Targeting IGF2BP1 Enhances Immune Checkpoint Therapy in High-grade Serous Ovarian Cancer [small RNA-seq]
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: MiTRAP experiments using 3´UTR of PD-L1 or MS2 control RNA were essentially performed as described recently (Busch et al. 2016). MBP-MS2BP (maltose-binding protein – MS2 binding protein) was coupled to amylose resin. Bait RNA was in vitro transcribed form linearized templates containing MS2 loops and coupled to the pre-coupled MBP-MS2BP. Cell-free extracts of 5x106 ES-2 cells per condition generated in binding buffer (20mM Tris, pH 7.5, 150mM NaCl, 1.5mM MgCl2, 8.6% glycerol and 0.05% NP40) supplemented with protease inhibitor cocktail (1:200; Sigma Aldrich) were cleared by centrifugation and incubated with bait-coupled resin and supplements (11 mg/ml heparin, 1mM DTT and 400 U/ml RNAsin (Promega)) for 30 min at room temperature. After three washing steps with heparin-supplemented binding buffer, protein–RNA complexes were eluted twice in 150 µl binding buffer supplemented with 15mM maltose. Protein enrichment was tested by Western blotting. To identify associated microRNAs, RNA was isolated by phenol-chloroform extraction and quality checked by a Bioanalyzer.  Library preparation and single-end short-read RNA sequencing was performed on Illumina HiSeq 1500 platform at Novogene (Hong Kong).