CoREST Complex Inhibition Alters RNA Splicing to Promote Neoantigen Expression and Enhance Tumor Immunity [scRNA-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. 6-10 week-old female C57BL/6 female mice (Jackson Lab) were inoculated with 2.5x105 B16F10 cells. Mice were treated with 200 µg/mice of Corin (HY-111048, MedChemExpress) or 200 µl vehicle control (5% DMSO/PBS) by daily intraperitoneal (i.p.) injections starting from day 6 after the tumor injection. For Anti-PD1 treatment, mice were treated with 150 µg/mice anti-PD1 (clone 29F.1A12, BioLegend, 135248) or isotype control (clone RTK2758, BioLegend, 400566) 3 times/week starting from day 7 post-tumor grafts. 10 mice per treatment group were used. Minced tumor biopsies were incubated with 200uL 20mg/mL collagenase, 200uL 20mg/mL hyaluronidase, and 5uL DNase for 20 minutes to 1 hour with agitation until the tumor fully dissociated. Single-cell suspension was passed through a 100um strainer and washed with 1X PBS by centrifugation. Cells were resuspended in 1ml of freezing media frozen down. Cryopreserved single-suspension tumor samples were thawed and dead cells were removed using the Dead Cell Removal Kit (Miltenyi, 130-090-101) following manufacturer instructions. Live cells from the flow-though were used for CD45+ cell isolation using CD45 mouse MicroBeads (Miltenyi, 130-052-301) following the recommendations. CD45+ cells were counted and resuspended in PBS + 0.04% BSA at 1000 cells/µL for scRNA-seq.