Abstract NG12: From cholesterol regulation to tumor suppression: Pcsk9 as a novel target for cancer immunotherapy
Authors: Bao Xuhui , Liu Xinjian , Hu Mengjie , Chang Hanman , Jiao Meng , Cheng Jin , Xie Liyi , Huang Qian , Li Fang , Li Chuan-Yuan 
Published: July 2021

Abstract
Background: Despite its great success, immunotherapy is still of limited efficacy in many cancer patients. Numerous efforts are underway to develop novel strategies to enhance immune checkpoint inhibition therapy. Proprotein convertase subtilisin/kexin type 9 (Pcsk9), as a key regulator of cholesterol metabolism, mainly functions to down-regulate cell surface low-density lipoprotein receptor (LDLR), which keeps plasma LDL-cholesterol levels high. In addition to LDLR, Pcsk9 is also able to regulate the expression of many other surface proteins. Here we report that Pcsk9 inhibition can boost anti-tumor immunity, albeit through a mechanism independent of cholesterol regulation, by increasing the expression of major histocompatibility protein class I (MHC I) on tumor cell surface. Methods: Pcsk9 inhibition was achieved by genetic deletion using CRISPR-Cas9 technology or neutralizing antibodies (evolocumab and alirocumab). Immunodeficient and syngeneic mouse tumor models were established to evaluate anti-tumor efficacy of Pcsk9 inhibition and its combination with immune checkpoint inhibitors. Flow cytometry analysis, qRT-PCR, co-immunoprecipitation, western blot, and immunofluorescence staining were used to determine immune infiltration, protein-protein interaction, surface protein expression, and cellular protein distribution. T-cell receptor (TCR) repertoire analysis was used to determine the intratumoral T-cell diversity. The Cancer Genome Atlas (TCGA) datasets were analyzed to examine the association of Pcsk9 expression with overall survival in human malignancies. Results: I. Pcsk9 depletion significantly attenuates tumor growth in syngeneic mice. Deletion of Pcsk9 in cancer cells dramatically attenuated their growth and improved tumor-free long-term survival in mouse 4T1 breast cancer (50% complete remission, CR), B16 melanoma (17% CR), CT26 (60% CR) and MC38 (20% CR) colon cancer models, although genetic Pcsk9 deletion did not alter the in vitro growth rates of tumor cells. Those mice with CR after initial tumor cell challenge could resist a re-challenge with parental tumor cells. However, Pcsk9 deficiency had no effect on 4T1 and B16 tumor growth in immunodeficient NCG mice or in Rag1-deficient mice. The subsequent animal studies indicated that depletion of CD8+ T cells instead of CD4+ T cells or NK cells completely abolished the tumor growth delay of Pcsk9-deficient B16 tumors in vivo. II. Pcsk9 inhibition overcomes tumor resistance to anti-PD1 therapy. Genetic Pcsk9 deletion also significantly enhanced the efficacy of anti-PD1 immunotherapy in all those four murine cancer syngeneic models, especially for B16 melanoma (71% CR) and MC38 colon cancer (100% CR). Meanwhile, clinically approved Pcsk9-neutralizing antibodies could synergize with anti-PD1 therapy in MC38 murine colon cancer models to achieve a promising CR rate (40% CR for anti-PD1+alirocumab and 50% CR for anti-PD1+evolocumab) compared to 100% tumor progression of either single arm of treatment. Moreover, in the anti-PD1 resistant MC38R colon cancer model, MC38R tumors only responded to evolocumab single treatment or combined with anti-PD1 therapy instead of anti-PD1 therapy alone. III. Pcsk9 depletion enhances robust intratumoral immune infiltration. Immune effector cells in control and Pcsk9-deficient B16 tumors were quantified by immunofluorescence staining and flow cytometry analysis. Immunofluorescence staining indicated that PCSK9 depletion led to an overall increase in intratumoral infiltration of CD45+ leukocytes and CD8+ cells. Of particular interest was the observation of CD8+ cells scattering in the periphery in control tumors but infiltrating tumor cell-rich areas in Pcsk9-deficient tumors. Flow cytometry analyses confirmed significant increases in intratumoral CD8+ cytotoxic T cells (CTLs), CD4+ T helper cells, γδT cells, and NK cells in Pcsk9-deficient tumors. In contrast, no significant increases in CD4+Foxp3+ regulatory T (Treg) cells were observed. The ratios of CTLs/Tregs in the Pcsk9-deficient tumors were significantly increased. Consistently, both the numbers and percentages of IFNγ+, or Granzyme B+ CTLs also significantly increased in Pcsk9-deficient tumors, as well as the intratumoral expression of IFNG and GZMB by qRT-PCR. On the other hand, T cell exhaustion markers (e.g. PD1, TIGIT, and CTLA4) showed no significant changes. Furthermore, TCR repertoire analyses revealed that total TCR counts and the number of unique TCRs were significantly increased in Pcsk9-deficient tumors than control tumors. IV. Pcsk9 depletion prevents lysosome-mediated degradation of MHC I in tumor cells. Our data indicated Pcsk9 inhibition led to a significant increase of surface MHC I expression in murine B16 and 4T1 tumor cells and human MDA-MB-231 breast cancer cells, while genetic depletion of H2-K1 (mouse MHC I) completely abrogated tumor growth delay induced by Pcsk9 inhibition. To examine if Pcsk9 could regulate MHC I by direct interaction, we first created recombinant mouse Pcsk9 mutants with various internal deletions or truncations based on domain structure. Each of those deleted Pcsk9 genes was then co-transduced into 293T cells together with a full-length H2-K1 gene. Our analysis showed that deletion of M2 domain within the C-terminal region of Pcsk9 showed almost complete loss of binding to H2-K1. On the other hand, deletion mapping of H2-K1 showed that absence of the α1 region (aa66-100) and in particular, of amino acids aa68-70, which sits in the loop right before the helix structure of the α1 domain, completely abolished its binding to Pcsk9. Further, re-introduction of the wild type (WT) Pcsk9 abolished the Pcsk9KO B16 tumor growth delay while that of Pcsk9ΔM2 (Pcsk9 with deletion of the M2 region) had no such effect. In comparison, re-introduction of WT H2-K1 gene attenuated the tumor-forming abilities of both H2-K1 deficient and Pcsk9/H2-K1 double KO cells. Nevertheless, re-introduction of the H2-K1Δ68-70 did not slow down tumor growth. Subsequently, immunofluorescence co-staining of exogenously expressed H2-K1 and/or Pcsk9 were carried out in B16F10 cells with Pcsk9 knockout or Pcsk9 over-expression (OE). In Pcsk9OE cells, more H2-K1 was localized in the lysosome and not in the plasma membrane. On the contrary, in Pcsk9KO cells, H2-K1 staining indicated a dominant plasma membrane localization. Western blot analysis of fractionated cellular lysates further confirmed the immunofluorescence staining results. In the lysosome fraction, Pcsk9OE caused an increase in H2-K1 protein while Pcsk9KO induced a decrease. In contrast, in the membrane fraction, Pcsk9OE reduced the relative abundance of the H2-K1 protein while Pcsk9KO increased it. V. Pcsk9 deficiency predicts significantly prolonged overall survival in cancer patients. TCGA analyses demonstrated that low expression of Pcsk9 predicted a significantly prolonged overall survival in nine malignancies including liver hepatocellular carcinoma (LIHC), pancreatic adenocarcinoma (PAAD), skin cutaneous melanoma (SKCM), uveal melanoma (UVM), bladder urothelial carcinoma (BLCA), lung adenocarcinoma (LUAD), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and ovarian carcinoma (OV). Conclusions: Our study revealed Pcsk9 as a promising target for cancer immunotherapy. Pcsk9 deficiency could significantly recruit intratumoral immune infiltration, suppress tumor growth, improve tumor-free survival, and synergize with immune checkpoint inhibitors, by promoting the surface expression of MHC I molecules. Considering the availability of clinically approved anti-Pcsk9 antibodies, we believe that combining Pcsk9 inhibition with immune checkpoint blockade may offer new options for some cancer patients in the future.
Citation Format: Xuhui Bao, Xinjian Liu, Mengjie Hu, Hanman Chang, Meng Jiao, Jin Cheng, Liyi Xie, Qian Huang, Fang Li, Chuan-Yuan Li. From cholesterol regulation to tumor suppression: Pcsk9 as a novel target for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG12.