DNA Damage–Mediated Innate Immunity and Related Liquid Biopsybased Biomarkers Uncovering Novel Features of Immune-Responsiveness in Small Cell Lung Cancer

Small cell lung cancer (SCLC) remains one of the most aggressive types of lung cancer. Its poor prognosis is strictly related to rapid tumor growth, early dissemination, high molecular heterogeneity, and treatment adaptability due to subtype plasticity and immune evasion. Although SCLC accounts for approximately 15% of all lung cancers, it is responsible for a high number of lung cancer–related deaths, with five-year survival rates remaining poor despite recent advances obtained from the introduction of immunotherapy in clinical settings. Standard front-line therapy for extensive-stage SCLC (ES-SCLC) consists of four cycles of platinum-based chemotherapy combined with anti–PD-L1 immunotherapy, followed by maintenance with immunotherapy alone. However, patient response to this regimen is highly heterogeneous, with a significant fraction exhibiting primary resistance, and even in responding patients, the benefits are often not durable due to the subsequent acquisition of mechanisms of secondary resistance. This therapeutic gap underscores the urgent need for mechanistic studies to design novel therapeutic strategies to overcome both intrinsic and acquired resistance. A major challenge in SCLC is the absence of targetable oncogenic drivers and reliable predictive biomarkers. Moreover, although SCLC is often characterized by a high tumor mutational burden (TMB), it fails to consistently predict immunotherapy responsiveness, suggesting that immune evasion mechanisms extend beyond mutation-derived antigenicity, due to low antigen-presenting machinery. A recent transcriptomic characterization refined SCLC classification into four molecular subtypes (ASCL1+, NEUROD1+, POU2F3+, and YAP1+) proposing an additional “inflamed” subtype instead of YAP1+ subtype, characterized by high immune infiltration, particularly by cytotoxic T cells and natural killer (NK) cells. Even if this subtype is associated with better clinical responses to chemo-immunotherapy (as demonstrated in trials like Impower-133), the high heterogeneity and dynamic nature of ES-SCLC subtypes requires a deeper understanding of the underlying mechanisms to define why only a subset of patients benefits from immune-based therapies. The present doctoral research aims to investigate the molecular and immunological pathways involved in treatment response/resistance in SCLC through the investigation of three main biological axis: the DNA damage response (DDR), epithelial–mesenchymal transition (EMT), and innate immune activation. The first part of this work focuses on studying the effect of DNA-damaging therapies (namely chemotherapy, radiotherapy or DDR inhibitors) as well as the transcriptional consequences of MYC amplification and MAX co-expression in engineered SCLC models. The results presented in this thesis demonstrates for the first time that sequential administration of a novel DDR inhibitor, specifically DNA-PKi, or radiotherapy following chemotherapy significantly enhanced innate immune signaling through cGAS–STING and MAVS pathways. DNA-PKi can induce a sustained immune response in SCLC through concomitant upregulation of multiple innate immune cytosolic dsDNA/RNA sensors, through the activation of a non-canonical STING-MAVS axis. Moreover, non-neuroendocrine (non-NE) subtypes displayed higher expression of immune markers and stronger responsiveness to combined treatment compared to neuroendocrine (NE) tumors. This subtype-dependent variability in immune activation suggests that the therapeutic response in SCLC is not only determined by tumor-intrinsic effects by genotoxic damages but also by the immune reactivity of the tumor. Transcriptomic profiling revealed a MYC-dependent reprogramming of DDR and immune signaling pathways. Functionally, MYC/MAX-overexpressing cells displayed higher sensitivity to DNA-damaging agents and showed enhanced activation of STING–IRF3 signaling following sequential DNA-PKi after cisplatin treatment. Therefore, MYC amplification primes SCLC cells for both increased DNA damage vulnerability and innate immune activation. The translational part of this work focuses on correlation studies involving peripheral immune components of therapy response, looking for new non-invasive approaches for biomarkers identification. Specifically, ex vivo study of peripheral blood mononuclear cells (PBMCs) showed that cGAS–STING expression correlated with clinical response to anti–PD-L1 therapy. Rare germline variants analysis through whole exome sequencing of PBMCs revealed the presence of pathogenic DDR variants among best responders, suggesting that DDR machinery defects may modulate immunotherapy efficacy. In parallel, the isolation and characterization of exosomes derived from PBMCs (PBMC-EXs) from best responders showed an enrichment in immune sensors such as STING and MAVS. Functionally, these PBMC-EXs were able to reduce viability in SCLC cells in vitro, indicating that circulating immune-derived exosomes actively participate in tumor–immune communication. Finally, the characterization of NK cell heterogeneity and function in SCLC patients undergoing chemo-immunotherapy was performed. Results showed an enrichment of activated NK cells (NK1 and NK3 subsets) in best responders, characterized by upregulation of interferon-stimulated genes, granzyme B, and perforin. Conversely, non-responders exhibited exhausted NK cell profiles represented by NKint and NK2 subsets. Collectively, the results presented in the present thesis establishes novel mechanistic and translational insights into how DDR activation, MYC signaling, and innate immune pathways intersect to determine treatment response in SCLC. It positions PBMC-based assays, exosomes, and NK cell phenotyping as practical tools for real-time monitoring of therapy response. Taken together, these findings suggest we need to change the way we think about SCLC. It is important to consider its immunological complexity and heterogeneity to design more effective, biomarker-informed combination therapies that can change immune-cold tumours into immune-responsive disease.