Abstract 5569: The impact of ADI-PEG20 on PDL1 expression in ASS1 deficient uveal melanoma
Authors: Khadeir Ramsay S. , Phillips Melissa M. , Sagoo Mandeep , Cohen Victoria , Thuang Caroline , Szlosarek Peter W. 
Published: July 2017

Abstract
Uveal melanoma (UM), involving the iris, choroid and ciliary body, is the commonest intraocular tumor in adults. Half of patients develop metastasis with a high mortality despite currently available systemic therapies including immune checkpoint blockade. Studies show that while CTLA4 antagonism has a modest effect in UM, PD1/PDL1 blockade is largely ineffective whereas trials of combination checkpoint blockade are yet to report. We confirmed previously that deficiency of argininosuccinate synthetase 1 (ASS1), a key enzyme involved in arginine synthesis, sensitizes UM cells to arginine deprivation using pegylated arginine deiminase or ADI-PEG20. Early trials of ADI-PEG20 in UM have shown safety and efficacy in the clinic and thus we tested the potential for a combined immunometabolic strategy. ASS1-deficient UM cell lines (OMM1, OMM2.5, MEL270) were analyzed for the immune checkpoint protein PDL1 along with their sensitivity to arginine depletion. We also tested metastatic UM for ASS1 and PDL1 expression, and the impact of ADI-PEG20 treatment using samples from a current clinical trial (NCT02029690). We showed that ASS1 and PDL1 protein expression were absent in the 3 UM cell lines and in a majority of primary tumors (75/102 for ASS1 and 83/102 for PDL1; 5% threshold of expression) and all metastatic tumor biopises (n=16/16 for both). Transfection of ASS1 in OMM1, OMM2.5, MEL270 led to an increase in PDL1 expression by qPCR, western blotting and FACS, which was reversible following knockdown of ASS1. Induction of PDL1 expression by ASS1 was accompanied by interferon (IFN)-alpha and beta (but not IFN-gamma) release into the cell supernatant, and was abrogated using the pan-STAT inhibitor ruxolitinib. Next, PDL1 expression was significantly increased in the 3 ASS1-negative UM cell lines with ADI-PEG20 treatment by 24hrs and was associated with Type I IFN signaling which waned along with PDL1 expression by 48hrs. The ADI-PEG 20 induction of PDL1 was abolished using ruxolitinib, indicating that the upregulation of Type I IFNs is critical for regulation of the PDL1 checkpoint protein. While analysis of UM biopsies of patients progressing on ADI-PEG20 revealed upregulation of ASS1 (n=2/2; and thus resistance to ADI-PEG20) a concomitant increase in PDL1 was not observed (n=0/2). Collectively, our data show that ASS1 is absent in a majority of patient biopsies of primary and metastatic UM tumors and is tightly correlated with PDL1 expression. UM cells lines displayed sensitivity to ADI-PEG20, which upregulated levels of PDL1 expression via Type 1 interferon signaling that may enhance the currently limited efficacy of checkpoint blockade in UM. Further studies are ongoing of the IFN-mediated signaling between ASS1 and PDL1 in UM in response to arginine deprivation with ADI-PEG20.
Citation Format: Ramsay S. Khadeir, Melissa M. Phillips, Mandeep Sagoo, Victoria Cohen, Caroline Thuang, Peter W. Szlosarek. The impact of ADI-PEG20 on PDL1 expression in ASS1 deficient uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5569. doi:10.1158/1538-7445.AM2017-5569