Small cell lung cancer (SCLC) is a highly aggressive form of lung cancer with limited treatment options. Patients often respond well to initial chemo-immunotherapy but relapse quickly, necessitating new strategies to enhance immune responsiveness. Recent research explores combining DNA-damaging therapies with immunotherapy to activate the STING pathway and improve the antitumor immune response. The addition of DNA Damage Repair (DDR) inhibitors, such as DNA-PKcs inhibitors, after chemotherapy has shown promise in activating innate immune sensors and enhancing CD8+ T cell and NK cell pathways in SCLC models. This approach could potentially reshape the tumor microenvironment and sustain an antitumor immune response, offering a maintenance strategy for SCLC treatment.

Given the aggressive nature of small cell lung cancer (SCLC) and the frequent relapse after initial chemotherapy, this work aims to identify novel approaches to enhance the immune response. DNA-dependent protein kinase (DNA-PK) is a targetable enzyme involved in non-homologous end joining (NHEJ) and antiviral responses. DNA-PK plays a role during cGAS/STING dependent and independent viral DNA sensing pathway. Thus, we investigated the role of DNA-PK inhibition (DNA-PKi) as a maintenance strategy following cisplatin treatment in SCLC patients, focusing on its ability to enhance antitumor immune responses. We included different models of SCLC, in particular SCLC patient-derived peripheral blood mononuclear cells (PBMCs), SCLC cell lines and cocultures of PBMCs with SCLC cells and SCLC tumor spheroids. SCLC cells were treated in vitro with low-dose cisplatin (0.5 µM), and blood was collected from SCLC patients on day 3 of cisplatin treatment. SCLC cells and PBMCs were subjected to transcriptomic sequencing and gene expression profiling to assess the effect of DNA-PKi on PBMC subsets (CD8+ T cells, NK cells and monocytes). Gene set enrichment analysis (GSEA) of RNA-seq data revealed that cisplatin treatment enriched several DDR pathways and upregulated immune-related pathways in both SCLC cell lines and patient-derived PBMCs. Sequential treatment with DNA-PKi after cisplatin significantly activated CD8+ T-cell and NK-cell pathways, as well as enhancing interferon-related pathways and gene signatures associated with viral infections. In vitro treatment with DNA-PKi resulted in increased expression of pro-inflammatory markers in PBMCs, promoting M1 polarization of macrophages (TNF-α, IL-6 and CXCL10) while inhibiting M2 polarization (IL-10, IL4 and CCL22). This shift towards an anti-tumor phenotype was confirmed by enhanced NK cell-mediated cytotoxicity against SCLC cell lines. In addition, combination therapy with DNA-PKi and anti-PD-L1 resulted in increased immune cell infiltration into tumor spheroids, demonstrating a synergistic effect on anti-tumor immunity. In conclusion, our data suggests that DNA-PKi may improve immune responsiveness and potentially prolong antitumor effects in SCLC patients. Future investigations are warranted to further elucidate these mechanisms and to evaluate the clinical applicability of DNA-PKi in combination with existing therapies for SCLC. Luisa Amato, Caterina De Rosa, Concetta Tuccillo, Francesca Iommelli, Viviana De Rosa, Virginia Tirino, Federica Papaccio, Kavya Ramkumar, Qi Wang, Jing Wang, Alberto Servetto, Floriana Morgillo, Lauren Averett Byers, Fortunato Ciardiello, Carminia Maria Della Corte. Efficacy of DNA-PK inhibitor as maintenance strategy after cisplatin induction in SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2895.

BackgroundBreast cancer manifests as a heterogeneous pathology marked by complex metabolic reprogramming essential to satisfy its energy demands. Oncogenic signals boost the metabolism, modifying fatty acid synthesis and glucose use from the onset to progression and therapy resistant-forms. However, the exact contribution of metabolic dependencies during tumor evolution remains unclear.MethodsIn this study, we elucidate the connection between FASN and LDHA, pivotal metabolic genes, and their correlation with tumor grade and therapy response using datasets from public repositories. Subsequently, we evaluated the metabolic and proliferative functions upon FASN and LDHA inhibition in breast cancer models. Lastly, we integrated metabolomic and lipidomic analysis to define the contributions of metabolites, lipids, and precursors to the metabolic phenotypes.ResultsCollectively, our findings indicate metabolic shifts during breast cancer progression, unvealling two distinct functional energy phenotypes associated with aggressiveness and therapy response. Specifically, FASN exhibits reduced expression in advance-grade tumors and therapy-resistant forms, whereas LDHA demonstrates higher expression. Additionally, the biological and metabolic impact of blocking the enzymatic activity of FASN and LDHA was correlated with resistant conditions.ConclusionsThese observations emphasize the intrinsic metabolic heterogeneity within breast cancer, thereby highlighting the relevance of metabolic interventions in the field of precision medicine.

Background: Lung cancer (LC), including both non-small (NSCLC) and small (SCLC) subtypes, is currently treated with a combination of chemo- and immunotherapy. However, predictive biomarkers to identify high-risk patients are needed. Here, we explore the role of peripheral blood mononuclear cells (PBMCs) as a tool for novel biomarkers searching. Methods: We analyzed the expression of the cGAS-STING pathway, a key DNA sensor that activates during chemotherapy, in PBMCs from LC patients divided into best responders (BR), responders (R) and non-responders (NR). The PBMCs were whole exome sequenced (WES). Results: PBMCs from BR and R patients of LC cohorts showed the highest levels of STING (p < 0.0001) and CXCL10 (p < 0.0001). From WES, each subject had at least 1 germline/somatic alteration in a DDR gene and the presence of more DDR gene mutations correlated with clinical responses, suggesting novel biomarker implications. Thus, we tested the effect of the pharmacological DDR inhibitor (DDRi) in PBMCs and in three-dimensional spheroid co-culture of PBMCs and LC cell lines; we found that DDRi strongly increased cGAS-STING expression and tumor infiltration ability of immune cells in NR and R patients. Furthermore, we performed FACS analysis of PBMCs derived from LC patients from the BR, R and NR cohorts and we found that cytotoxic T cell subpopulations displayed the highest STING expression. Conclusions: cGAS-STING signaling activation in PBMCs may be a novel potential predictive biomarker for the response to immunotherapy and high levels are correlated with a better response to treatment along with an overall increased antitumor immune injury.

Osimertinib is a third-generation tyrosine kinase inhibitor clinically approved for first-line treatment of EGFR-mutant non-small cell lung cancer (NSCLC) patients. Although an impressive drug response is initially observed, in most of tumors, resistance occurs after different time and an alternative therapeutic strategy to induce regression disease is currently lacking. The hyperactivation of MEK/MAPKs, is one the most common event identified in osimertinib-resistant (OR) NSCLC cells. However, in response to selective drug pressure, the occurrence of multiple mechanisms of resistance may contribute to treatment failure. In particular, the epithelial-to-mesenchymal transition (EMT) and the impaired DNA damage repair (DDR) pathways are recognized as additional cause of resistance in NSCLC thus promoting tumor progression. Here we showed that concurrent upregulation of ITGB1 and DDR family proteins may be associated with an increase of EMT pathways and linked to both osimertinib and MEK inhibitor resistance to cell death. Furthermore, this study demonstrated the existence of an interplay between ITGB1 and DDR and highlighted, for the first time, that combined treatment of MEK inhibitor with DDRi may be relevant to downregulate ITGB1 levels and increase cell death in OR NSCLC cells.

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.

Small cell lung cancer (SCLC) is a highly invasive and rapidly proliferating lung tumor subtype. Most patients respond well to a combination of platinum-based chemotherapy and PD-1/PDL-1 inhibitors. Unfortunately, not all patients benefit from this treatment regimen, and few alternative therapies are available. In this scenario, the identification of new biomarkers and differential therapeutic strategies to improve tumor response becomes urgent. Here, we investigated the role of exosomes (EXs) released from the peripheral blood mononuclear cells (PBMCs) of SCLC patients in mediating the functional crosstalk between the immune system and tumors in response to treatments. In this study, we showed that PBMC-EXs from SCLC patients with different responses to chemoimmunotherapy showed different levels of immune (STING and MAVS) and EMT (Snail and c-Myc) markers. We demonstrated that PBMC-EXs derived from best responder (BR) patients were able to induce a significant increase in apoptosis in SCLC cell lines in vitro compared to PBMC-EXs derived from non-responder (NR) SCLC patients. PBMC-EXs were able to affect cell viability and modulate apoptotic markers, DNA damage and the replication stress pathway, as well as the occurrence of EMT. Our work provides proof of concept that PBMC-EXs can be used as a tool to study the crosstalk between cancer cells and immune cells and that PBMC-EXs exhibit an in vitro ability to promote cancer cell death and reduce tumor aggressiveness.

Background & aims: Non-alcoholic fatty liver disease is increasingly gaining epidemiological ground in liver diseases. Among the proposed non-pharmacologic interventions, dietary interventions have been widely used. Several patients suffering from it complain of gastrointestinal symptoms unrelated to organic gastrointestinal tract disease. However, the role of drinking water quality modifications in this regard has not been investigated in depth.Methods: Patients with upper or lower functional gastrointestinal symptoms were enrolled and divided into groups based on bright liver ultrasound relief's presence (SP) or absence (NSP). These patients were asked to drink bicarbonate-sulphate-calcium-magnesium and sodium-low drinkable water (Fonte Essenziale & REG;) for six months. Participants were assessed at baseline (T0), at the end of six months of drinking water intake (T6), and after an additional six months of washout (T12) by questionnaires designed to evaluate lower and upper gastrointestinal symptoms (Leeds dyspepsia score, short form) severity and frequency.Results: A total of 61 patients were enrolled. In the SP population, the severity of lower gastrointestinal symptoms improved between T0-T6 (Z: -2.437; ES: 0.312) and worsened after the water washout (Z: -2.492; ES: 0.319). The same was for the Leeds score severity sub score in T0-T6 (Z: -2.850; ES: 0.364) and T6-T12 (Z: -2.921; ES: 0.374). These improvements seem unrelated to the severity of liver steatosis at baseline. Furthermore, no safety issues were recorded while taking the water nor during the six-month follow-up afterwards.Conclusion: Regular six-month intake of 400 mL of Fonte Essenziale & REG; water was associated, in the absence of dietary regimen modifications, with an improvement in some qualitative and quantitative features of upper and lower functional gastrointestinal symptoms in both an SP and NSP sample.& COPY; 2023 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.

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.