Abstract B011: Oncogenic neomorphic ATRX-in-frame fusion mutations mechanistically enforce a new immunotherapeutic vulnerability through altered epigenomic regulation

Abstract

Children with high-risk neuroblastomas (NB) marked by in-frame fusion mutations in the ATRX chromatin remodeler (“ATRX-IFF”) display highly chemoresistant disease. As a result, despite multi-modality therapies, these children have poor overall survival. At present, there are no molecularly-targeted agents for these tumors. Importantly, these tumors are slowly growing, as compared with non-ATRX-IFF neuroblastomas, and are marked by an immune-rich microenvironment, suggesting the potential utility of immunotherapeutic approaches for tumor control. To understand the function of mutant and wild-type ATRX, we first performed shRNA knockdown assays, followed by RNAseq, which demonstrated that the ATRX-IFF functions as a transcriptional activator. Therefore, we hypothesized that the ATRX-IFF protein may directly control the expression of potential targets for immunotherapeutic capitalization. We performed epigenomic assays in both cell lines and PDXs, including ChIP-seq to ATRX-IFF, ATAC-seq for chromatin accessibility, CUT&RUN-seq to identify active gene enhancers and promoters, and integrated this with RNAseq to identify regulated genes. These data were fused with quantitative mass spectrometry-based cell surface biotinylated proteomics to identify potential targets under direct control of ATRX-IFF. We identified that ATRX-IFF neuroblastomas display a unique epigenomic and transcriptomic landscape, with newly established gene enhancers at sites of ATRX-IFF binding in regions of open chromatin that result in transcriptional upregulation of target genes. shRNA-based knockdown of ATRX-IFF results in loss of these gene enhancers and chromatin accessibility, along with suppression of associated gene expression. Importantly, the ATRX-IFF, but not wild-type ATRX, directly binds to and nucleates a super-enhancer at the PTK7 locus, thereby driving extremely high-level cell surface expression of this cell surface pseudokinase receptor. PTK7 shRNAs resulted in loss of ATRX-IFF neuroblastoma growth, suggesting a role for PTK7 in tumor growth in this specific subtype of neuroblastoma. PTK7 is the target of experimental CAR-T cell and ADCC therapies in active preclinical development. Antigen densitometry assay shows markedly higher surface expression of PTK7 in ATRX-IFF NB cell lines and orthotopic PDXs and anti-PTK7 CAR-T cells exhibit higher cytotoxicity toward ATRX-IFF models in an ATRX-IFF-dependent manner in vitro compared to wildtype samples. These data establish a transcriptionally activating neomorphic function for ATRX-IFF and identifies a new mutated oncogene-regulated cell surface protein dependency poised for potential therapeutic capitalization in this high-risk subtype of neuroblastoma. Ongoing work is expanding this work to understand whether this mutationally-derived dependency is relevant in in vivo models of ATRX-IFF neuroblastoma.