Mevalonate kinase inhibits anti-tumor immunity by impairing the tumor cell-intrinsic interferon response in microsatellite instability colorectal cancer

doi:10.21203/rs.3.rs-4819905/v1

Download PDF

Article

Mevalonate kinase inhibits anti-tumor immunity by impairing the tumor cell-intrinsic interferon response in microsatellite instability colorectal cancer

https://doi.org/10.21203/rs.3.rs-4819905/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

Inadequate tumor cell-intrinsic interferon response leads to limited efficacy of immune checkpoint blockade (ICB) therapy, such as anti-PD-1. Cholesterol metabolism can sharply regulate anti-tumor immune response. However, the mechanism of cholesterol synthesis affects the tumor cell-intrinsic interferon response in microsatellite instability (MSI) colorectal cancer (CRC) remains unclear.

Method

Small interfering RNA(siRNA) libraries and GSEA enrichment analysis are employed to screen out the key molecular which affects the tumor cell-intrinsic interferon response in MSI CRC. Mass cytometry and multiple immunofluorescence (mIF) for detecting changes in tumor micro-environment. The confocal immunofluorescence (IF), truncated protein construction, and co-immunoprecipitation (co-IP) were utilized to investigate the mechanism. The efficacy of immunotherapy were assessed in subcutaneous transplantation tumor models and human peripheral blood mononuclear cells-patient derived xenografts(hPBMC-PDX) models.

Results

Using the siRNA library and GSEA analysis, we revealed that mevalonate kinase (MVK) notably impairs the tumor cell-intrinsic interferon response in MSI CRC cells. After MVK gene knockout, the levels of Th1 type chemokines (CXCL9 and CXCL10) and the abundance of CD8⁺T cells were increased in tumor, and tumor growth was significantly slowed in mice with intact immune systems. Mechanistically, MVK interacts with the transcriptional activation domain (TAD) of signal transducer and activator of transcription 1 (STAT1), a key transcription factor in the interferon response. This interaction leads to reduced nuclear translocation of STAT1, ultimately impacting interferon reactivity. In the analysis of the hPBMC-PDX model and the MSI CRC clinical cohort, we observed that a low level of MVK in tumors is associated with a significant efficacy of anti-PD-1 therapy.

Conclusion

MVK is the crucial medium in the cholesterol metabolism to inhibit the tumor cell-intrinsic interferon response of tumor cells. Moreover, targeting MVK is promising to increase the efficacy of ICB therapy by increasing the interferon response in MSI CRC.

Biological sciences/Cancer/Oncogenes

Biological sciences/Cancer/Tumour immunology/Immunosurveillance

Colorectal cancer

Tumor cell-intrinsic interferon response

Mevalonate kinase

STAT1 phosphorylation

Immune checkpoint blockade (ICB) by the anti-PD-1 monoclonal antibody or anti-CTLA-4 monoclonal antibody, has been approved for treating microsatellite instability colorectal cancer (MSI CRC) ^[1]. Clinical trials of KEYNOTE-177 and CHECKMATE-142 indicate that half of MSI CRC patients experience limited efficacy from single or dual immunotherapy regimens ^[2–3]. Tumor cell-intrinsic interferon response significantly affects anti-tumor immune resistance and the efficacy of immunotherapy ^[4–7]. Within the tumor microenvironment, activated T cells and NK cells release IFN-γ, triggering JAK1 and STAT1 phosphorylation and enhancing the transcription of Th1 type cytokines (CXCL9, CXCL10, and CXCL11) and antigen-presenting molecules. This activation pathway can recruit anti-tumor immune cells and bolstering cytotoxic activity ^[8–9]. Absence of IFN-γ receptors or mutations in JAK1/STAT1 in tumor cells can suppress T cell-mediated immune responses and lead to immune evasion ^[10]. Furthermore, clinical cohorts of melanoma, non-small cell lung cancer, and MSI CRC all revealed that JAK1 and B2M gene mutations are correlated with the efficacy of ICB therapy ^[11–14].

Abnormally activated cholesterol synthesis is a metabolic hallmark in colorectal cancer and a crucial mechanism of promoting tumor immune evasion ^[15–16]. However, the potential crosstalk between the cholesterol synthesis and tumor cell-intrinsic interferon response remains unclear. Mevalonate pathway is the main source of cholesterol synthesis in vivo. Besides the metabolic synthesis functions, several enzymes of mevalonate pathway have been proved affect the conduction of signaling pathways. 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) promotes MYC phosphorylation by stabilizing the activity of RacGTPase, thereby promoting the development of hepatocellular carcinoma ^[17]. Cytochrome P450 family 27 subfamily A member 1 (CYP27A1) overexpresses in various cancers and produces 27-hydroxycholesterol (27HC), 27HC has been demonstrated that can induce AKT activation and the secretion of IL-6, vascular endothelialgrowth factor (VEGF), resulting in CRC development ^[18]. In addition, the knockdown of sterol regulatory element binding transcription factor (SREBP) could suppress tumor growth in xenograft models of colon cancer and downregulate the expression of genes associated with cell stem ^[19]. These findings indicate that cholesterol metabolizing enzymes may impact interferon reactivity through non-biochemical mechanisms.

In this study, we identified MVK was the key medium in the crosstalk between cholesterol metabolism and the tumor cell-intrinsic interferon response. We sought to demonstrate the underlying mechanism by which MVK affects interferon reactivity in both vitro and vivo. Finally, the expression level of MVK and the efficacy of ICB therapy were demonstrated in animal models and clinical cohorts. Our current study is promising to explain the limited effectiveness of ICB therapy in some MSI CRC patients from the perspective of abnormal cholesterol metabolism affecting interferon reactivity. This is expected to provide theoretical support for the development of targeted MVK-sensitizing ICB therapies.

Cell culture, Reagents, and antibodies

Human cell lines (CCD-841CoN,RKO,HCT15,HT29,HCT116,SW48,SW620,SW480 SW948,DLD1) and mouse cell lines(MC38,CT26) were acquired from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China).These cell lines were routinely authenticated by quality examinations of morphology and short tandem repeat (STR) markers by the supplier.All cells were mycoplasma-free and cultured in RPMI 1640 medium (Gibco, USA) or DMEM medium (Gibco, USA), containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gibco United States). All cells were kept at 37°C in a humidified incubator supplied with 5% CO2.

The CRISPR/Cas9 technology were using to create CT26 Msh2-knockout cells. Plasmids containing GFP, Cas9 and puromycin resistance markers, and single guide RNAs (sgRNA) targeting exon 2 of mouse MSH2 (the sequences are sg1:5’-CCTGGGTCTTGAACACCTCG-3’; sg2:5’-GGGCTTCGTGCGCTTCTTTG-3’) were cotransfected into CT26 cells. The transfected cells were treated with puromycin (2 µg/ml) and selected by FACS. Cell clones were enriched in 96-wells plates. The qRT-PCR and western blot analysis were used to verify the expression of MSH2 .

The antibodies involved are listed in Supplementary Table S1,and drugs utilised in this work include Atorvastatin (S5715, Selleck), IFN-γ (I17001/I4777, Sigma), Anti-mouseCD8α-inVivo(A2102, Selleck), Anti-mousePD1-inVivo(A2122, Selleck).

Patients and Clinical Specimens

The colorectal tumours and paired normal tissues were collected from 68 MSIH-H CRC patients who underwent radical resection in the Department of Colorectal Surgery, Harbin Medical University Affiliated Cancer Hospital. The patients detailed statistics are provided in Table S5. Serum samples were obtained from 3 patients of CRC. Informed written consents were obtained from all subjects.

None of the patients in this study had undergone prior surgeries or been taking any medication. The research protocol involving human conforms to the principles of the Helsinki Declaration and was approved by the Clinical Research Ethics Committee of the Cancer Hospital affiliated with Harbin Medical University.(Approval number:KY2023-57)

Plasmid and truncated protein

The full-length myc/flag-tagged human MVK/STAT1 cDNA were obtained from General Biology(Anhui, China). The myc/flag-tagged mouse Mvk cDNA are synthesized in laboratory based on the transcripts from NCBI database, using the pCDH-EF1-MCS-CMV-copGFP-T2A-Puro vector General Biology(Anhui, China).Sequences of the transcriptss used for plasmid construction are listed in Supplementary Table S4.

Using the plasmid of wildtype human STAT1 as the template, truncate five structural domains separately(TAD:between amino acids from 683 to 750)(SH2:between amino acids from 481 to 683)(DBD:between amino acids from 317 to 481)(CCD:between amino acids from 138 to 317)(NT:between amino acids from 1 to 138). All sequences of truncated proteins are listed in Supplementary Table S4.

Additionally, the siRNAs of human MVK, MVD, FDFT1, ACAT2, GGPS1, FDPS, PMVK, HMGCR, HMGCS1 and IDI1 were obtained from General Biology(Anhui,China). (Supplementary Table S2-siRNA sequences).

Lentivirus transduction and CRISPR‒Cas9 gene editing

We used pCDH-EF1-MCS-CMV-copGFP-T2A-Puro lentiviral vectors obtained from General Biology(Anhui,China) to clone MVK sequences. The lentivirus were subsequently used to package the vectors and infected cells for six hours. The MVK overexpressing cells were selected by medium containing puromycin (Sigma).

The CRISPR/Cas9 technology were using to create MVK-knockout cells. Plasmids containing GFP, Cas9 and puromycin resistance markers, and single guide RNAs (sgRNA) targeting exon 2 of mouse MVK were cotransfected into cells for 24h. The transfected cells were treated with puromycin (2 µg/ml) and selected by FACS. Cell clones were enriched in 96-wells plates. The qRT-PCR and western blot analysis were used to verify the expression of MVK after 14 − 20 days.(Supplementary Table S4-sgRNA sequences).

Co-immunoprecipitation

For co-immunoprecipitation (Co-IP), the 1mL lysis buffer were used to treat 10⁶ cells on ice for 20 min and centrifuged at 12,000 rpm for 15 min at 4℃. The supernatant was incubated with 50µL anti DYKDDDDK magnetic agarose suspension (A36798, ThermoFisher) for 30 minutes at room temperature, followed by collecting magnetic agarose with Magnetic frame.After washing by lysis buffer, magnetic agarose were boiled with 5x loading buffer for 10 min followed by western blotting analyse.

The plasmid of flag-tagged STAT1 and flag-tagged truncations, were transiently transfected with transfection reagent for 48 h. Cells were lysed bt radioimmunoprecipitation assay (RIPA) buffer in the presence of complete protease inhibitors and used anti DYKDDDDK magnetic agarose for immunoprecipitation.

Multi-color IHC (mIHC)

Formalin-fixed paraffin-embedded tissue (FFPE) sections from subcutaneous transplant tumor tissue were cut in 4µm serial sections. Conduct antigen retrieval using the immunohistochemical protocol until the primary antibodies were incubated (Primary antibodies included CD3 and CD8a). Subsequently, multi-color IHC was performed using the five-color multiplex fluorescent immunohistochemical staining kit (abs50013, ABSIN). The position of the primary antibodies binding specific antigen were paired to TSA fluorophore from the kit. All fluorophores and DAPI were prepared according to manufacturer guidelines. For multiple fluorescent staining, sections were processed starting from the antigen retrieval step to remove binding antibodies, and then they were incubated with another primary antibody. This was repeated until all antigens were stained. Finally, counterstaining was performed with DAPI and anti-fluorescence quenching blocker was added dropwise. Images were captured using an Olympus BX53 microscope under suitable laser excitation conditions.

Mass Cytometry

Stain 6 to 11 million Percoll-enriched cells with 0.5mM cisplatin in 1mL PBS (without Ca2 + and Mg2+) at room temperature for 2 minutes. Add 2mL of cell staining buffer (Fluidigm, catalog number 201068) to halt the reaction, and centrifuge the mixture at 500 × g for 5 minutes. Following Fc blockade (BioLegend, Cat # 422302), the cells were incubated with a cocktail of metal-labeled antibodies, including 41 types (Supplementary Table S1.), at room temperature for 30 minutes. After washing with the cell staining buffer, the cells were incubated with 125mM Intercator Ir (Fluidigm, Cat # 201192A) in fixed and osmotic buffer (Fluidigm, Cat # 201067) at room temperature for 1 hour. Subsequently, wash the cells twice with Maxpar water (Fluidigm, Cat # 201069), mix with EqBeads (Fluidigm, Cat # 201078), and analyze on a CyTOF Helios machine. Refer to Supplementary Table S1 for the detailed staining protocol.

Construction of hPBMC-PDX model and therapeutic experiments in vivo

Immediately following surgical removal, fresh colorectal cancer tumor samples were collected and transported in cold complete culture medium. The tissue was then sectioned into 2-3mm pieces and implanted into NYG mice from Charles River (Beijing, China), which are immunodeficient due to knockout mutations in Prkdc and Il2rg genes. The growth of the xenografts was monitored twice weekly using calipers, and subsequent passaging commenced once the tumor volume reached approximately 0.1 cm³.Upon reaching the third passage, inject human PBMCs (approximately 5 x 10⁶) into NYG mice, to create a humanized PBMC-PDX model. Suspend the human PBMCs at a density of 4 x 10⁶ cells per 0.1 ml in sterile phosphate-buffered saline and inject them intraperitoneally with a 1-cc tuberculin syringe.NYG mice can receive donor PBMCs that match or do not match the PDX implanted in their bodies. Donor blood samples do not require HLA typing.

Approximately 2 weeks after injection, perform flow cytometry analysis to assess the human cells in the mice. Mice with a human CD45 + cell percentage of > 1% are included in the experimental group. Monitor the health status of mice daily following human cell implantation. Based on the growth rate of huPBMC-PDX, mice were randomly added to treatment group and control group when the size of tumour reached 0.1 cm³ approximately. The hPBMC-PDX model was raised in the pathogen-free environment. Treatment groups received the following: Anti mouse PD-1 monoclonal antibody in vivo (0.2 mg/mice, twice a week, i.p.) or PBS. Each experimental group includes 3 mice with Unilateral implanted tumors. Record tumor growth every 3 to 4 days and calculate tumor volume using the formula V=(length x width²). If the tumor's size exceeds 1.5 centimeters in any dimension, the mouse will be humanely euthanized.(Approval number:KY2023-57)

Subcutaneous transplantation tumor model in mice and therapeutic experiments

Six-week-old female C57BL/6,BALB/c,and BALB/c-nude mice obtained form Charles River(Beijing,China) were subcutaneously injected with 1 × 10⁶ cells into the right flank.Upon tumour formation, treatment was initiated with Anti mouse PD-1 monoclonal antibody in vivo (0.2 mg/mice, twice a week, i.p.); Atorvastatin (0.2 mg/mice daily, p.o.); Anti mouse PD-1 monoclonal antibody in vivo (0.2 mg/mice, twice a week, i.p.) + Atorvastatin (0.2 mg/mice daily, p.o.).Tumour dimensions were consistently monitored every other day using Vernier callipers, and the volume was calculated using the formula: V = [length × (width)²]/2. Mice were euthanised if the tumour lengths exceeded 1.5 cm in any direction. (Approval number:KY2023-57)

Statistical analysis

The mean ± standard deviation denotes the presentation of quantitative data. GraphPad Prism (RRID: SCR_002798) was employed to assess statistical significance using a two-tailed unpaired Student's t-test. Statistical significance is defined as p-values less than 0.05: p < 0.05, p < 0.01, and p < 0.001. At least three independent biological replicates were used in each experiment. The R value is determined by Spearman correlation analysis.

1.MVK impairs the tumor cell-intrinsic interferon response in MSI CRC

To identify the specific mediators involved in cholesterol synthesis and the tumor cell-intrinsic interferon response, we conducted a screening of the key enzymes within the mevalonate pathway, using both siRNA library and GSEA (Fig. 1A, Sup1A). We assessed the gene expression levels of these enzymes and their enrichment with the interferon signaling pathway from the TCGA-CRC cohort, identifying the MVK having the highest enrichment score (Fig. 1B). Additionally, we employed siRNA to interfere with the expression of the enzymes in the MSI CRC cell line HCT116 (Sup1B), and assessed alterations in the expression levels of Th1 type cytokines (CXCL9 and CXCL10) using qRT-PCR and flow cytometry. The findings revealed that, disrupting MVK expression led to the most pronounced elevation in CXCL9 and CXCL10 levels (Fig. 1C, 1D). Subsequently, we gathered paraffin-embedded tumor samples of 68 MSI CRC patients, along with adjacent normal specimens, and discovered that within MSI CRC tumor tissues, higher MVK expression levels correlated with lower levels of CXCL9 and CXCL10, and reduced infiltration of CD8⁺T cells (Fig. 1E). Analysis of transcriptome data from the TCGA-CRC database further validated these findings (Sup1C). We assessed the basal expression of MVK in various human MSI CRC cell lines and three enterocyte lines (Sup1E). We selected two cell lines with high basal MVK expression along with two mouse colorectal cancer cell lines, and used CRISPR-Cas9 to knock out the MVK gene (Sup1H). In these four MVK-knockout cell lines, we observed a significant increase in the transcription levels of CXCL9 and CXCL10 following IFN-γ stimulation, compared to the MVK wild-type (Fig. 1F). Regarding the basal expression of MVK, the results were consistent with the variations noted in the TCGA-CRC database (Sup1D), the WB and IHC analysis also revealed that MVK expression was significantly higher in colorectal cancer tissues than in normal tissues (Sup1F-G).

2.MVK influences the growth of MSI CRC via the tumor cell-intrinsic interferon response

To verify the potential influence of MVK on the progression of CRC cells, we compared the growth and migration of CRC cell lines in vitro with CCK-8 and cell migration assay. The results indicate that no differences in proliferation and migration of these MSI CRC cells following MVK gene knockout (Sup2A-H). In vivo experiments, we established mouse subcutaneous transplant tumor models (Fig. 2A, H) using the MC38-Mvk^WT/KO and CT26^Msh2−/−-Mvk^WT/KO cell lines. We observed that the tumors generated by the Mvk^KO cell lines were significantly suppressed in both growth rate and tumor weight compared to the control cell lines (Mvk^WT) (Fig. 2B-D, Fig. 2I-K). To reconfirm the impact of MVK on the tumor cell-intrinsic interferon response in vivo, the IHC revealed a significant increase in the number of CD8⁺T cells and expression of CXCL9 and CXCL10 within all subcutaneous transplant tumors derived from the Mvk^KO cell lines(Fig. 2E-G,Fig. 2L-N). The qRT-PCR experiments demonstrate that the transcription level of the CXCL9 and CXCL10 in subcutaneous transplanted tumors is significantly elevated as a result of MVK knockout (Sup2I-J). However, when we injected the MC38-Mvk^WT/KO cell lines into BALB/c-nude mice which lacking intact immune system (Fig. 2O), we observed no differences in tumor growth among these immunodeficient mice (Fig. 2P-R), indicating that the inhibition of MVK on MSI CRC growth is dependent on the intact immune system.

3.MVK significantly modifies the cytotoxic T lymphocytes (CTL) in MSI CRC tumor

To investigate the specific impact of MVK on MSI CRC, we conducted mass cytometry analysis (Fig. 3A-B) on MC38-Mvk^WT/KO subcutaneous transplanted tumors from C57BL/6 mice. These tumor-infiltrating lymphocytes were segmented into distinct subgroups based on the cell surface markers (Fig. 3A-B). Within the Mvk^KO group, we observed a significant elevation in the number of T cell subset, particularly CD8⁺T cells (Fig. 3C). Additionally, these T cells displayed a notable upregulation in the expression of effector molecules, such as IFN-γ and GZMB (Fig. 3D). We also stained the subcutaneous transplanted tumors from each group and detected a marked increase in CD8⁺T cells within the MVK knockout group via flow cytometry (Sup3A). Concurrently, mIF revealed that, compared to MVK wild-type tumors, the MVK knockout group exhibited a substantial rise in the numbers of CD3 and CD8 cells within the tumor center, though these differences were not apparent at the tumor margins (Fig. 3E). To further validate the role of MVK in influencing tumor growth via CD8 + T cells, we administered anti-CD8a antibodies to the Mvk^WT/KO group, effectively depleting CD8⁺T cells (Sup3E). Our results demonstrate that, following the depletion of CD8 + T cells, the subcutaneous transplanted tumors in the MVK knockout group resumed their growth (Fig. 3F-I). To exclude the interference caused by potential changes in tumor immunogenicity, we employed immunofluorescence to assess γ-H2AX expression and whole exome sequencing to monitor alterations in genomic mutation levels in both Mvk^WT and Mvk^KO cell lines. These experiments revealed no significant differences in DNA damage or exon mutation rates in cells with MVK knocked out (Sup3B-D).

4.MVK impairs the phosphorylation of STAT1 within the interferon response

We further explored the mechanism of MVK regulating the interferon response. Initially, we transfected the Flag-MVK plasmid into the human cell line RKO and the mouse cell line MC38, and verified the interactions among key proteins in the interferon signaling pathway using CO-IP experiments (Sup4A). The WB revealed an interaction between MVK and STAT1 protein (Fig. 4A). Subsequently, when we transfected the Flag-STAT1 plasmid for CO-IP experiments, we have observed the same result (Fig. 4B). Additionally, using IF we demonstrated the co-localization of STAT1 and MVK proteins in cytoplasm (Fig. 4C). STAT1 comprises five distinct functional domains ^[20]. To clarify how MVK influences STAT1's function, we reconstructed five truncated forms of STAT1 (Fig. 4D). The CO-IP experiments revealed that MVK binds to the TAD region of STAT1. Using the AlphaFold2 website to predict protein-protein interactions, we discovered a high level of interaction between MVK and the TAD region of STAT1(Sup4B). We transfected the Flag-STAT1 plasmid into both MVK knockout and wild-type of RKO and MC38. Co-IP experiments revealed a significant increase in the binding levels of STAT1 and JAK1 proteins in the MVK knockout strains (Fig. 4E). Following stimulation with IFN-γ, a notable elevation in the phosphorylation levels of STAT1 was observed in the MVK knockout strains (Fig. 4F). Additionally, through Lucifer and IF assays, we observed that the nuclear accumulation of pSTAT1 was significantly higher in MVK-knockout cells compared to the control group after IFN-γ stimulation (Sup4C- D). Intriguingly, after adding atorvastatin, a clinically prescribed cholesterol-suppressing drug, to the culture medium for colorectal cancer cells, we observed a dose-dependent reduction in MVK expression levels. And subsequently led to heightened levels of STAT1 and JAK1 protein binding (Sup4E).

5.MVK replenishment in MVK-deficient tumor cells re-suppresses the interferon response

To verified the function of MVK impacting interferon response, we leveraging the Mvk^KO cell lines established in Fig. 1 to generate stable MVK over-expressing lines through lentivirus. We observed that rescuing MVK significantly reduced the phosphorylation levels in cells stimulated with IFN-γ, without affecting the baseline expression of STAT1 (Fig. 5A). To further confirm the tumor cell-intrinsic interferon response in MSI CRC, we utilized that rescuing MVK significantly attenuated the upregulation of CXCL9 and CXCL10 RNA levels following interferon stimulation (Fig. 5B). In vivo experiments, we observed that subcutaneous transplanted tumors with the wild-type MVK experienced accelerated growth following MVK overexpression. Subcutaneous transplanted tumors with MVK knockdown resumed growth following restoration of MVK expression (Fig. 5C-J). We then performed mIF experiments on the subcutaneous transplanted tumors. Within the tumor microenvironment, the restoration of MVK expression led to a significant reduction in the numbers of CD3 and CD8⁺T cells (Fig. 5K-L). The IHC experiments have also demonstrated similar alterations in the level of CXCL9, CXCL10 and CD8⁺T, potentially contributing to the resumed tumor growth in mice (Sup5A-D).

6.Suppression of MVK improves the effectiveness of immunotherapy in MSI CRC

We postulate whether inhibiting MVK expression might enhance T cell infiltration and improve the effectiveness of ICB immunotherapy. For this purpose, we established subcutaneous transplant tumor models with Mvk^WT/KO cells in C57BL/6 and BALB/C mice, and treated them with anti-PD-1 monoclonal antibodies (Fig. 6A, Sup6A). The results revealed that in these mouse tumor models, the MVK knockout group demonstrated significantly greater anti-PD-1 therapeutic efficacy compared to the wild-type group (Fig. 6B-C and Sup6B-C). We further analyzed tumor tissues using IHC and flow cytometry, and consistent with our prior findings, the MVK knockout group displayed increased levels of Th1 chemokines and CTL cells, which contribute to the superior immune therapeutic response (Fig. 6D-E and Sup6D-E). Considering the inhibitory effect of statins on MVK (Sup4E), we unexpectedly discovered that atorvastatin suppresses the expression of MVK. Consequently, we conducted experiments in vivo to investigate the sensitizing effect of atorvastatin on ICB efficacy (Fig. 6F and Sup6F), revealing that, while atorvastatin lacks intrinsic antitumoral activity, it significantly enhances the therapeutic efficacy of anti-PD-1 monoclonal antibodies (Fig. 6G-J and Sup6G-J).

7.The expression of MVK influences the immunotherapy response in MSI CRC patients

Based on the experimental results above, we constructed a hPBMC-PDX model with human immune system to further assess the impact of MVK on the immunotherapy for MSI CRC patients. The hPBMC-PDX model was randomly assigned to either the treatment or control group and treated with PD-1 monoclonal antibody (Fig. 7A). The results demonstrated that the hPBMC-PDX models with low MVK expression exhibited superior immunotherapy efficacy compared to those with high MVK expression (Fig. 7B-D). Analysis of the tumor micro-environment revealed a significant increase in the proportion of CLT cells and Th1 type chemokines in the tumor tissues of mice with low MVK expression levels (Fig. 7E-F). Furthermore, our follow-up observations on the immunotherapy efficacy in the three parental patients. The imaging results indicate that compared with patients with high MVK expression, patients with low MVK expression have a more significant reduction in liver metastasis and exhibit better treatment responsiveness after anti-PD-1 treatment(Fig. 7G). To further assess the clinical relevance, we gathered imaging examination and paraffin-embedded tumor tissue samples from 14 MSI CRC patients who undergone immunotherapy at our center. The therapeutic efficacy was assessed using CT scans, and patients were categorized based on their MVK immunohistochemical scores (Fig. 7F). Our findings revealed that patients with low MVK expression demonstrated significantly greater sensitivity to immunotherapy compared to those with high MVK expression, with notably elevated proportions of PR and CR patients (Fig. 7H-I).

Tumor cells influences the anti-tumor immune response through abnormal cholesterol metabolism. For instance, cholesterol induces the expression of molecules like LAG-3 and PD-1 on CD8⁺ T cells, leading to T cell exhaustion and subsequently diminishing immunotherapy outcomes in melanoma ^[21]. In thymus, cholesterol specifically downregulates T cell signaling and impedes T cell maturation through a complex with TCR-CD3 ^[22]. Besides the direct impact of cholesterol on cancer immunotherapy, numerous key enzymes within mevalonate pathway are also implicated in crosstalk with several signaling pathways. Knockdown of the geranylgeranyl diphosphate synthase 1 (GGPS1) gene markedly influences the membrane localization and activity of KRAS in CRC cells ^[23]. HMGCS1 activates the pyroptosis pathway in ovarian cancer, thereby enhancing the effectiveness of immunotherapy ^[24]. Sterol regulatory element binding transcription factor 2 (SREBF2) enhances liver metastasis of rectal cancer via the activation of the PI3K-AKT-mTOR signaling pathway ^[25].

Resistance to ICB therapy is partly due to the inadequate intrinsic interferon response in tumor cells ^[26–29]. Exome sequencing in melanoma reveals that patients resistant to ICB frequently display mutations in the JAK1, JAK2, and β-2 microglobulin(B2M) genes. The mutated JAK1 in these patients fails to upregulate the expression of ISG genes following IFN-γ stimulation ^[30–31]. Furthermore, reduced expression levels of genes like IFNGR1, JAK1, and STAT1 can significantly impact the survival of melanoma patients ^[7]. Liao et al. also discovered that USP4 disrupts the interferon response in colorectal cancer cells, resulting in ineffective immunotherapy ^[32]. Therefore, an inadequate intrinsic interferon response in tumor cells represents a limiting factor for ICB treatment. In our research, employing the small interfering RNA library, we identified MVK, a key enzyme in the mevalonate pathway, significantly impacting the intrinsic interferon response of tumor cells.

MVK has been demonstrated to be highly expressed in gastric cancer and prostate cancer, and significantly impacting patients' 5-year overall survival ^[33–34]. In this study, we discovered that MVK expression is markedly elevated in CRC, yet its effect does not depend on influencing tumor cell proliferation or invasion. The mouse subcutaneous tumor models also indicate that MVK primarily affects MSI CRC via the intact immune system. Mechanistically, MVK knockout markedly enhanced the abundance of CD8⁺T cells. Prior research has indicated that the increase of CD8 + T cells within the tumor micro-environment correlates with the generation of neoantigens, and both DNA damage and exon mutations in tumor cells have the potential to elevate neoantigen levels ^[35–36]. We also clariid that there are no differences in neoantigen levels. We found that MVK binds to the TAD of STAT1, disrupting the phosphorylation cascade between JAK1 and STAT1. This interaction subsequently diminishes the nuclear translocation of STAT1. Ultimately, this impaired the interferon response within tumor cells, rendering the ICB treatment ineffective. Furthermore, we observed a significant correlation between MVK expression levels and the effectiveness of immunotherapy for MSI CRC in clinical cohorts.

As the inhibitor of mevalonate pathway, statins inhibit tumor cell proliferation, promote tumor cell apoptosis, modulate inflammation, endothelial function, and angiogenesis ^[37–38]. Mechanistically, when statins inhibit the mevalonate pathway, they concurrently decrease the production of intermediates such as isoprene, farnesyl pyrophosphate, and geranyl pyrophosphate. These products are closely linked to the activation of small G proteins like Ras and Rho ^[39–40]. In CRC, using statins either alone or in conjunction with celecoxib, has been shown to decrease polyp formation in genetically susceptible mice with multiple intestinal neoplasia(Min) mouse ^[41–42]. Leveraging the pleiotropic properties of statins, our study discovered that these drugs can suppress the expression of the MVK. We proposed the scientific hypothesis for combined statin and immunotherapy, which was further validated through enhanced therapeutic efficacy observed in mouse subcutaneous tumor model. Nonetheless, this study mainly focuses on atorvastatin, while other statin drugs have not been included. In addition, the precise mechanism by which statins inhibit MVK expression also worth further exploration.

To clarify the mechanism of cholesterol in modulating the interferon response of MSI CRC cells. We identified the MVK, a key enzyme in cholesterol synthesis, disrupts the interferon response of colorectal cancer cells via its non-biochemical function. This crosstalk ultimately diminishes the effectiveness of ICB therapy. Consequently, this research offers the novel targets to enhance the efficacy of immunotherapy for MSI CRC, also positioning MVK as a potential biomarker for predicting the immunotherapeutic prognosis in MSI CRC.

These authors contributed equally

Conflicts of Interest

The authors declare they have no conflicts of interest

Acknowledgements:

We express our heartfelt gratitude to Professor Shuijie Li of Harbin Medical University for his guidance and invaluable advice on this work. Funding for this work was provided by grants from the National Natural Science Foundation of China (nos. U22A20330, 82173233, 82373372, and 82102858), the Key Project of Research and Development Plan in Heilongjiang Province (no. 2022ZX06C01, JD2023SJ40), the Natural Science Funding of Heilongjiang (no. YQ2022H017) and Haiyan Research Fund of Harbin Medical University Cancer Hospital (JJJQ2024-02).

Asaoka Y, Ijichi H, Koike K. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med. 2015;373(20):1979.
Diaz LA Jr, Shiu KK, Kim TW, Jensen BV, Jensen LH, Punt C, et al. Pembrolizumab versus chemotherapy for microsatellite instability-high or mismatch repair-deficient metastatic colorectal cancer (KEYNOTE-177): final analysis of a randomised, open-label, phase 3 study. Lancet Oncol. 2022;23(5):659–670.
Lenz HJ, Van Cutsem E, Luisa Limon M, Wong KYM, Hendlisz A, Aglietta M, et al. First-Line Nivolumab Plus Low-Dose Ipilimumab for Microsatellite Instability-High/Mismatch Repair-Deficient Metastatic Colorectal Cancer: The Phase II CheckMate 142 Study. J Clin Oncol. 2022;40(2):161–170.
Pitt JM, Vétizou M, Daillère R, Roberti MP, Yamazaki T, Routy B, et al. Resistance Mechanisms to Immune-Checkpoint Blockade in Cancer: Tumor-Intrinsic and -Extrinsic Factors. Immunity. 2016;44(6):1255–1269.
Vesely MD, Zhang T, Chen L. Resistance Mechanisms to Anti-PD Cancer Immunotherapy. Annu Rev Immunol. 2022;40:45–74.
Gao J, Shi LZ, Zhao H, Chen J, Xiong L, He Q, et al. Loss of IFN-γ Pathway Genes in Tumor Cells as a Mechanism of Resistance to Anti-CTLA-4 Therapy. Cell. 2016;167(2):397–404.e9.
Sucker A, Zhao F, Pieper N, Heeke C, Maltaner R, Stadtler N, et al. Acquired IFNγ resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions. Nat Commun. 2017;8:15440.
Nagarsheth N, Wicha MS, Zou W. Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy. Nat Rev Immunol. 2017;17(9):559–572.
Tokunaga R, Zhang W, Naseem M, Puccini A, Berger MD, Soni S, et al. CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation - A target for novel cancer therapy. Cancer Treat Rev. 2018;63:40–47.
Shin DS, Zaretsky JM, Escuin-Ordinas H, Garcia-Diaz A, Hu-Lieskovan S, Kalbasi A, et al. Primary Resistance to PD-1 Blockade Mediated by JAK1/2 Mutations. Cancer Discov. 2017;7(2):188–201.
Thompson JC, Davis C, Deshpande C, Hwang WT, Jeffries S, Huang A, et al. Gene signature of antigen processing and presentation machinery predicts response to checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma. J Immunother Cancer. 2020;8(2):e000974.
Zaretsky JM, Garcia-Diaz A, Shin DS, Escuin-Ordinas H, Hugo W, Hu-Lieskovan S, et al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N Engl J Med. 2016;375(9):819–829.
Bortolomeazzi M, Keddar MR, Montorsi L, Acha-Sagredo A, Benedetti L, Temelkovski D, et al. Immunogenomics of Colorectal Cancer Response to Checkpoint Blockade: Analysis of the KEYNOTE 177 Trial and Validation Cohorts. Gastroenterology. 2021;161(4):1179–1193.
Zhang C, Li D, Xiao B, Zhou C, Jiang W, Tang J, et al. B2M and JAK1/2-mutated MSI-H Colorectal Carcinomas Can Benefit From Anti-PD-1 Therapy. J Immunother. 2022;45(4):187–193.
Jun SY, Brown AJ, Chua NK, Yoon JY, Lee JJ, Yang JO, et al. Reduction of Squalene Epoxidase by Cholesterol Accumulation Accelerates Colorectal Cancer Progression and Metastasis. Gastroenterology. 2021;160(4):1194–1207.e28.
Ma X, Bi E, Lu Y, Su P, Huang C, Liu L, et al. Cholesterol Induces CD8 + T Cell Exhaustion in the Tumor Microenvironment. Cell Metab. 2019;30(1):143–156.e5.
Hu J, Liu N, Song D, Steer CJ, Zheng G, Song G. A positive feedback between cholesterol synthesis and the pentose phosphate pathway rather than glycolysis promotes hepatocellular carcinoma. Oncogene. 2023;42(39):2892–2904.
Xiao W, Hu C, Ni Y, Wang J, Jiao K, Zhou M, et al. 27-Hydroxycholesterol activates the GSK-3β/β-catenin signaling pathway resulting in intestinal fibrosis by inducing oxidative stress: effect of dietary interventions. Inflamm Res. 2024;73(2):289–304.
Wen YA, Xiong X, Zaytseva YY, Napier DL, Vallee E, Li AT, et al. Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer. Cell Death Dis. 2018;9(3):265.
Philips RL, Wang Y, Cheon H, Kanno Y, Gadina M, Sartorelli V, et al. The JAK-STAT pathway at 30: Much learned, much more to do. Cell. 2022;185(21):3857–3876.
Ma X, Bi E, Lu Y, Su P, Huang C, Liu L, et al. Cholesterol Induces CD8 + T Cell Exhaustion in the Tumor Microenvironment. Cell Metab. 2019;30(1):143–156.e5.
Wang F, Beck-García K, Zorzin C, Schamel WW, Davis MM. Inhibition of T cell receptor signaling by cholesterol sulfate, a naturally occurring derivative of membrane cholesterol. Nat Immunol. 2016;17(7):844–850.
Jia WJ, Jiang S, Tang QL, Shen D, Xue B, Ning W, et al. Geranylgeranyl Diphosphate Synthase Modulates Fetal Lung Branching Morphogenesis Possibly through Controlling K-Ras Prenylation. Am J Pathol. 2016;186(6):1454–1465.
Zhou W, Liu H, Yuan Z, Zundell J, Towers M, Lin J, et al. Targeting the mevalonate pathway suppresses ARID1A-inactivated cancers by promoting pyroptosis. Cancer Cell. 2023;41(4):740–756.e10.
Zhang KL, Zhu WW, Wang SH, Gao C, Pan JJ, Du ZG, et al. Organ-specific cholesterol metabolic aberration fuels liver metastasis of colorectal cancer. Theranostics. 2021;11(13):6560–6572.
Albacker LA, Wu J, Smith P, Warmuth M, Stephens PJ, Zhu P, et al. Loss of function JAK1 mutations occur at high frequency in cancers with microsatellite instability and are suggestive of immune evasion. PLoS One. 2017;12(11):e0176181.
Sade-Feldman M, Jiao YJ, Chen JH, Rooney MS, Barzily-Rokni M, Eliane JP, et al. Resistance to checkpoint blockade therapy through inactivation of antigen presentation. Nat Commun. 2017;8(1):1136.
Du W, Hua F, Li X, Zhang J, Li S, Wang W, et al. Loss of Optineurin Drives Cancer Immune Evasion via Palmitoylation-Dependent IFNGR1 Lysosomal Sorting and Degradation. Cancer Discov. 2021;11(7):1826–1843.
Middha S, Yaeger R, Shia J, Stadler ZK, King S, Guercio S, et al. Majority of B2M-Mutant and -Deficient Colorectal Carcinomas Achieve Clinical Benefit From Immune Checkpoint Inhibitor Therapy and Are Microsatellite Instability-High. JCO Precis Oncol. 2019;3:PO.18.00321.
Zaretsky JM, Garcia-Diaz A, Shin DS, Escuin-Ordinas H, Hugo W, Hu-Lieskovan S, et al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N Engl J Med. 2016;375(9):819–829.
Garcia-Diaz A, Shin DS, Moreno BH, Saco J, Escuin-Ordinas H, Rodriguez, et al. Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression. Cell Rep. 2017;19(6):1189–1201.
Shi D, Wu X, Jian Y, Wang J, Huang C, Mo S, et al. USP14 promotes tryptophan metabolism and immune suppression by stabilizing IDO1 in colorectal cancer. Nat Commun. 2022;13(1):5644.
Pisanti S, Picardi P, Ciaglia E, D'Alessandro A, Bifulco M. Novel prospects of statins as therapeutic agents in cancer. Pharmacol Res. 2014;88:84–98.
Buhaescu I, Izzedine H. Mevalonate pathway: a review of clinical and therapeutical implications. Clin Biochem. 2007;40(9–10):575–584.
Giannakis M, Mu XJ, Shukla SA, Qian ZR, Cohen O, Nishihara R, et al. Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma. Cell Rep. 2016;17(4):1206.
Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene-induced DNA damage model for cancer development. Science. 2008;319(5868):1352–1355.
Ahmadi M, Amiri S, Pecic S, Machaj F, Rosik J, Łos MJ, et al. Pleiotropic effects of statins: A focus on cancer. Biochim Biophys Acta Mol Basis Dis. 2020;1866(12):165968.
Demierre MF, Higgins PD, Gruber SB, Hawk E, Lippman SM. Statins and cancer prevention. Nat Rev Cancer. 2005;5(12):930–942.
Weissenrieder JS, Reilly JE, Neighbors JD, Hohl RJ. Inhibiting geranylgeranyl diphosphate synthesis reduces nuclear androgen receptor signaling and neuroendocrine differentiation in prostate cancer cell models. Prostate. 2019;79(1):21–30.
Chang WC, Cheng WC, Cheng BH, Chen L, Ju LJ, Ou YJ, et al. Mitochondrial Acetyl-CoA Synthetase 3 is Biosignature of Gastric Cancer Progression. Cancer Med. 2018;7(4):1240–1252.
Swamy MV, Patlolla JM, Steele VE, Kopelovich L, Reddy BS, Rao CV, et al Chemoprevention of familial adenomatous polyposis by low doses of atorvastatin and celecoxib given individually and in combination to APCMin mice. Cancer Res. 2006;66(14):7370–7377.
Teraoka N, Mutoh M, Takasu S, Ueno T, Yamamoto M, Sugimura T, et al. Inhibition of intestinal polyp formation by pitavastatin, a HMG-CoA reductase inhibitor. Cancer Prev Res (Phila). 2011;4(3):445–453.

There is NO conflict of interest to disclose.

Download PDF

Editorial decision: revise
18 Sep, 2024
Review #2 received at journal
10 Sep, 2024
Review #1 received at journal
01 Sep, 2024
Reviewer #2 agreed at journal
26 Aug, 2024
Reviewer #1 agreed at journal
16 Aug, 2024
Reviewers invited by journal
12 Aug, 2024
Submission checks completed at journal
31 Jul, 2024
First submitted to journal
30 Jul, 2024
Unknown event
29 Jul, 2024
Editor assigned by journal
29 Jul, 2024

You are reading this latest preprint version

Mevalonate kinase inhibits anti-tumor immunity by impairing the tumor cell-intrinsic interferon response in microsatellite instability colorectal cancer

Status:

Version 1

Abstract

Background

Method

Results

Conclusion

Figures

Introduction

Material and Methods

Cell culture, Reagents, and antibodies

Patients and Clinical Specimens

Plasmid and truncated protein

Lentivirus transduction and CRISPR‒Cas9 gene editing

Co-immunoprecipitation

Multi-color IHC (mIHC)

Mass Cytometry

Construction of hPBMC-PDX model and therapeutic experiments in vivo

Subcutaneous transplantation tumor model in mice and therapeutic experiments

Statistical analysis

Results

1.MVK impairs the tumor cell-intrinsic interferon response in MSI CRC

2.MVK influences the growth of MSI CRC via the tumor cell-intrinsic interferon response

3.MVK significantly modifies the cytotoxic T lymphocytes (CTL) in MSI CRC tumor

5.MVK replenishment in MVK-deficient tumor cells re-suppresses the interferon response

6.Suppression of MVK improves the effectiveness of immunotherapy in MSI CRC

7.The expression of MVK influences the immunotherapy response in MSI CRC patients

Discussion

Declarations

Conflicts of Interest

Acknowledgements:

References

Additional Declarations

Supplementary Files

Status:

Version 1