Lung cancer remains the leading cause of cancer-related mortality worldwide. Small cell lung cancer (SCLC), which accounts for roughly 15% of all lung cancer, is the deadliest with a dismal 5-year overall survival rate of approximately 5%. SCLC is particularly aggressive for its rapid proliferation and early widespread metastasis. Most SCLCs have high prevalence of somatic mutations, but these tumors display remarkably low levels of neoantigen expression or completely absent. Despite recent promising advances in immunotherapy, majority of SCLC patients remain unresponsive to immunotherapeutic approaches alone. The combination of poor survival and therapy resistance underscores the unmet need to identify novel and effective targeted therapies to treat SCLC. To better understand the molecular underpinnings of SCLC’s aggressiveness, proteomic studies have revealed that SCLC enriches for spliceosome and RNA transport protein expression, suggesting a dependency on RNA-processing machinery. The spliceosome is a dynamic multiprotein complex that is critical to the RNA splicing fidelity, and essential for tumorigenesis. Numerous studies reported that cancers deregulate splicing to promote tumor-specific transcriptome and proteome for disease progression. Furthermore, studies shown that highly proliferative MYC-driven tumors, including SCLC, have heightened dependency on splicing components, such as SF3B1, suggesting that targeting the core spliceosome component may represent a broadly applicable therapeutic vulnerability. Spliceosome inhibition has emerged as a durable anti-cancer strategy, and recent studies demonstrate that spliceosome-targeted therapies (STTs) trigger antitumor immune responses in triple-negative breast cancer (TNBC) by inducing the accumulation of right-handed double-stranded (ds)RNA (A-RNA), thereby activating mitochondrial antiviral signaling protein (MAVS) and Type I interferon (IFN)-driven responses. To evaluate whether spliceosome inhibition is a promising therapeutic vulnerability in SCLC, we treated human SCLC cell lines (H82 and H446) with pladienolide B (PlaB), a small molecule SF3B1 inhibitor. PlaB significantly impaired SCLC proliferation capacity and induced cell death, indicating that SCLC are hypersensitive to spliceosome perturbation. We demonstrated that spliceosome perturbation triggers robust tumor-intrinsic antiviral immunity and Type-I interferon (IFN) response through the accumulation of A-RNA. To characterize the extent of spliceosome perturbation induced dsRNA, we performed immunofluorescence (IF) staining and discovered that spliceosome inhibition also induced uncommon left-handed Z-RNA, a potent necroptosis-activating ligand. Z-RNA induction upon spliceosome perturbation was confirmed in SCLC and immortalized mouse embryonic fibroblasts (MEFs) in vitro by immunofluorescence staining, flow cytometry, and RNA dot-blot assay. We further categorized the Z-RNA species generated through RNA immunoprecipitation followed by sequencing (RIP-Seq) and revealed that the intron-retained RNAs were the main source of endogenous Z-nucleic acid, implicating spliceosome inhibition-induced intron-retention as the driver of necroptosis ligands, Z-RNAs. Z-nucleic acids (Z-NAs) are activators of Z-nucleic acid-sensor Z-DNA binding protein 1 (ZBP1), which trigger the downstream necroptosis cascade. To identify and define the cell death mechanism triggered by spliceosome perturbation, we inhibited the spliceosome pharmacologically and through genetic neutralization of SF3B1 in immortalized necroptosis-competent and necroptosis-incompetent mouse embryonic fibroblasts (MEFs), and mouse SCLC cell lines. Spliceosome inhibition induced necroptosis in necroptosis-competent cells via ZBP1 activation, indicating that necroptosis-ligands generated upon spliceosome disruption is sufficient to drive ZBP1-dependent necroptosis. We further determined that spliceosome inhibition promotes potent ZBP1-dependent cell death in cancer-associated fibroblasts (CAFs), a key cell population within the tumor microenvironment (TME) contributing to therapy resistance. Inducing ZBP1-driven necroptosis in CAFs induced a more immunogenic TME, enhancing immunotherapy response in a SCLC syngeneic immunocompetent mouse model. To determine whether ZBP1 is required for the antitumor effects of spliceosome inhibition in vivo, we compared tumor burden in Zbp1+/+ (Wild-Type, WT) and Zbp1-/- (Knockout, KO) mice treated with PlaB with or without anti-programmed death-1 (anti-PD-1) antibody. Surprisingly, we revealed that both the therapeutic efficacy of PlaB and the ability to potentiate anti-PD-1 treatment were completely abolished in Zbp1-/- mice, demonstrating for the first time that ZBP1 is indispensable for spliceosome inhibition-mediated tumor suppression. To further define how spliceosome perturbation shapes tumor-intrinsic antiviral signaling, we examined Type-I IFN responsive genes by quantitative real-time polymerase chain reaction (qRT-PCR) and by flow cytometry. We evaluated surface expression of human leukocyte antigens ABC (HLA-ABC) and programmed death ligand 1 (PD-L1), key determinants of favorable immune checkpoint blockade (ICB) efficacy. Spliceosome inhibition upregulated Type-I IFN, immunogenicity, antigenicity, and PD-L1 in SCLC cell lines. In addition, we observed that spliceosome perturbation induced a significant reduction in receptor-interacting serine/threonine-protein kinase 1 (RIPK1) protein expression and apoptosis, suggesting that RIPK1 also contributes to the tumor-intrinsic death pathways upon spliceosome disruption. Collectively, these findings demonstrate that spliceosome inhibitors efficiently induce A-RNA and Z-RNA, activating both RIPK1-dependent apoptosis in SCLC cells and on-demand ZBP1-dependent cell death in cells of the TME. This dual mechanism offers an effective therapeutic strategy to overcome immunotherapy resistance in SCLC. More broadly, our results position spliceosome inhibition as a powerful catalyst to trigger antiviral immune response, and a broadly applicable therapeutic target for immunotherapeutic strategies extending beyond SCLC to other ‘cold’ and therapy resistant tumors in which splicing deregulation is prevalent. Further investigations are needed to define how spliceosome perturbation results in RIPK1-dependent cell death and how RIPK1 contributes to acquired resistance to STTs, as well as to identify additional TME cell populations susceptible to ZBP1-driven necroptosis upon spliceosome inhibition.
Xinpei Jiang (Thu,) studied this question.