Targeting IGF2BP1 Enhances Immune Checkpoint Therapy in High-grade Serous Ovarian Cancer [polyA RNA-seq, sorted co-cultures]
High-grade serous ovarian cancer (HGSC) accounts for the vast majority (>70%) of ovarian cancer-associated deaths with minor therapeutic improvements. Among four proposed molecular subtypes of HGSC, the C5 subtype is distinguished by high proliferative potential and elevated immune evasion which is indicated by an unfavorable MHCI/PD-L1 ratio. However, the underlying key drivers of the C5 subtype remained elusive. Here we identify oncofetal RNA-binding proteins (RBPs) promoting the immune evasion of C5-HGSC. The IGF2 mRNA binding protein 1 (IGF2BP1) enhances expression of the E3 ligase MDM2, which induces degradation of IRF1 resulting in reduced MHCI presentation. Concomitantly, IGF2BP1 elevates PD-L1 synthesis by impairing its microRNA-directed silencing. This shifts the intra-tumoral MHCI/PD-L1 ratio, limits immune cell infiltration, and promotes evasion of tumor cells from cytotoxic T cells (CTLs) in human and syngeneic mouse models of ovarian cancer. The small molecule inhibitor BTYNB impairs IGF2BP1-directed regulation of MHCI/PD-L1 ratios, promotes CTL-directed killing as well as activation, and strongly synergizes with immune checkpoint inhibition (ICI) by Nivolumab in vitro and in vivo. Overall design: Co-culture of ES-2 cells with PBMCs with BTYNB treatment ES-2 cells (2.5x105) we treated with 10 µM BTYNB 24h before HLA-matched human PBMCs in a 1:2 ratio together with CD3/CD28 activation beads (Thermo Fisher) were added for additional 36h. ES-2 cells and PBMCs were separated by FACS sorting before RNA was isolated by TRIZOL. RNA integrity was examined using a BioAnalyzer 2100 (Agilent) before poly(A) RNA enrichment was performed with the poly(A) RNA Selection kit V1.5 (Lexogen). The CORALL Total RNA-Seq V1 (Lexogen) was used for strand-specific library generation. Library preparation and sequencing of paired-end reads was performed on an Illumina NextSeq 1000 platform at the Core Facility Imaging (MLU Halle).