Abstract 400: MEK inhibition enhances immune checkpoint blockade treatment of murine models of neuroblastoma
Authors: Muthugounder Sakunthala , Hung Long , Chan Randall , Kim Jin , Shirinbak Soheila , Shimada Hiroyuki , Asgharzadeh Shahab 
Published: August 2015

Abstract
Background: The tumor microenvironment (TME) plays an important role in neuroblastoma (NBL) biology. Infiltrations of tumor-associated macrophages with alterations in patterns of pro-inflammatory genes are associated with poor prognosis in NBL. However, the role of regulatory T-cells in NBL remains unknown. As several genes in MAPK pathway are among recurrent mutations in neuroblastomas, and the production of pro-inflammatory mediators in immune cells is MAPK-dependent, we hypothesized targeted kinase inhibitor in combination with check-point blockade could exert synergistic effects on the TME's response to tumor cells. Therefore, we assessed the efficacy of combined therapy using a transgenic MYCN non-amplified neuroblastoma murine model driven by SV40 large T antigen (NB-Tag).
Methods: Three human neuroblastoma cell lines and two NB-Tag derived mouse cell-lines were used for in vitro cell proliferation assay. For in vivo tumor growth models, combinations of cyclophosphamide, topotecan, trametinib, anti-CTLA4, and anti-PD1 therapies were studied in NB-Tag transgenic and transplantable subcutaneous (NB-SQ) murine models.
Results: In vitro studies demonstrated trametinib had the highest anti-proliferative activity compared to other kinase inhibitors, and it effectively blocked cell cycle arrest in G1 phase. These anti-proliferative effects could not be rescued by co-culturing tumor cells with murine or human macrophages. In NB-Tag mice, which develop neuroblastoma spontaneously at 12 weeks, daily oral administration of trametinib (0.6mg/Kg) at pre-tumoral age (10 wk) significantly impaired tumor growth by 17 weeks (1424 mm3 in controls vs. 43 mm3 in treated mice). Treatment of 15 week-old NB-Tag mice (visible tumor by MRI) with trametinib after chemotherapy administration (5-days of Cyclophosphamide + Topotecan) also significantly impaired tumor regrowth (volume four weeks post-chemo, 491 vs. 42 mm3, p = 0.037), and more importantly, treatment increased the survival of NB-Tag mice compared to control (median survival: control = 24.5 wk, treated = 35 wk, p&lt;0.0001). Trametinib treatment also showed similar results in the NB-SQ model. Addition of immune checkpoint blockade of NB-SQ mice with anti-CTLA4 and anti-PD1 to treatment with trametinib further increased survival compared to controls (p&lt;0.0001). Tumors and macrophages collected from mice treated with trametinib showed effective block in phosphorylation of ERK. Transgenic animals that grew despite trametinib treatment showed strong STAT3 phosphorylation suggesting this pathway as an escape mechanism.
Conclusions: Our results provide strong evidence that MEK inhibition combined with checkpoint blockade significantly inhibited tumor formation in a syngeneic subcutaneous model. These findings indicate opportunities to enhance antitumor immunity with the potential to produce durable clinical responses in children with neuroblastomas.
Citation Format: Sakunthala Muthugounder, Long Hung, Randall Chan, Jin Kim, Soheila Shirinbak, Hiroyuki Shimada, Shahab Asgharzadeh. MEK inhibition enhances immune checkpoint blockade treatment of murine models of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 400. doi:10.1158/1538-7445.AM2015-400