Targeting MYCN upregulates L1CAM tumor antigen in MYCN-dysregulated neuroblastoma to increase CAR T cell efficacy

Abstract

AbstractBackgroundCurrent treatment protocols have only limited success in pediatric patients with neuroblastomas harboring amplifications of the central oncogene,MYCN. Adoptive T cell therapy presents an innovative strategy to improve cure rates. However, L1CAM-targeting CAR T cells achieved only limited response against refractory/relapsed neuroblastoma in an ongoing phase I trial to date. Here, we investigate how oncogenic MYCN levels influence tumor cell response to CAR T cells, as one possible factor limiting success in trials.MethodsHigh MYCN levels were induced in SK-N-AS cells harboring the normal diploidMYCNcomplement using a tetracycline-inducible system. The inducible MYCN cell model orMYCN-amplified neuroblastoma cell lines were cocultured with L1CAM-CAR T cells. CAR T cell effector function was assessed via activation marker expression (flow cytometry), cytokine release and tumor cytotoxicity (biophotonic signal assessment). The cell model was characterized using RNA sequencing, and our data compared to publicly available RNA and proteomic data sets from neuroblastomas. ChIP-sequencing data was used to determine transcriptionalL1CAMregulation by MYCN using public data sets. Synergism between CAR T cells and the MLN8237 AURKA inhibitor, which indirectly inhibits MYCN activity, was assessedin vitrousing the Bliss model andin vivoin an immunocompromised mouse model.ResultsInducing high MYCN levels in the neuroblastoma cell model reduced L1CAM expression and, consequently, L1CAM-CAR T cell effector function (activation, cytokine release and cytotoxicity)in vitro. Primary neuroblastomas possessing highMYCNlevels expressed lower levels of both theL1CAMtranscript and L1CAM tumor antigen. Indirectly inhibiting MYCN via AURKA using MLN8237 treatment restored L1CAM expression on tumor cellsin vitroand restored L1CAM-CAR T cell effector function. Combining MLN8237 and L1CAM-CAR T cell treatment synergistically increased neuroblastoma-directed killing in MYCN-overexpressing cellsin vitroandin vivoconcomitant with severein vivotoxicity.ConclusionWe shed new light on a primary resistance mechanism in MYCN-driven neuroblastoma against L1CAM-CAR T cells via target antigen downregulation. These data suggest that combining L1CAM-CAR T cell therapy with pharmacological MYCN inhibition may benefit patients with high-risk neuroblastomas harboringMYCNamplifications.