The RNA-binding Protein MEX3B Mediates Resistance to Cancer Immunotherapy by Downregulating HLA-A Expression

Abstract

Purpose: Cancer immunotherapy has shown promising clinical outcomes in many patients. However, some patients still fail to respond, and new strategies are needed to overcome resistance. The purpose of this study was to identify novel genes and understand the mechanisms that confer resistance to cancer immunotherapy.Experimental Design: To identify genes mediating resistance to T-cell killing, we performed an open reading frame (ORF) screen of a kinome library to study whether overexpression of a gene in patient-derived melanoma cells could inhibit their susceptibility to killing by autologous tumor-infiltrating lymphocytes (TIL).Results: The RNA-binding protein MEX3B was identified as a top candidate that decreased the susceptibility of melanoma cells to killing by TILs. Further analyses of anti-PD-1-treated melanoma patient tumor samples suggested that higher MEX3B expression is associated with resistance to PD-1 blockade. In addition, significantly decreased levels of IFNγ were secreted from TILs incubated with MEX3B-overexpressing tumor cells. Interestingly, this phenotype was rescued upon overexpression of exogenous HLA-A2. Consistent with this, we observed decreased HLA-A expression in MEX3B-overexpressing tumor cells. Finally, luciferase reporter assays and RNA-binding protein immunoprecipitation assays suggest that this is due to MEX3B binding to the 3' untranslated region (UTR) of HLA-A to destabilize the mRNA.Conclusions: MEX3B mediates resistance to cancer immunotherapy by binding to the 3' UTR of HLA-A to destabilize the HLA-A mRNA and thus downregulate HLA-A expression on the surface of tumor cells, thereby making the tumor cells unable to be recognized and killed by T cells. Clin Cancer Res; 24(14); 3366-76. ©2018 AACRSee related commentary by Kalbasi and Ribas, p. 3239.

Figure. 1. MEX3B was identified from an ORF screen to mediate resistance to T-cell–mediated tumor cytotoxicity.

MEX3B was identified from an ORF screen to mediate resistance to T-cell–mediated tumor cytotoxicity. A, Schematic illustration of the ORF screen in patient-derived melanoma cells. B, The comboscore was calculated using the following formula: [(Apoptosis_ORF+TILs – Apoptosis_ORF)/(Apoptosis_GFP+TILs – Apoptosis_GFP)]². The top 20 candidates with the lowest comboscores are shown. MEX3B is one of these top candidates (highlighted in red). C, The relative mRNA expression of MEX3B (using Z score as the readout) from 15 responders and 13 nonresponders to anti–PD-1 immunotherapy was analyzed based on the RNA-Seq data from a published anti–PD-1–treated melanoma patient cohort. Top: The MEX3B Z scores of all samples are ranked from highest to lowest and represented in gradually changing colors from blue to orange. Each column represents the response information (purple, responder; gray, nonresponder) and the MEX3B mRNA expression (blue, high; orange, low) of each patient. Bottom: The Z scores (as a readout for relative MEX3B expression) of each responder or nonresponder are shown. ***, P < 0.001 by Mann–Whitney test. D, TCGA analysis of the correlation of the expression of MEX3B with cytolytic score in patients with skin cutaneous melanoma. MEX3B high (above median expression, n = 236) group: median cytolytic score is 5.66, with an SD of 19.05. MEX3B low (below median expression, n = 236) group: median cytolytic score is 8.41, with an SD of 41.80. ***, P < 0.001 by Mann–Whitney test. E, TCGA analysis of the correlation of lymphocyte score (LS) with the expression of MEX3B in patients with melanoma. LS low (scores 0, 1, and 2; n = 167) group: median MEX3B expression is 2.87, with an SD of 5.69. LS high (scores 3, 4, 5, and 6; n = 163) group: median MEX3B expression is 1.93, with an SD of 3.42. *, P < 0.05 by Mann–Whitney test. F, TCGA analysis of the correlation between the expression of MEX3B and CD8A. Each dot represents log₁₀-transformed mRNA expression of each tumor sample (n = 472). Pearson correlation coefficient = −0.104, P = 0.0237.

Figure 2.. Overexpression of MEX3B in tumor cells decreases their susceptibility to T-cell–mediated cytotoxicity. qRT-PCR validation of overexpression of MEX3B in 2549 (A) and 2559 (C) melanoma cells.

Overexpression of MEX3B in tumor cells decreases their susceptibility to T-cell–mediated cytotoxicity. qRT-PCR validation of overexpression of MEX3B in 2549 (A) and 2559 (C) melanoma cells. GFP-MEX3B-overexpressing or GFP control 2549 (B) or 2559 (D) melanoma cells were incubated with autologous 2549 TILs (B) or 2559 TILs (D) at different E:T ratios for 3 hours, followed by quantification of apoptosis by flow cytometry analysis of activated caspase-3. Data are represented as mean ± SEM. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student t test.

Figure 3.. Overexpression of MEX3B in melanoma cells decreases IFNγ release from autologous TILs.

A, GFP-MEX3B–overexpressing or GFP control 2549 cells were incubated with autologous 2549 TILs at different E:T ratios for 24 hours, followed by measurement of IFNγ levels in supernatants by ELISA. B, GFP-MEX3B–overexpressing or GFP control 2559 cells were incubated with autologous 2559 TILs at different E:T ratios for 24 hours, followed by measurement of IFNγ levels in supernatants by ELISA. Data are represented as mean SEM. ***, P < 0.001; ****, P < 0.0001 by Student t test.

Figure 4.. Decreased surface HLA-A levels in MEX3B-overexpressing melanoma cells is responsible for the decreased IFNγ release from TILs.

A, GFP-MEX3B–overexpressing or GFP control 2559 cells were pulsed with MART-1 for 1 hour and incubated with MART-1–specific TILs at different E:T ratios for 24 hours, followed by measurement of IFNγ levels in supernatants by ELISA. B, GFP-MEX3B–overexpressing or GFP control 2559 cells, as well as those cells overexpressing exogenous HLA-A2, were pulsed with MART-1 for 1 hour and incubated with MART-1–specific TILs at different E:T ratios for 24 hours, followed by measurement of IFNγ levels in supernatants by ELISA. Data are represented as mean ± SEM. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student t test. C, GFP-MEX3B–overexpressing or GFP control 2559 cells were treated with or without IFNγ for 24 hours and stained for HLA-A2, followed by flow cytometry analysis. The representative histograms of each cell line and unstained control, as well as their mean fluorescent intensity (MFI) of HLA-A2, are shown. This graph is representative of three independent experiments.

Figure 5.. Overexpression of MEX3B inhibits HLA-A by binding to the 3′ UTR.

A, The correlation between MEX3B and HLA-A mRNA expression in the skin cutaneous melanoma TCGA database is shown. Overexpression of MEX3B inhibits HLA-A by binding to the 3′ UTR. B, The correlation between MEX3B and HLA-A mRNA expression based on the RNA-Seq data from a published anti–PD-1–treated melanoma patient cohort that is the same as in Fig. 1C. Each dot represents relative mRNA expression of each tumor sample (n = 28). C, qRT-PCR analysis of HLA-A mRNA expression in GFP-MEX3B–overexpressing or GFP control 2549 or 2559 cells. D and E, Dual-luciferase reporter assays were performed using a ffLuc reporter construct or the same construct with its C-terminus fused with the 3′ UTR of HLA-A2 mRNA as well as a Renilla luciferase reporter construct to control the transfection efficiency. These two luciferase reporter constructs, together with GFP-expressing or GFP-MEX3B–expressing plasmids (D) or pCDNA3.1 vector or pCDNA3.1-MEX3B (wild type) or pCDNA3.1-MEX3B-mutKH plasmids (E), were transiently transfected into 293T cells. Two days after transfection, dual-luciferase reporter assays were performed. The relative luciferase activity (from the HLA-A2 3′ UTR–containing ffLuc construct relative to the control ffLuc construct) in cells transfected with GFP-expressing or GFP-MEX3B–expressing plasmids was compared by Student t test (D). The experiment was done in triplicate each time (i.e., for each combination of plasmids, three wells of 293T cells were transfected) and repeated three independent times. Similarly, the relative luciferase activity in cells transfected with pCDNA3.1 vector, pCDNA3.1-MEX3B (wild type), or pCDNA3.1-MEX3B-mutKH plasmids was compared by ANOVA plus the Tukey multiple comparison test (E). The experiment was done in triplicate each time and repeated two independent times. Each dot represents the relative luciferase activity of one well from one experiment. F and G, A RIP assay was performed on 2549 (F) or 2559 (G) melanoma cells. Cell lysates were immunoprecipitated (IP) with mouse anti-MEX3B antibody or mouse IgG as a control. Coimmunoprecipitated RNA was recovered, and qRT-PCR was performed to amplify HLA-A. MEX3B-associated HLA-A levels were normalized to control mouse IgG–associated HLA-A levels. Data are represented as mean ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by Student t test.