosis as in (F). Quantitative results in (A, D, F–I) were expressed as means ± s.d. of three independent experiments. NS, P > 0.05; *; P < 0.05.

Fig. 2

Fig. 2. OSW-1-induced necroptosis requires p53-mediated transcriptional upregulation of PUMA.

A Comparison of OSW-1 IC50 of _p53_-wildtype (WT) and _p53_-mutant CRC cell lines analyzed in Fig. 1A. B, C HCT116 cells were treated with (B) OSW-1 (0.5 nM) for indicated time, or (C) OSW-1 at indicated concentrations for 24 h. Upper, real time RT-PCR analysis of PUMA mRNA expression; lower, western blotting of indicated proteins in whole cell lysates (WCL) and HMGB1 in 20-μl cell culture medium (M). D, E Western blotting of indicated proteins in D WT and _p53_-knockout (_p53_-KO), E WT and _PUMA_-KO HCT116 cells treated with OSW-1 (0.5 nM) for 24 h. F MTS analysis of WT, _p53_-KO, and _PUMA_-KO HCT116 cells treated with OSW-1 at indicated concentrations for 48 h. G-I WT, _p53_-KO, and _PUMA_-KO HCT116 cells were treated with OSW-1 as in (D) and analyzed by (G) crystal violet staining of viable cells, H annexin-V/PI staining followed by flow cytometry, I measuring ATP levels, and J TEM. In (J), representative TEM images are shown. White arrowheads indicate plasma membrane, and black arrowheads denote mitochondria. Scale bars, 2 μm. K, L WT and _PUMA_-KO HCT116 cells with or without infection with an adenovirus expressing PUMA (Ad-PUMA) or control BH3-deleted PUMA (Ad-control) were treated with OSW-1 as in (D). K Crystal violet staining of viable cells. L Western blotting of indicated proteins. Quantitative results in (B, C, F, I) were expressed as means ± s.d. of two or three independent experiments. *, P < 0.05; **, P < 0.01**; *****, P < 0.001.

Fig. 3

Fig. 3. OSW-1 stabilizes p53 and promotes its K120 acetylation to selectively upregulate PUMA transcription and induce necroptosis.

A Western blotting of indicated proteins in HCT116 cells treated with OSW-1 (0.5 nM) at indicated time points. B, C HCT116 cells were treated with OSW-1 (0.5 nM) along with the translation inhibitor cycloheximide (CHX; 10 μg/mL). B Western blotting of indicated proteins at indicated time points. C Quantification of western blots by NIH Image J and normalized to the loading control β-actin. D HCT116 cells with or without OSW-1 (0.5 nM) treatment for 24 h were subjected to immunoprecipitation (IP) to pull down Mdm2 followed by western blotting of indicated proteins. IgG was used as a negative control. E, F HCT116 cells were treated for 24 h with OSW-1 (0.5 nM) alone or in combination with the GSK3 inhibitor CHIR99021 (10 μM). E Western blotting of indicated proteins in whole cell lysates (WCL) and HMGB1 in 20-μl cell culture medium (M). F ATP levels in treated cells. G–I _p53_-KO HCT116 cells were transfected with control empty vector, WT, K164R, or K120R mutant p53 were treated with OSW-1 as in (D). G Western blotting of indicated proteins. H Crystal violet staining of viable cells. I ATP levels in treated cells. Quantitative results in (F) and (I) were expressed as means ± s.d. of four independent experiments. **, P < 0.01; ***, P < 0.001.

Fig. 4

Fig. 4. OSW-1 induces OSBP and ORP4 protein degradation and promotes ER stress to induce p53 K120 acetylation and necroptosis.

A Western blotting of indicated proteins in HCT116 cells treated with OSW-1 (0.5 nM) at indicated time points. B–D HCT116 cells transfected with empty vector, OSBP, ORP4, or both OSBP and ORP4 were treated with OSW-1 (0.5 nM) for 24 h. B Crystal violet staining of viable cells. Western blotting of indicated proteins in whole cell lysates (WCL) and HMGB1 in 20-μl cell culture medium (M) from cells transfected with C OSBP or D ORP4. E Western blotting of indicated proteins in HCT116 cells transfected with control (Ctr), OSBP, ORP4, or both OSBP and ORP4 siRNAs for 48 h. F TEM analysis of HCT116 cells treated with OSW-1 as in (B). Upper, representative TEM pictures with arrowheads indicating ERs (Scale bars, 1 μm); lower, quantification of dilated, fragmented, and normal ERs. Results were expressed as means ± s.d. from counting ERs in six randomly selected fields. G, H HCT116 cells were treated for 24 h with OSW-1 (0.5 nM) alone or in combination with Salubrinal (5 μM). G Crystal violet staining of viable cells. H Western blotting of indicated proteins. ** P < 0.01; *** P < 0.001.

Fig. 5

Fig. 5. OSW-1-induced necroptosis is executed by CaMKIIδ-mediated MLKL phosphorylation.

A Western blotting of indicated proteins in HCT116 cells treated with OSW-1 (0.5 nM) at indicated time points. B Western blotting of indicated proteins in HCT116 cells treated with OSW-1 at indicated concentrations for 24 h. C, D Western blotting of indicated proteins in (C) WT and _p53_-KO, D WT and _PUMA_-KO HCT116 cells treated with OSW-1 (0.5 nM) for 24 h. E MTS analysis of HCT116 cells treated for 48 h with OSW-1 at indicated concentrations alone or in combination with the CaMKII inhibitor KN93 (1 μM). F–I HCT116 cells were treated for 24 h with OSW-1 (0.5 nM) alone or in combination with KN93 (1 μM). F Crystal violet staining of viable cells. G ATP levels in treated cells. H LDH release from treated cells. I Western blotting of indicated proteins in whole cell lysates (WCL) and HMGB1 in 20-μl cell culture medium (M). J, K WT and _CaMKIIδ_-KO J HCT116 and K RKO cells were treated with OSW-1 as in (C). Necroptosis was analyzed by measuring ATP levels (upper), Western blotting of indicated proteins (middle), and crystal violet staining (lower). L HCT116 cells treated with OSW-1 as in (C) were subjected to IP to pull down CaMKII (left) or MLKL (right), followed by western blotting of indicated proteins. IgG was used as a negative control. Quantitative results in (E, G, H, J, K) were expressed as means ± s.d. of three independent experiments. *, P < 0.05; **, P < 0.01**; *****, P < 0.001.

Fig. 6

Fig. 6. The in vivo antitumor activity of OSW-1 is mediated by p53/PUMA/MLKL-dependent necroptosis.

A–D C57BL/6 mice were injected s.c. with 5 × 105 WT, _p53_-KO, or _PUMA_-KO MC38 cells (n = 9–10 in each group). After 7 days, mice were treated with OSW-1 (i.p.; 12.5 μg/kg) every other day as indicated by arrows. A Tumor volume at indicated time points after treatment with statistical significance for indicated comparisons. B Representative tumors at the end of the experiment. Scale bars, 10 mm. C H&E staining of indicated organ tissues. Scale bars, 100 μm. D Western blotting of indicated proteins. E Paraffin-embedded tumor tissues from mice treated as in (A) and resected at day 11 were analyzed by immunostaining for CD8. Left, representative staining pictures with arrows indicating example cells with positive staining (Scale bars, 20 μm); right, quantification of CD8+ cells (n = 5 in each group). At least 300 nuclei from 3 randomly selected fields were counted for each tumor. F–H C57BL/6 mice were injected with WT or _MLKL_-KO MC38 cells and treated with OSW-1 as in (A) (n = 7 in each group). F Tumor volume at indicated time points after treatment with statistical significance for indicated comparisons. G Representative tumors at the end of the experiment. Scale bars, 10 mm. H Western blotting of indicated proteins. In (D, H), pooled lysates from three randomly selected tumors in each group were analyzed. **, P < 0.01; ***, P < 0.001.

Fig. 7

Fig. 7. OSW-1 combined with anti-PD-1 antibody enhances tumor suppression and antitumor immune response.

A–F C57BL/6 mice were injected s.c. with 5 × 105 WT MC38 cells. After tumor growth for 7 days, mice were treated with OSW-1 (i.p.; 5 μg/kg), anti-mouse-PD-1 (i,p.; 150 μg/dose), or their combination as indicated in (A). A Tumor volume at indicated time points with statistical significance for indicated comparisons (n = 11–12 in each group). B Representative tumors at the end of the experiment. Scale bars, 10 mm. C–F Flow cytometry analysis of infiltrating lymphocytes in tumors resected on day 11 (n = 6 in each group): C CD3+/CD45+, D CD8+/CD45+, E CD11c+/CD45+, and F CD25+/FoxP3+/CD4+. G–L C57BL/6 mice were injected s.c. with 5 × 105 WT, _p53_-KO, or _PUMA_-KO MC38 cells. After tumor growth for 7 days, mice were treated with OSW-1 (i.p.; 5 μg/kg), anti-mouse-PD-1 (i,p.; 150 μg/dose), or their combination as indicated in (G). G Tumor volume at indicated time points with statistical significance for indicated comparisons (n = 7–12 in each group). H Representative tumors at the end of the experiment. Scale bars, 10 mm. I–L Flow cytometry analysis of infiltrating lymphocytes in tumors resected on day 11 (n = 6 in each group): I CD3+/CD45+, J CD8+/CD45+, K CD11c+/CD45+, and L CD25+/FoxP3+/CD4+. *, P < 0.05; **, P < 0.01; **, P < 0.001.

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