Abstract 5073: NT219 induces tumor PD-L1 expression and potentiates anti-PD-1 efficacy
Authors: de Azevedo Ricardo Alexandre , Reuveni Hadas , Bar-Eli Menashe , Curran Michael 
Published: April 2023

Abstract
While the advent of immune checkpoint blockade (ICB) has dramatically improved the prognosis of many immune-infiltrated cancers, for others, unfortunately, these benefits have yet to be realized. The major challenge before the field, then, is to identify combination therapies that act both to combat evolved resistance. NT219 is a novel dual inhibitor of insulin receptor substrates 1 and 2 (IRS) and STAT3. NT219 demonstrated antitumor effects against both in situ and metastatic human melanoma models in mice as a stand-alone treatment and in combination with mutated BRAF and MEK inhibitors. The potential of NT219 to overcome resistance and increase efficacy was demonstrated in PDX models with multiple drug classes. Collectively, these findings provided preclinical proof-of-concept NT219 as a promising novel cancer therapy. Given this data, the goal of our study was to assess the efficacy of combining NT219 with anti-PD-1 and anti-CTLA-4 ICB, and test capacity of the combination to overcome immune resistance. To that end, we have examined PD-L1 expression levels in vitro following NT219 treatment in two different strains of melanoma cells, B16-tdTomato (TMT) and B16 3I-F4, ICB sensitive and ICB resistant derivative lines, respectively. In addition, to evaluate the combination effect of NT219 with ICB therapy in ICB-resistant PDX model, we used a humanized PDX model of pembrolizumab-resistant gastroesophageal tumor (GEJ). These mice were injected with PBMCs from the same patient. Using multiple syngeneic immunocompetent models of melanoma (TMT and 3I-F4 cells) and the humanized PDX model, we investigated the potential of each ICB with NT219. Our findings showed that NT219 increased PD-L1 expression levels on both melanoma cell lines in vitro, however, the PD-L1 expression levels were much more elevated 3I-F4 than TMT cells. Interestingly, the levels of both IRS and activated pSTAT3, the targets of NT219, which consist well known resistance mechanisms, were higher in the resistant cells as compared to the sensitive line. In accordance we found that NT219 was able to restore anti-PD-1 sensitivity in 3I-F4 model (TGI = 58%) in the syngeneic model, promoting tumor rejection and increasing survival rates. The combined approach between NT219 and anti-CTLA-4 displayed a moderate effect (TGI = 37%). In the TMT, NT219 combined with anti-PD-1 showed greater efficacy compared to anti-PD-1 alone (TGI = 63% and TGI = 47%, respectively). These results were confirmed using the humanized ICB-resistant PDX model, where impressive synergy between NT219 and the anti-PD-1 (TGI = 98%) was demonstrated. To summarize, we found a significant synergistic effect of NT219 combined with anti-PD-1 therapy, supported by a mechanism of PDL-1 induction making ICB resistant tumors amenable to ICB treatment. Collectively, these findings demonstrated that NT219 has the potential to reverse ICB resistance in both human PDX and murine syngeneic tumor model systems
Citation Format: Ricardo Alexandre de Azevedo, Hadas Reuveni, Menashe Bar-Eli, Michael Curran. NT219 induces tumor PD-L1 expression and potentiates anti-PD-1 efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5073.