SCLC is a highly aggressive cancer that remains challenging to treat. Combining DNA damaging therapies (chemo-, radiotherapy or DNA damage repair inhibitors, DDRi) with immunotherapy (IO) can enhance the antitumor immune response by activating the stimulator of interferon genes (STING). However, only a small proportion of SCLCs, called "inflamed", benefit from immunotherapy, so there is a need for novel strategies to improve the immune response. We hypothesized that DDRi might sensitize immune resistant SCLC by simultaneously activating multiple innate immune pathways. RNA-seq data available in the cBio Cancer Genomics Portal were used to analyze STING1 mRNA expression in SCLC tumors (n=196 samples). DDR mRNA expression was assessed in n= 60 SCLC cell lines using the SclcCellMinerCDB database. SCLC patients derived PBMCs (n=10 samples) were whole exome sequenced for 58 DDR gene panels. These two models were used to assess the DNA-PK inhibition effect on immune mediated cytotoxicity and DNA/RNA sensors activation. High STING1-expressing SCLC tumours were characterized by increase of other innate immune pathways and DNA/RNA sensors (IFI-16, DDX60/DDX60L and MAVS activator RIG-I), as well as the effector kinases IRF1/7. RNAseq data also showed an inverse correlation between STING1 and DDR protein expression, suggesting that the dysregulation of the DDR system suppresses the innate immune mediated anti-tumour activity. Higher DDR expression of Non-Homologous End-Joining (NHEJ) components was observed in SCLC cell lines, as compared to other DDR pathways. In parallel, whole exome sequencing of PBMC samples from lung cancer patients was performed. Each subject had at least 1 germline alteration in a DDR gene. DNA-PK inhibitor (DNA-PKi) significantly increased the expression of STING and MAVS in SCLC cell lines and PBMCs. Increased mitochondrial recruitment of STING together with increased NK cell-mediated cytotoxicity was found in DNA-PKi treated immune cells. DNA-PKi also increased lymphocytes infiltration into tumour spheroids, resulting in tumour disruption. Collectively, our results elucidate a mechanism of anti-tumor immune response and support DNA-PKi as a novel therapeutic strategy to improve IO response in SCLC.

Although immunotherapy (IO) offers significant advances in the clinical management of NonSmall Cell Lung Cancer (NSCLC), durable benefits are not achieved in all patients. Mechanisms of resistance to IO in NSCLC are complex, mainly comprising tumor and infiltrating immune cells. However, resistance landscape against PD-1 blockade remains unclear. More recently, some evidence has provided evidence for metformin overcoming primary resistance to PD-1 inhibitor in mutant NSCLC models. Considering this, we aimed to assess the impact of the combined treatment of metformin plus atezolizumab on antitumor immune response from NSCLC patients-derived peripheral blood immune cells (PBMCs). mRNA and protein expression of cGAS-STING downstream pathway and monocyte M1 marker levels were measured in PBMCs from NSCLC patients. MTS assay was used to assess cell viability of NSCLC cell lines co-cultured with PBMCs or monocyte-conditioned medium. Evaluation of senescence phenotype was also performed based on a histochemical stain for the detection of β-galactosidase activity. Colony-forming ability of NSCLC cell lines co-cultured with immune cells derived from NSCLC patients (pre-treated with metformin 2 mM + atezolizumab 10 μg/ml for 48h) was also assessed. Metformin ± atezolizumab was able to activate the cGAS-STING pathway in PBMC-derived lymphocytes from NSCLC patients and reduce in vitro cell viability of NSCLC cells in 2D co-culture. Colony formation ability of NSCLC cell lines was reduced by activated lymphocytes pre-treated with metformin ± atezolizumab. Evaluation of the senescent phenotype in PBMC-isolated monocytes showed the greatest reduction in blue staining in the presence of the combined treatments. We also observed a reduction in cell viability by MTS assay of NSCLC cells cultured with monocyte-conditioned media treated with metformin ± atezolizumab. Furthermore, we found increased levels of M1 markers (TNFα, IL1β) in monocytes treated with the combined therapy. Our results provide a novel role for synergistic effects of metformin combined with IO in enhancing the anti-tumor activity of multiple immune cells subsets from NSCLC patients.

Small cell lung cancer (SCLC) is characterised by TP53 and RB1 loss and amplification of MYC in approximately 30% of patients. Our group and others have shown that DNA damaging therapies (chemotherapy, radiotherapy, or DDR inhibitors, DDRi) synergize with immune checkpoint inhibition in SCLC in vivo models by activating a Stimulator of Interferon Genes (STING)-mediated innate immune response. Also, the transcription factor MYC is known to be positively correlated with the expression of CXCL10 and CCL5, the two downstream chemokines of STING pathway, in SCLC clinical datasets. Based on these findings, we hypothesised that the expression of MYC family members defines distinct molecular states that may be associated with unique responses to treatment with DDRi. We explored the landscape of DDR pathways, STING, and immune-related cytokines expression both at mRNA and protein expression levels among a panel of n=9 SCLC cell lines classified as MYChigh or MYClow. We then tested the two SCLC cell line subsets for micronucleus (MN) formation and innate immune activation in response to cisplatin 0.5 μM and/or DNA-PK inhibitor (DNA-PKi) 2 μM. STING and PD-L1 mRNA expression were higher in MYChigh cells as compared to MYClow SCLC cells (p=0.02). Also, in cisplatin-treated SCLC panel, we found that DDR pathway promoted STING upregulation preferentially in MYChigh subset. In parallel, in vitro treatment with DNA-PKi further increased STING expression in MYChigh cells while reduced or had no effect on STING in MYClow SCLC cells. MN assay showed increased MN formation, particularly in DNA-PKi treated MYChigh cells, resulting in cytosolic DNA release and STING pathway activation. Furthermore, DNA damage accumulation by DNA-PKi promoted tumor recognition and NK cell activity by inducing tumor cell display of NKG2D ligands MICA/B and ULBP1/2/3. Overall, MYChigh cells were more responsive to innate immune cell activity possibly due to higher baseline levels of replication stress. Our study report that c-MYC expression levels in SCLC cells differentially impact DNA-PK inhibition-mediated activation of STING pathway and presents STING as a downstream of targetable DDR pathway that could offer therapeutic benefit in a subset of MYChigh SCLC patients.

In the past decade, radiotherapy (RT) has been widely practised for treating extensive-stage small cell lung cancer (ES-SCLC). Despite this, it has been discouraged in phase III trials of first line chemoimmunotherapy. Instead, robust rationales have emerged in favour of using RT as a booster to promote a sustained anti-tumour immune response. On basis of published data defining a subtype of SCLC patients who respond to chemotherapy (CT) classified as "inflamed" and expressing high innate immune genes such as the STimulator of INterferon pathway (STING) pathway and based on further preclinical data supporting STING levels as potential biomarkers of response to combined chemo- and immunotherapy, we hypothesised that innate immune activation may provide an opportunity to evaluate anti-tumour immune activity in vitro. We explored the landscape of STING, Mitochondrial antiviral-signaling protein (MAVS), Gamma-interferon-inducible protein (IFI-16) and immune-related cytokines expression both at mRNA and protein expression levels among two SCLC cell lines, namely H82 and H524. We then tested the effect of in vivo treatment with CT and RT (4 Gy dose) on PBMC subpopulations derived from SCLC patients by LDH cytotoxicity assay. We performed sequential in vitro treatment with CT and RT (4 Gy) in selected SCLC cell lines and we investigated changes in STING pathway, as indicator of DNA damage induced by CT and RT. cGAS, STING and downstream protein p-TBK1 and p-IRF3 were upregulated by CT plus RT in both cell lines. Interestingly, these increments were accompanied by high levels of DNA damage, as suggested by increased levels of H2A.X, ATM, ATR, and DNA-PK. IFI-16 DNA sensor was also increased at both protein and mRNA levels. Moreover, inflammatory T cells recruiting the chemokines IL6, IFN-β, CXCL5, CXCL10, were also significantly increased by sequential treatment with CT and RT. The SCLC patients-derived immune cells activity patients were also significantly increased post-RT as measured by LDH cytotoxicity assay. We demonstrated in cellular models of SCLC a positive modulation of innate immune pathways with CT and RT sequential treatment, suggesting a possible boost role of RT in SCLC patients treated with chemoimmunotherapy.