CoREST Complex Inhibition Alters RNA Splicing to Promote Neoantigen Expression and Enhance Tumor Immunity [PRO-seq]

Epigenetic macromolecular enzyme complexes tightly regulate gene expression at the chromatin level and have recently been found to colocalize with RNA splicing machinery during active transcription1; however, the precise functional consequences of these interactions are uncertain. Here, we identify unique interactions of the CoREST repressor complex (LSD1-HDAC1-CoREST) with components of the RNA splicing machinery and their functional consequences in tumorigenesis. Using mass spectrometry, in vivo binding assays, and cryo-EM we find that CoREST complex-splicing factor interactions are direct and perturbed by the CoREST complex selective inhibitor, corin2–5, leading to extensive changes in RNA splicing in melanoma and other malignancies. Moreover, these corin-induced splicing changes are shown to promote global effects on oncogenic and survival-associated splice variants leading to a tumor-suppressive phenotype. Using predictive machine learning models, MHC IP-MS, and ELISpot assays we identify thousands of neopeptides derived from unannotated splice sites which generate corin-induced splice neoantigens that are demonstrated to be immunogenic in vitro. Corin is further shown to reactivate the response to immune checkpoint blockade and promote dramatic expansion of cytotoxic T cells in an immune cold melanoma tumor model, effectively sensitizing them to anti-PD1 immunotherapy. These data position CoREST complex inhibition as a unique therapeutic opportunity in cancer which perturbs oncogenic splicing programs across broad tumor types while also creating tumor-associated neoantigens that enhance the immunogenicity of current therapeutics and may be readily translated to the clinic. Overall design: SKMEL5 cells were treated for 24h with 2.5uM corin or DMSO and permeabilized as described. All sample preparation was conducted on ice (4°C). Cells were washed in ice cold 1x PBS and resuspended in wash buffer (10 mM Tris-HCl pH 8.0, 10% glycerol, 250 mM sucrose, 10 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 1mM EGTA, Halt protease inhibitor cocktail (Thermo Scientific), and 4 u/mL RNase inhibitor [SUPERaseIN, Invitrogen]). Then cells (2x10^7) were gently permeabilized in permeabilization buffer (10 mM Tris-HCl pH 8.0, 10% glycerol, 250 mM sucrose, 10 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 0.1% Igepal, protease inhibitors cocktail (Roche), 4 u/mL RNase inhibitor [SUPERaseIN, Invitrogen]) for 5 minutes. Cells were recovered by centrifugation (400 x g for 8 minutes) and the supernatant was carefully removed. Cells were washed with 10 mL of wash buffer and then centrifuged again under the same conditions. Finally, cells were resuspended in 400uL of freeze buffer (50 mM Tris-HCl pH 8.0, 40% glycerol, 5 mM MgCl2, 0.5 mM DTT, 4 u/mL RNase inhibitor [SUPERaseIN, Invitrogen]) and stored at -80°C.