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Cited by 490Cancer Immunotherapy and BiomarkersImmunotherapy and Immune ResponsesT-cell and B-cell Immunology

Inhibition of PCSK9 potentiates immune checkpoint therapy for cancer

Open access
Liu, Xinjian, Bao, Xuhui, Hu, Mengjie, Chang, Hanman, Jiao, Meng, Cheng, Jin, Xie, Liyi, Huang, Qian +2 more·2020·Nature
DOI 10.1038/s41586-020-2911-7PMC PMC7770056
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Abstract

Despite its success in achieving the long-term survival of 10-30% of treated individuals, immune therapy is still ineffective for most patients with cancer 1,2. Many efforts are therefore underway to identify new approaches that enhance such immune 'checkpoint' therapy 3-5 (so called because its aim is to block proteins that inhibit checkpoint signalling pathways in T cells, thereby freeing those immune cells to target cancer cells). Here we show that inhibiting PCSK9-a key protein in the regulation of cholesterol metabolism 6-8-can boost the response of tumours to immune checkpoint therapy, through a mechanism that is independent of PCSK9's cholesterol-regulating functions. Deleting the PCSK9 gene in mouse cancer cells substantially attenuates or prevents their growth in mice in a manner that depends on cytotoxic T cells. It also enhances the efficacy of immune therapy that is targeted at the checkpoint protein PD1. Furthermore, clinically approved PCSK9-neutralizing antibodies synergize with anti-PD1 therapy in suppressing tumour growth in mouse models of cancer. Inhibiting PCSK9-either through genetic deletion or using PCSK9 antibodies-increases the expression of major histocompatibility protein class I (MHC I) proteins on the tumour cell surface, promoting robust intratumoral infiltration of cytotoxic T cells. Mechanistically, we find that PCSK9 can disrupt the recycling of MHC I to the cell surface by associating with it physically and promoting its relocation and degradation in the lysosome. Together, these results suggest that inhibiting PCSK9 is a promising way to enhance immune checkpoint therapy for cancer.

AI snapshot

Written from the paper’s full text.

TL;DR

PCSK9 inhibition, via genetic deletion or clinically approved antibodies, potentiates anti-PD1 immune checkpoint therapy in murine tumors by increasing tumor-cell surface MHC I and cytotoxic T-cell infiltration, independent of cholesterol/LDLR regulation. PCSK9 physically promotes MHC I lysosomal degradation, and high tumor PCSK9 mRNA correlates with worse overall survival in TCGA cohorts.

Key findings

  • PCSK9 knockout in B16F10, 4T1, MC38, and CT26 murine cancer cells attenuated tumor growth in syngeneic mice; this effect was absent in T/B/NK-deficient NCG mice and Rag1-deficient mice, and CD8+ T-cell depletion completely abolished the tumor growth delay.
  • PCSK9 deficiency synergized with anti-PD1 therapy in all four tumor models; evolocumab or alirocumab plus anti-PD1 suppressed MC38 tumor growth and produced long-term survival in some mice, and evolocumab was effective in anti-PD1-resistant MC38R tumors but had no effect on PCSK9-deficient MC38 tumors.
  • PCSK9 inhibition increased tumor cell surface MHC I (H2-Kb/SIINFEKL, H2b, H2d, HLA-A2), intratumoral CD8+ CTLs, CD4+ Th cells, γδT cells, NK cells, CTL/Treg ratios, IFNγ+ and GZMB+ CTLs, and TCR counts/diversity, without significant increases in Tregs or exhaustion markers.
  • Mechanistically, PCSK9 bound H2-K1 via its M2 domain and H2-K1 aa68–70, promoting MHC I relocation to the lysosome and degradation; LDLR knockdown did not affect MHC I surface levels or the growth suppression caused by PCSK9 deficiency, and high PCSK9 mRNA was associated with worse overall survival in 9 TCGA cancer cohorts.

Methods

  • Murine cancer cell lines (B16F10, 4T1, MC38, CT26) and human MDA-MB-231 cells were modified with CRISPR/Cas9 PCSK9 knockout, LDLR knockdown/knockout, or H2-K1 knockout/overexpression; in vitro growth and soft agar assays were used, and in vivo experiments used syngeneic C57BL/6J or Balb/c mice, NCG mice, Rag1−/− mice, LDLR−/− mice on high-fat diet, and OT-1 transgenic mice.
  • Tumor growth delay, survival, in vivo competition, and re-challenge experiments were performed; treatments included anti-PD1 (clone RMP1–14), evolocumab, alirocumab, isotype controls, and antibody-mediated depletion of CD4+, CD8+, or NK cells.
  • Analyses included flow cytometry, immunofluorescence, qRT-PCR, TCR sequencing, co-immunoprecipitation, cell fractionation, cycloheximide chase, and bafilomycin treatment; human correlations used GENT for PCSK9/CD8A and TCGA/cBioPortal for PCSK9 mRNA and overall survival, with two-way ANOVA, log-rank tests, and unpaired two-sided t tests.

Limitations

  • The paper states that no randomization or blinding was used in the tumor growth delay experiments.
  • The paper has no explicit limitations section; evident from the text, the efficacy and mechanistic findings are primarily preclinical in murine tumor models and cell lines, and the human evidence is correlative (GENT/TCGA), not from a clinical trial of PCSK9 inhibition plus checkpoint blockade.
  • Also evident, several mechanistic domain-mapping experiments rely on overexpression or deletion mutants in 293T/B16F10 cells, which may not fully reflect endogenous PCSK9–MHC I interactions.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41586-020-2911-7

Cites

47 of 47 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

4 of 6 cited sources checked · 1 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    Deletions of the M1, M3, and aa367-386 domains of PCSK9 showed reduced H2-K1 binding.

    “deletions of the other two domains (M1 & M3) and the aa367–386 domain, which was involved in LDLR binding34, in the catalytic region, also showed reduced H2-K1 binding”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [34] Duff, C. J. et al. Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor. Biochem. J. 419, 577–584 (2009). source
  • Premisecitation

    Previous studies demonstrated that the amount of active T-cells is positively correlated with the success of immune checkpoint blockade therapy.

    “Previous studies have demonstrated that the amount of active T-cells is positively correlated with the success of immune checkpoint blockade therapy36,37”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [36] Zhang, L. et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N. Engl. J. Med. 348, 203–213 (2003). source

      The ‘Intratumoral T Cells and Clinical Outcome’ section reports that patients with intratumoral T cells had longer progression-free and overall survival, and the discussion describes evidence that those T cells were activated. The paper does not examine immune checkpoint blockade or show that the amount of active T cells predicts its success; its finding concerns T-cell presence and outcomes in ovarian cancer. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [37] Carstens, J. L. et al. Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer. Nat. Commun. 8, 15095 (2017). source

      The Abstract links endogenous antitumour T cells to the efficacy of therapies combined with checkpoint blockade. But it does not report a positive correlation between the amount of active T cells and checkpoint-blockade success. The Discussion states that T-cell functional status was not measured; page numbers are unavailable in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Findingcitation

    Anti-PD1 therapy was ineffective in the 4T1 model.

    “anti-PD1 therapy was ineffective, as reported before29”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [29] Kim, K. et al. Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells. Proc. Natl Acad. Sci. USA 111, 11774–11779 (2014). source

      The Effects of Immune Checkpoint Blockade section describes 4T1 tumors as resistant to immunotherapy, and the Discussion says combined anti-PD-1/anti-CTLA-4 treatment generally could not cure them. The copy does not explicitly report the outcome of anti-PD-1 alone in 4T1 mice; moreover, 3 of 10 mice had complete primary-tumor regression with the combination, so “ineffective” needs qualification. (LLM, conf=0.79)

      Experimental — accuracy not yet measured
  • Findingown data

    PCSK9 deletion enhanced the efficacy of anti-PD1 immune checkpoint therapy significantly.

    “It also enhanced the efficacy of anti-PD1 immune checkpoint therapy significantly.”
    Quote in the paperSubstantial claim
  • Findingown data

    Clinically approved PCSK9-neutralizing antibodies synergize with anti-PD1 therapy in suppressing tumor growth in murine tumor models.

    “clinically approved PCSK9-neutralizing antibodies could synergize with anti-PD1 therapy in suppressing tumor growth in murine tumor models”
    Quote in the paperSubstantial claim
  • May combine two claims. 14
  • The quote only partly states this claim. 12
  • Classified as probably not a substantial claim. 19

  • May combine two claims.

    Findingown data

    PCSK9 knockout did not alter the morphology or in vitro growth rates of tumor cells.

    “PCSK9 knockout (PCSK9KO) did not alter the morphology or the in vitro growth rates of tumor cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both anti-PD1 and anti-PCSK9 antibodies boosted the numbers of CTLs inside the 4T1 tumor.

    “both anti-PD1 and anti-PCSK9 antibodies could boost the numbers of CTLs inside the tumor”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    PCSK9-deficient B16F10 melanoma cells were more susceptible to killing by CTLs in vitro.

    “Our results indicated that PCSK9 more susceptible to killing by CTLs”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Re-introduction of wild type PCSK9 abolished the tumor growth delay while PCSK9ΔM2 had no effect.

    “re-introduction of the wild type PCSK9 abolished the tumor growth delay while that of PCSK9ΔM2 had no effect”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    H2-K1 expression significantly attenuated tumor growth alone or in combination with anti-PD1.

    “H2-K1 expression could significantly attenuate tumor growth alone or in combination with anti-PD1”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Bafilomycin caused HLA-ABC levels to increase significantly in control cells but not change much in PCSK9KO cells.

    “when the cells were exposed to bafilomycin to inhibit lysosome function, the levels of HLA-ABC increased significantly in control cells but did not change much in PCSK9KO cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Evolocumab treatment was effective either alone or in combination with anti-PD1 treatment for MC38R tumors.

    “Evolocumab treatment was effective either alone or in combination with anti-PD1 treatment for MC38R tumors”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the membrane fraction, PCSK9OE reduced the relative abundance of the H2-K1 protein while PCSK9 knockout increased it.

    “In contrast, in the membrane fraction, PCSK9OE reduced the relative abundance of the H2-K1 protein while PCSK9 knockout increased it”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CD8+ cells stayed mostly in the periphery in control tumors but moved into tumor cell-rich areas in PCSK9-deficient B16F10 tumors.

    “CD8+ cells staying mostly in the periphery in control tumors (Extended Data Fig. 6b, top panels) but moving into tumor cell-rich areas in the PCSK9-deficient B16F10 tumors”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the lysosome fraction, PCSK9 OE caused an increase in H2-K1 protein while PCSK9KO induced a decrease.

    “In the lysosome fraction, PCSK9 OE caused an increase in H2-K1 protein while PCSK9KO induced a decrease”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The efficacy of anti-PCSK9 antibodies was enhanced significantly when combined with anti-PD1, with long-term survival of some host mice.

    “their efficacies were enhanced significantly when combined with an anti-PD1 antibody, with long-term survival of some host mice”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    PCSK9 neutralization may promote intratumoral T cell infiltration and render tumors more responsive to immune checkpoint therapy.

    “It suggests that PCSK9 neutralization may promote intratumoral T cell infiltration and thus render tumors more responsive to immune checkpoint therapy”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Both the number and diversity of mature T-cells were significantly elevated in PCSK9-deficient tumors.

    “These results suggest that both the number and diversity of mature T-cells were significantly elevated in PCSK9-deficient tumors”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    PCSK9 plays important roles in regulating cell surface MHC I levels and thus influences intratumoral immune infiltration.

    “The finding that PCSK9 plays important roles in regulating cell surface MHC I levels (see Extended Data Fig. 10 for a summary schema) and thus influences intratumoral immune infiltration is novel mechanistically”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The IP-Western blot analysis of the interaction of HA-labeled mouse PCSK9-HA to full-length H2-K1 or H2-K1 with more limited deletions (aa 66-100, α1 domain; or aa68-70) was repeated twice with biologically independent samples with similar results.

    “The analyses were repeated twice with biologically independent samples with similar results.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All animal experiments were approved by Duke University Institutional Animal Use and Care Committee.

    “All animal experiments conducted in this study were approved by Duke University Institutional Animal Use and Care Committee (IACUC)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based). The quote only partly states this claim.

    Study design and conductown data

    The western blot analysis of lentivirus-mediated over-expression of HA-tagged H2-K1 in B16F10 cells was done once.

    “The analysis was done once.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Cell line identities were verified by the STR method.

    “Their identities were verified by the STR method”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All cell lines were subjected to mycoplasma testing periodically.

    “All cell lines were subjected to mycoplasma test periodically”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    PCSK9 knockout in infected cells was verified by western blot.

    “expression of target protein (PCSK9) in infected cells were detected by western blot to verify the knockdown”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    At least 5 mice per treatment group were included.

    “At least 5 mice per treatment group were included”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Original uncropped images highlighting the cropped areas were shown in Supplementary Data Fig. 1.

    “Original uncropped images highlighting the cropped areas were shown in Supplementary Data Fig. 1”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based). The quote only partly states this claim.

    Study design and conductown data

    The western blot analysis of CRISPR-Cas9 mediated knockdown of LDLR in B16F10 cells was done once.

    “The analysis was done once.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The fluorescence imaging experiments of tdTomato-labeled tumor cells with or without OVA antigen in the presence or absence of OVA-specific T cells were completed twice with similar results.

    “The experiments were completed twice with similar results.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The analysis of OVA antigen presentation by MHC-I in cultured B16F10 cells with PCSK9 deficiency used representative results from 4 sets of biologically independent samples.

    “Shown were representative results from analyses of 4 sets of biologically independent samples.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The western blot analysis of PCSK9 expression in control or PCSK9KO MDA-MB-231 cells was done twice.

    “The analyses were done twice.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The IP-Western blot analysis of the interaction between full-length Flag-labeled H2-K1 and full-length or partially deleted mouse PCSK9-HA was repeated twice with biologically independent samples with similar results.

    “The analyses were repeated twice with biologically independent samples with similar results.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Two independent experiments were carried out for the soft agar colony formation assay.

    “Two independent experiments were carried out”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The IP-Western blot analysis of the interaction between full-length HA-labeled mouse PCSK9-HA and full-length or partially deleted H2-K1-Flag (aa66-202) was repeated twice with biologically independent samples with similar results.

    “The analyses were repeated twice with biologically independent samples with similar results.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The western blot analysis of LDLR knockdown in control and PCSK9KO B16F10 tumor cells was done once.

    “The analysis was done once.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    In the absence of PCSK9, MHC I levels on the surface remain high and can present tumor-specific peptide antigens more efficiently to T cells.

    “In the absence of PCSK9, either because of genetic deletion or antibody neutralization, MHC I levels on the surface remains high and is thus able to present tumor-specific peptic antigens more efficiently to T cells (right panel).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The western blot analysis of exogenously transduced HA-tagged PCSK9 in PCSK9KO B16F10 cells was done once.

    “The analysis was done once.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The western blot analysis of PCSK9 expression in murine tumor lines with PCSK9 knockout was done twice with biologically independent samples.

    “Analysis was done twice with biologically independent samples.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    No randomization or blinding was used in the tumor growth delay experiments.

    “No randomization or blinding was used in our tumor growth delay experiments”
    Quote in the paperNot a substantial claim
34 of 34

  • The paper relies on this without support.

    Findings from murine tumor models and cell lines will translate to human cancer patients.

    “provide compelling rationales for conducting future clinical trials in human cancer patients”

  • The paper relies on this without support.

    Combining PCSK9 inhibitors with immune checkpoint blockade will not add side effects.

    “Given the well-known safety profiles of the anti-PCSK9 antibodies, PCSK9 inhibitors may enhance immune checkpoint blockade therapy without additional side effects.”
2 of 2

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • LDLR knockout is described as knockdown in the cited experiment

    Minor

    The text calls the LDLR intervention in Extended Data Fig. 8j a knockout, but the figure caption labels both relevant cell groups LDLR knockdown (LDLRKD).

  • Extended Data Fig. 8 assigns panel c to two experiments

    Minor

    The caption first assigns panel c to the H2-K1 overexpression survival curve, then assigns it to the separate H2-K1 knockdown/PCSK9 knockout survival curves, despite labeling that second experiment d-e.

2 of 2

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Related

Authors
Liu, X., Bao, X., Hu, M., Chang, H., Jiao, M., Cheng, J., Xie, L., Huang, Q., Li, F., & Li, C.-Y.
Affiliations
  • Sun Yat-sen University (CN)
  • Duke Medical Center (US)
  • Shanghai Jiao Tong University (CN)
  • Fudan University (CN)
  • Duke University (US)
Corresponding author
Li, Chuan-Yuan
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2020-11-11
Volume
588
Issue
7839
Pages
693-698
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-020-2911-7
PMC
PMC7770056
OpenAlex
W3103804121
Cited by
490
References
47
Citations by year
2026: 60 · 2025: 107 · 2024: 106 · 2023: 81 · 2022: 73 · 2021: 60 · 2020: 2
FWCI
17.32
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • National Institutes of Health (2p30ca014236-51)
  • National Cancer Institute (P30‐CA014236, R01 CA208852, R01CA216876)
  • National Institute of Environmental Health Sciences (R01ES024015)
Topics
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
Keywords
PCSK9 inhibition, immune checkpoint therapy, anti-PD-1 therapy, cancer immunotherapy, cytotoxic T lymphocytes, tumor surveillance, lysosomal degradation, antigen presentation, tumor growth inhibition
MeSH terms
Proprotein Convertase 9, Immune Checkpoint Inhibitors, PCSK9 Inhibitors, Animals, Disease Models, Animal, Female, Humans, Immunotherapy, Lysosomes, Neoplasms, T-Lymphocytes, Cytotoxic, Histocompatibility Antigens Class I, Xenograft Model Antitumor Assays, Cell Line, Tumor, Mice, Antibodies, Neutralizing, Programmed Cell Death 1 Receptor

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