Improving IL12 immunotherapy in glioblastoma by targeting the long noncoding RNA INCR1

Abstract

Purpose: The potent antitumor effects of interleukin 12 (IL12) gene therapy in glioblastoma (GBM) are significantly attenuated by the highly immunosuppressive microenvironment and the upregulation of the PD-1/PD-L1 immune checkpoint. However, combining IL12 gene therapy with PD-1/PD-L1 inhibitors failed to improve efficacy. This study aims to assess the effects of silencing the immunosuppressive long noncoding RNA INCR1 when combined with IL12 therapy.

Methods: RNAscope in situ hybridization was performed to analyze INCR1 and PD-L1 expression in tumor tissues from GBM patients pre- and post-IL12 gene therapy. Quantitative PCR was used to analyze immunosuppressive gene expression in patient-derived GBM cells co-cultured with immune cells stimulated with IL12. The effects of INCR1 and PD-L1 silencing on the expression of immunosuppressive genes were evaluated by RNA sequencing. 3D-cytotoxicity assays were performed to assess the activity of immune cells against GBM tumor cells.

Results: INCR1 and PD-L1 expression was upregulated in tumor tissue from GBM patients treated with IL12 gene therapy compared to the tumor tissue of the same patients before the IL12 treatment. Co-culture of patient-derived GBM cells with IL12-stimulated immune cells increased the expression of several immunosuppressive genes. Knocking down INCR1 was more effective than silencing PD-L1 in reducing the expression of multiple immunosuppressive genes. INCR1 silencing improved IL12-mediated immune cell antitumor activity compared to monoclonal antibodies targeting the PD-1/PD-L1 immune checkpoint signaling.

Conclusion: INCR1 silencing affects more immune evasive pathways than PD-L1. Targeting INCR1 may represent a valid approach to improve the efficacy of IL12 therapy in GBM.

Keywords: INCR1; GBM; IL12; Immunotherapy; PD-L1.

Fig. 1

INCR1 and PD-L1 expression levels increase in GBM patients treated with regulatable IL12 gene therapy: (A) INCR1 (left) and PD-L1 (right) RNA scope staining of FFPE tissues from GBM patients pre- and post-IL12 gene therapy. Red dots are indicative of INCR1 (left panels) and PD-L1 (right panels) signals. Scale bar: 20 μm. B-C Quantification of INCR1 (B) and PD-L1 (C) positive cells in five different GBM patients pre- and post-IL12 therapy. Data were analyzed by paired t-test: *p < 0.05

Fig. 2

IL12 treatment induces the expression of multiple immunosuppressive genes in patient-derived GBM cells (PDGCs). A Schematic representation of the experimental design. PDGCs in monoculture (left) or in co-culture with PBMCs were treated with 50 ng/ml IL12 for 72 h. B-E qPCR analysis of INCR1 (B), immunosuppressive checkpoints and surface molecules PD-L1, PD-L2, and LGALS9 (C), immunosuppressive enzymes and signaling pathways IDO1, SOCS1, TDO2, and NOS2 (D), immunosuppressive cytokines and receptors IL20RA and CSF1 (E) in unstimulated or IL12 stimulated cells. Data shown as mean ± SD of three replicates. Data were analyzed by unpaired t-test: *p < 0.05, ***p < 0.001, ****p < 0.0001

Fig. 3

Silencing INCR1 leads to increased PBMC-mediated tumor cell killing upon IL12 treatment. A Schematic representation of the experimental design. U251 stable transfected with shRNA control or an shRNA targeting INCR1 were cultured as tumor spheres and co-cultured with PBMCs and treated with 50 ng/ml IL12. B-C Representative fluorescent microscopy pictures of GFP-positive control and INCR1-knockdown U251 tumor spheres co-cultured with PBMCs for 96 h with or without IL12 treatment (B) and relative tumor sphere area (C). Data shown as mean ± SD of three replicates. Data were analyzed by unpaired t-test: **p < 0.01, ***p < 0.001

Fig. 4

Knocking down INCR1 reduced the expression of a larger number of immunosuppressive genes compared to PD-L1 silencing. A Volcano plot of differentially expressed mRNAs (RNA-seq) between U251 cells transfected with an siRNA control and U251 cells transfected with two different siRNAs targeting PD-L1. Differentially expressed genes (DEGs) with an FDR < 0.01 and a fold-change ≥ 1.5 are depicted in red (up-regulated) and blue (down-regulated). B GSEA Hallmark analysis of enriched gene sets in PD-L1 knockdown cells compared to control cells (FDR q-value < 0.05). A negative NES indicates enrichment in the control cells. C The Venn diagram shows the number of DEGs in PD-L1 knockdown cells relative to INCR1 knockdown cells. D Gene ontology analysis of genes commonly deregulated in INCR1 knockdown cells compared to PD-L1 knockdown cells. Analysis was performed using DAVID Bioinformatics Tool. E Heatmap of the expression levels of immunosuppressive genes in INCR1 knockdown cells compared to control (n = 3 biological replicates) and PD-L1 knockdown cells (n = 2 biological replicates)

Fig. 5

Silencing INCR1 promotes an increased IL12-mediated PBMC cytotoxicity activity against tumor cells than antibodies against PD-1 or PD-L1. A-B Representative fluorescent microscopy pictures of GFP-positive control (left) and INCR1-knockdown (right) U251 tumor spheres co-cultured with PBMCs for 5 days and treated with 50 ng/ml IL12, 10 µg/ml anti-PD-1, 10 µg/ml anti-PD-L1 (A) and tumorsphere area analysis (B). Data shown as mean ± SD of three replicates. Data were analyzed by unpaired t-test: **p < 0.01, ***p < 0.001, ****p < 0.0001