rs10204525 binding to miR-4717-3p modulates PD-1 expression and predicts the development of immune-related adverse events in patients with advanced cancer treated with anti-PD-1/PD-L1 therapy

Abstract

Background: Predictive biomarkers for anti-programmed cell death-1 (PD-1) and anti-programmed cell death ligand 1 (PD-L1) therapy are needed. Here, we validated the role of PD-1 single nucleotide polymorphisms (SNPs) in predicting the development of immune-related adverse events (irAEs) in patients with advanced cancer treated with anti-PD-1/PD-L1-based immunotherapy and defined the molecular mechanisms underlying the role of identified SNP candidate.

Methods: Blood samples, clinical-pathological characteristics, survival outcomes and irAEs were collected from two cohorts of patients: (1) patients with advanced cancer treated with anti-PD-1/PD-L1 alone and (2) patients with advanced non-small cell lung cancer (NSCLC) treated with anti-PD-1 in combination with platinum-based chemotherapy with or without anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) therapy. PD-1 SNPs including rs2227981, rs7421861, rs11568821, rs36084323, rs2227982 and rs10204525 were genotyped and correlated with clinical-pathological characteristics and irAEs. Putative miRNAs binding to PD-1 SNP candidate were identified by in silico analysis. Validation of miRNA binding to PD-1 SNP allele specificity as well as evaluation of the induced PD-1 modulation was performed in vitro using patient-derived peripheral blood mononuclear cells (PBMCs). Susceptibility of non-cancer cells to immune cells incubated with anti-PD-1 based on PD-1 SNP allele specificity and miRNA modulation was performed by co-culturing non-cancer human epidermal keratinocyte (HaCaT) cells and bronchial epithelial BEAS-2B cells with human leukocyte antigen (HLA)-matched PBMCs, obtained from patients with cancer treated with anti-PD-1/PD-L1-based immunotherapy and carrying a different PD-1 SNP.

Results: Most of the analyzed PD-1 SNPs were not associated with the development of irAEs. In contrast, rs10204525 exhibited a significant association with the occurrence of both grade 1-2 and 3-4 irAEs in both cohorts of patients. Specifically, patients carrying C/C had a higher rate of irAEs as compared with those carrying C/T. rs10204525 mapped on 3' untranslated region (3'-UTR) region of PD-1. miR-4717-3p bound to rs10204525 based on its allele specificity. Modulation of miR-4717-3p expression as well as of miR-4717-3p binding to rs10204525 differentially regulated PD-1 expression and induction in PMBCs harboring C/C or C/T genotypes as well as their ability to recognize and destroy HLA-matched HaCaT cells, even more in the presence of anti-PD-1 therapy. Specifically, PBMCs carrying a C/T genotype displayed a significantly lower ability to recognize and destroy non-cancer cells as compared to those carrying C/C. These results were further validated by co-culturing of both BEAS-2B and HaCaT non-cancer cells with PBMCs carrying differential rs10204525 genotypes, isolated from additional patients with cancer, incubated with anti-PD-1 or anti-PD-1 in combination with anti-CTLA-4 therapy.

Conclusions: These findings have high clinical relevance since they define rs10204525 binding to miR-4717-3p-mediated PD-1 expression and induction as a mechanism modulating the reactivity of immune cells to non-cancer cells as well as a novel biomarker for predicting irAEs in patients with advanced cancer treated with anti-PD-1/PD-L1-based immunotherapy.

Keywords: Biomarker; Immune Checkpoint Inhibitor; Immune related adverse event - irAE; Immunotherapy.

Figure 1. PFS and OS of patients with advanced cancer treated with anti-PD-1/PD-L1 mAbs, atezolizumab, nivolumab and pembrolizumab. At a median follow-up of 21.93 months (5.53–46.67 months), median PFS and OS were 4.15 months (A) and 10.70 months (B), respectively. PFS and OS analysis were performed using the Kaplan-Meier method. mAbs, monoclonal antibodies; PD-1, programmed cell death-1; PD-L1, programmed cell death ligand 1; PFS, progression-free survival; OS, overall survival.

Figure 2. Modulation of PD-1 expression by miR-4717–3 p based on rs10204525 allele specificity in human PBMCs under basal conditions or following IFN-ɣ treatment. Both PBMCs^C/C and PBMCs^C/T were transfected with specific miR-4717–3 p mimic, inhibitor, TSB and a combination of miR-4717–3 p mimic with TSB for 48 hours. Negative miRNA mimic was used as a control. Both transfected PBMCs^C/C and PBMCs^C/T were seeded into 24-well plates at a density of 2×10⁵ cells per well and incubated with IFN-ɣ (100 ng/mL). Following a 24 hours incubation at 37°C in a 5% CO₂ atmosphere, expression levels of (A) miR-4717–3 p normalized to miR-103–3 p and (B) PD-1 mRNA normalized to GAPDH were evaluated by RT-PCR. The performances of each miR-4717–3 p mimic, inhibitor or TSB were compared with negative miRNA mimic and expressed as means±SD of the results obtained in three independent experiments (***p≤0.001). (C) Following a 48 hours incubation at 37°C in a 5% CO₂ atmosphere, PBMCs were harvested, lysed and analyzed by western blot with PD-1-specific Ab. GAPDH was used as a loading control. The performances of each miR-4717–3 p mimic, inhibitor or TSB were compared with negative miRNA mimic and plotted below their representative images as means±SD of the results obtained in three independent experiments (***p≤0.001). (D) Following a 48 hours incubation at 37°C in a 5% CO₂ atmosphere, PBMCs were harvested and cell surface stained with PE anti-PD-1 IgG1k Ab. PE anti-mouse IgG1k was used as a specificity control. Data, expressed as representative histogram profiles of MFI-PD-1, are the results obtained in three independent experiments. Ab, antibody; IFN, interferon; MFI, mean fluorescence intensity; mRNA, messenger RNA; PBMC, peripheral blood mononuclear cell; PD-1, programmed cell death-1; PE, phycoerythrin; RT-PCR, real-time PCR; TSB, target site blocker.

Figure 3. Modulation by rs10204525 allele-specificity of in vitro activity of anti-PD-1 nivolumab on transfected PBMCs co-cultured with HLA-matched HaCaT cells. HaCaT cells were seeded into 24-well plates at a density of 2×10⁵ cells per well. Following a 48 hours incubation at 37°C in a 5% CO₂ atmosphere, cells were co-cultured with HLA-matched PBMCs^C/C and PBMCs^C/T isolated from Patient #1 and #2 transfected with miRNA specific constructs (miR-4717–3 p mimic, inhibitor and a combination of the mimic and TSB) in a 1:5 ratio and treated with 10 µg/mL of anti-PD-1 nivolumab and its human isotype IgG4 for 24 hours. Negative miRNA mimic transfected PBMCs^C/C and PBMCs^C/T were used as controls. (A) Following a 48 hours incubation after transfection, PBMCs were harvested and cell surface stained with PE anti-PD-1 IgG1k Ab. PE anti-mouse IgG1k was used as a specificity control. Data, expressed as MFI, are representative of the results obtained in three independent experiments. (B) HaCaT cell viability was determined by CCK-8 assay. Data are expressed as mean percentages of the viability of co-cultured HaCaT cells±SD as compared with HaCaT cells alone. The mean percentage of cell viability and SD were calculated from three independent experiments; each of them was performed in triplicate. (C) IFN-γ levels in the medium harvested from co-cultured cells were measured by an ELISA Max Deluxe Set Human IFN-γ kit. Data are expressed as a means of IFN-γ levels±SD of the results obtained in three independent experiments; each of them performed in triplicate. (D) The induction of apoptosis of HaCaT cells was determined by flow cytometry analysis of annexin V and PI staining. The levels of apoptosis are plotted and expressed as a mean fraction of annexin V+cells±SD of the results obtained in three independent experiments. *p<0.05. **p<0.01. ***p<0.001. ns indicates not statistically significant. Ab, antibody; CCK-8, cell counting kit-8; HaCaT, human epidermal keratinocyte; IFN, interferon; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; PD-1, programmed cell death-1; PE, phycoerythrin; PI, propidium iodide; TSB, target site blocker.

Figure 4. Differential immune response by rs10204525 genotype in PBMCs from patients with NSCLC co-cultured with non-cancer cells on anti-PD-1 treatment. HaCaT and BEAS-2B cells were seeded into 24-well plates at a density of 2×10⁵ cells per well. Following a 24 hours incubation at 37°C in a 5% CO₂ atmosphere, cells were co-cultured with HLA-matched PBMCs^C/C and PBMCs^C/T isolated from Patients #1–4 in a 1:5 ratio and treated with 10 µg/mL of anti-PD-1 nivolumab and its human isotype IgG4 for 48 hours. Monocultured HaCaT and BEAS-2B cells were used as controls. (A) Following a 48 hours incubation, non-cancer viability was determined by CCK-8 assay. Data are expressed as mean percentages of the viability of co-cultured HaCaT and BEAS-2B cells±SD as compared with HaCaT or BEAS-2B cells alone. The mean percentage of cell viability and SD were calculated from three independent experiments; each of them was performed in triplicate. (B) IFN-γ levels in the medium harvested from co-cultured cells were measured by an ELISA Max Deluxe Set Human IFN-γ kit. Data are expressed as means of IFN-γ levels±SD of the results obtained in three independent experiments; each of them performed in triplicate. (C) The induction of apoptosis of HaCaT and BEAS-2B cells was determined by flow cytometry analysis of annexin V and PI staining. The levels of apoptosis are plotted and expressed as a mean fraction of annexin V+cells±SD of the results obtained in three independent experiments. *p<0.05. **p<0.01. ***p<0.001. ns indicates not statistically significant. BEAS-2B, bronchial epithelial; CCK-8, cell counting kit-8; HaCaT, human epidermal keratinocyte; IFN, interferon; NSCLC, non-small cell lung cancer; PBMC, peripheral blood mononuclear cells; PD-1, programmed cell death-1; PI, propidium iodide.

Figure 5. Enhanced immune response in rs10204525 C/C PBMCs isolated from patients with NSCLC on anti-PD-1 or combined anti-PD-1/CTLA-4 treatment in co-culture with non-cancer cells. HaCaT and BEAS-2B cells were seeded into 24-well plates at a density of 2×10⁵ cells per well. Following a 24 hours incubation at 37°C in a 5% CO₂ atmosphere, cells were co-cultured with HLA-matched PBMCs^C/C and PBMCs^C/T isolated from Patients #5 and #6 in a 1:5 ratio and treated with 10 µg/mL of anti-PD-1 nivolumab alone or in combination with 3.3 µg/mL of anti-CTLA-4 ipilimumab for 48 hours. Human IgG was used as an isotype control. Monocultured HaCaT and BEAS-2B cells were used as controls. (A) Following a 48 hours incubation, non-cancer cell viability was determined by CCK-8 assay. Data are expressed as mean percentages of the viability of co-cultured HaCaT and BEAS-2B cells±SD as compared with HaCaT or BEAS-2B cells alone. The mean percentage of cell viability and SD were calculated from three independent experiments; each of them was performed in triplicate. (B) IFN-γ levels in the medium harvested from co-cultured cells were measured by an ELISA Max Deluxe Set Human IFN-γ kit. Data are expressed as means of IFN-γ levels±SD of the results obtained in three independent experiments; each of them performed in triplicate. (C) The induction of apoptosis of HaCaT and BEAS-2B cells was determined by flow cytometry analysis of annexin V and PI staining. The levels of apoptosis are plotted and expressed as a mean fraction of annexin V+cells±SD of the results obtained in three independent experiments. *p<0.05. **p<0.01. ***p<0.001. ns indicates not statistically significant. CCK-8, cell counting kit-8; CTLA-4, cytotoxic T-lymphocyte antigen 4; HaCaT, human epidermal keratinocyte; IFN, interferon; NSCLC, non-small cell lung cancer; PBMC, peripheral blood mononuclear cells; PD-1, programmed cell death-1; PI, propidium iodide.

Figure 6. Graphical representation of the influence of rs10204525 genotype on PD-1 expression and increased sensitivity of HaCaT cells to anti-PD-1 therapy. 3’-UTR, 3′ untranslated region; GzmB, Granzyme B; HaCaT, human epidermal keratinocyte; IFN, interferon; PBMC, peripheral blood mononuclear cells; PD-1, programmed cell death-1; TNF, tumor necrosis factor.