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Abstract
Background The level of expression of a mutation-associated neoantigen (MANA) is a critical determinant of effective T cell priming.1 Thus, interventions that increase the expression of a protein containing a MANA could increase responses to immune checkpoint inhibitors (ICI), especially for tumors with relatively low mutation burden like microsatellite stable (MSS) colorectal cancer (CRC).1 We have shown that radiation therapy (RT) upregulates the expression of genes containing MANA in a preclinical model, resulting in enhanced presentation of the derived MANA.2 We also found expansion of CD8 T cells against a MANA upregulated in expression by RT in a lung cancer patient with complete response to RT+ipilimumab.3 Here we set out to test if MANA upregulation is a common response of human tumors to RT.
Methods Patient-derived tumor organoids (PDO) from colorectal (CRC) (n=3) (figure 1a) and lung (n=4) cancer were characterized for radiation response4 (figure 1b). PDOs were then irradiated with doses of 5-8 Gy daily for 3 days (RT) or left untreated (UT) and RNAseq performed 24h post-RT. Differentially expressed genes were identified using DESeq2 differential expression analysis. Ingenuity Pathway Analysis (IPA) was used to identify pathways modulated by RT.
Results In the CRC PDO, 2034 and 362 genes were found to be concordantly upregulated and downregulated, respectively, by RT (figure 1c). Upregulated genes were associated with p38 MAPK signaling and innate immune signaling pathways (figure 1d). This RT-modulated gene set was used to interrogate predicted MANA dataset from CRC patients [1]. We found that ≥1 MANA-encoding gene was predicted to be significantly upregulated by RT in 263/266 patients from the TCGA COADREAD MSS cohort (figure 2a). The median number/patient of upregulated MANA was 11 (range 0-79), while for downregulated MANA it was 0 (range 0-15, figure 2b). In the lung PDO, 439 and 117 genes were found to be concordantly upregulated and downregulated, respectively, by RT. Upregulated genes were largely associated with DNA damage response pathways. We found that ≥1 MANA-encoding gene was predicted to be significantly upregulated by RT in 29/35 patients from a lung cancer dataset.5 The median number/patient of upregulated MANA was 3 (range 0-25), while for downregulated MANA it was 0 (range 0-14).
Conclusions Our data support the novel concept that RT can uncloak neoantigens in tumors like MSS CRC where low neoantigen expression may preclude effective T cell priming and contribute to ICI resistance.
Acknowledgements The authors are grateful for the financial support of The NIH (R01CA198533, SD) and from the AACR (21-40-12-VANN, SV).
References
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a. Representative H&E images showing morphological similarity between carcinoma cells in the original resection and organoid culture for two CRC PDOs. b. Radiation response for each CRC organoid studied. Surviving fraction was evaluated based on ATP levels as measured using the CellTiter-Glo Assay, in irradiated compared to unirradiated PDO samples at 1 week following exposure to single radiation doses between 2 Gy and 12 Gy.4 c. Volcano plot showing 2,034 genes upregulated (red), 362 genes downregulated (blue) and 13,717 genes unchanged (grey) concordantly in all three PDOs at 24 hours following RT (5Gy x 3). Differential expression analysis was applied to a pooled dataset of three CRC PDOs, each with n=4 biological replicates per treatment condition. Cutoffs corresponding to a fold change in gene expression of 1.5 and adjusted p-value of 0.05 as determined from DESEQ2 were used to define the threshold between genes that are unchanged vs modulated by RT. d. Ingenuity Pathway Analysis (IPA) identified a total of 79 pathways enriched (-log(p-value)>2) by RT in the transcriptome, in all three CRC PDOs. Twelve representative pathways are shown.
a. Neoantigen burden associated with MSI-H (n=62), MSI-L (n=68) and MSS (n=266) patient subsets in the TCGA COADREAD cohort as reported by Westcott et al 1 and overlay of the RT-modulated gene set derived from CRC PDOs with MANA encoding genes on a patient-by-patient basis for the MSS subset. Only neoantigens with binding affinity (IC50)<500 nM and upper quartile normalized FPKM>0 are included. b. Box plots (outliers removed) showing the median and percentile range of MANA predicted to be upregulated and downregulated based on the PDO-derived RT-modulated gene set.