A05 ART1, a Mono-ADP-Ribosyltransferase, Regulates Tumor-Infiltrating CD8+ T Cells and Is Highly Expressed in EGFR Mutated Lung Cancers

Abstract

Introduction: ADP-ribosyltransferase 1 (ART1), a GPI-linked cell surface protein, is broadly expressed at the protein level in human tumors and has been linked to tumor progression in colon cancer and gliomas. ART1 may regulate the immune microenvironment through mono-ADP-ribosylation of the P2X7 receptor on CD8+ T cells, leading to T-cell apoptosis through NAD-induced cell death. P2X7R expression is prominent on tissue resident memory (Trm) CD8+ T cells, which have increasingly been recognized for their critical role in immune response. We evaluated the role of ART1 in an immune-competent murine model and sought to determine the expression of ART1 in human tumors, particularly EGFR mutated tumors, which are known to be poorly responsive to immunotherapy. Methods: Initial experiments were performed using the KP1 lung cancer cell line derived from lung tumors of KRASG12D/P53-/- mice. KP1-shART1 cells with doxycycline (DOX)-inducible knockdown of ART1 were generated by lentiviral constructs and used in flank and tail vein tumor models to determine ART1 effects on tumor growth. A lung adenocarcinoma tissue microarray (TMA) was then stained and scored for ART1 expression in order to determine ART1 expression in human tumors. We also evaluated ART1 expression in patient samples by whole-tumor RNAseq and IHC from a prospective clinical trial of neoadjuvant durvalumab +/- subablative radiation therapy (NCT02904954). Results: ART1 knockdown significantly decreased tumor burden in flank and tail vein tumor models. Populations of tumor-infiltrating CD8+ T cells and of P2X7R+/CD8+ T cells were higher with ART1 knockdown, suggesting that ART1 expression on tumor cells may regulate tumor-infiltrating T cells. In human lung cell lines, the EGFR+ cell line H1650 expressed significantly more cell surface ART1 than A549 cells or BEAS cells (2.7- and 6.9-fold, respectively). In the TMA, among 463 stage I patients, 257 patient tumors (55.5%) strongly expressed ART1. Among patients with EGFR mutated tumors (n=79), 69.6% strongly expressed ART1 compared to 52.9% of KRAS+ tumors (n=119, p=0.03). In NCT02904954, among patients with whole-tumor RNAseq performed from preoperative biopsy (n=21), relative ART1 expression was 3-fold higher in EGFR+ patients (n=6). Median post-treatment H-scores for ART1 staining in resected tumors were also higher in EGFR+ tumors (120 vs. 77.5, p<0.065). No patients with EGFR+ tumors (n=8) had a major pathologic response to neoadjuvant durvalumab +/- RT, compared to a 38% combined arm MPR rate in EGFR- tumors (n=34). P2RX7 was strongly expressed in post-treatment tumors by whole-tumor RNAseq and trended higher in responders. Conclusions: ART1 expression on lung cancer cells modulates tumor-infiltrating CD8 T cells. Knockdown of ART1 abrogates tumor growth, suggesting that ART1 may be a potential novel immune checkpoint and a therapeutic target. ART1 is particularly overexpressed in EGFR mutated lung cancers and may provide one mechanism to help explain their poor response to immunotherapy.