Cancer Gene Therapy

Lung adenocarcinomas frequently harbour actionable oncogenic mutations that are vulnerable to treatment with targeted therapies. While responses to targeted therapies are often initially dramatic, relapse is almost inevitable and prevents durable responses in advanced-stage patients. Relapse is, in part, caused by drug tolerant persister cells (DTPs) which are able to survive treatment by entering a reversible, dormant state. Although long non-coding RNAs (lncRNAs) regulate processes thought to allow DTPs to survive and become stably resistant, the potential roles of lncRNAs in DTPs are largely unknown. In this study, we sought to investigate the expression of lncRNAs in in vitro DTP models of lung adenocarcinoma. We found that the lncRNAs Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) were enriched in DTPs and that knocking down MALAT1 enhanced the effect of targeted therapies in both EGFR- and KRAS-mutant DTP models. To better understand pathways that MALAT1 might regulate in DTPs, bulk RNA-sequencing was performed and several pathways that may contribute to the actions of MALAT1 in DTPs were identified. Overall, our work describes a role for the lncRNA MALAT1 in DTPs in NSCLC and suggests that MALAT1 may be a novel target for the prevention of drug tolerance and subsequent resistance to targeted therapy in NSCLC.

While recent therapeutic advances have extended the survival of patients with non-small cell lung cancer (NSCLC), overcoming metastatic progression in the CNS remains a significant challenge. Some patients with NSCLC may require concurrent management of CNS and extracranial metastases, while others develop isolated brain metastasis or leptomeningeal disease. These heterogenous clinical outcomes are difficult to predict and diagnose for early intervention with current surveillance modalities. Herein, we comprehensively analyzed gene mutations, copy number variations, and DNA methylation of NSCLC brain metastasis tissue collected at the time of craniotomy, combined with ctDNA sequencing of paired plasma and CSF liquid biopsies. We confirmed a high concordance between the molecular features of brain metastasis tissue with ctDNA from CSF which were largely distinct from ctDNA alterations in paired plasma samples. Plasma ctDNA tumor fraction and ctDNA hypermethylation were most significantly associated with extracranial metastasis and overall survival. Alternatively, we identified specific hypermethylated DNA loci in brain metastasis tissue and CSF ctDNA as significant correlates of brain metastasis progression and risk of leptomeningeal disease. Our findings support the utility of integrating ctDNA testing from CSF and plasma, while revealing distinct epigenetic features and biomarkers of brain metastasis or leptomeningeal disease.

Background: Immune-oncology has revolutionized cancer treatment, but some patients fail to benefit due to primary resistance and tumour-immune evasion. Extracellular vesicles (EVs) are secreted by both tumour and immune cells and mediate communication between cancer cells and the immune system. Our study used proteomic profiling of circulating EVs collected from NSCLC patients treated with immune checkpoint inhibitors (ICI) to identify predictive biomarkers of response as well as immune evasion mechanisms related to treatment resistance. Methods: EVs were isolated from plasma collected prior to ICI treatment using peptide-affinity purification and high-throughput proteomics was performed using Proximal Extension Assay. Differentially expressed EV proteins between durable (DR) and non-durable responders (NDR) were identified and evaluated using Cox proportional hazards regression, survival analysis, sex-stratified analysis, as well as pathway and network analysis. Results: Proteomics analysis identified 116 differentially expressed EV proteins between DR and NDR. NDR was characterized by enrichment of inflammatory, angiogenic, and immune-suppressive EV proteins, such as IL1RL1, TFRC, IL6ST, galectins, TNF superfamily death receptors, chemokines, and PCSK9. Pathway analysis revealed enrichment of angiogenesis, chemotaxis, ECM remodeling, and neutrophil degranulation associated with poor progression-free survival (PFS). In contrast, DR to ICI treatment was associated with EV proteins related to T- and B-cell activation and adaptive immunity. Sex-related differences in abundance and association with PFS was observed for certain EV proteins, including IL1RL1 and TFRC. A six protein EV model (IL1RL1, TFRC, ERI1, CCN5, IGFBPL1, and TNFRSF13C) demonstrated good prognostic performance for identifying NDR (AUC = 0.907) and stratified patients into three discrete risk groups. Conclusions: High-plex EV proteomics revealed biologically coherent tumour-immune signaling programs that are associated with ICI treatment resistance. Profiling circulating EVs may improve our understanding of EV-mediated immune evasion mechanisms and identify protein signatures that reflect the tumour immune microenvironment and predict response to immune checkpoint blockade.

Long non-coding RNAs (lncRNAs) have attracted increasing interest because of their roles as modulators of tumor progression, acting either as oncogenic drivers or tumor suppressors, depending on the cellular context. LINC01133 has been implicated in regulation of multiple tumor-related mechanisms; however, its role in breast cancer, particularly in the triple-negative subtype, remains poorly characterized. In this study, we investigated the impact of LINC01133 depletion on malignant phenotypes and on the expression of migration- and invasion-associated genes using the Hs578T triple-negative breast cancer (TNBC) cell line, through comparative analyses of parental, control, and LINC01133-knockout cell lines, namely Hs578T_wt, Hs578T_ctr, and Hs578T_ko. Functional characterization included morphological analysis, growth assays, anchorage-independent colony formation, migration, invasion, and quantitative biomolecular experiments. Depletion of LINC01133 led to reduction of cell diameter, a significant increase in colony-forming capacity, and marked enhancement of migratory and invasive potential. At the molecular level, LINC01133 loss induced the expression of genes associated with extracellular matrix remodeling and cellular plasticity, including fibronectin, vimentin, integrins, FOXC1, and TWIST1, concomitant with reduced expression of ZEB1, TWIST2, and N-cadherin. Collectively, these data indicate that LINC01133 acts as a potential fine regulator of in vitro migration and invasion processes in TNBC, with its expression favoring a more asymptomatic mode of tumor progression, whereas its loss markedly enhances tumor malignancy.

Although KRAS G12C-specific inhibitors such as sotorasib have been approved by US FDA and currently used in clinic, treating non-G12C mutants and overcoming acquired resistance for these inhibitors remain critical challenges. Here, we introduce a reciprocal feedback blockade therapy combining the MEK inhibitor trametinib and the multi-tyrosine kinase inhibitor imatinib to overcome these limitations. Our study reveals their compensatory roles: trametinib suppresses MEK activity yet promotes tyrosine kinase signaling and angiogenesis, while imatinib, a pan-tyrosine kinase inhibitor unleashes the MEK/ERK pathway via phosphatase suppression. Combining these agents blocks the reciprocal survival signals, inducing robust cell death across diverse KRAS-mutant models. Mechanistically, this combination reprograms cellular metabolism, leading to autophagy-dependent lipid peroxidation accumulation and ferroptosis. This strategy was effective in sotorasib-resistant lung cancer cells and various mouse models, including pancreatic cancer patient-derived xenograft. Furthermore, a pilot clinical trial for KRAS-mutant pancreatic cancer yielded encouraging responses. Consequently, the trametinib-imatinib combination represents a promising, broad-spectrum therapeutic strategy to overcome the constraints of current KRAS-targeted therapies.

Resistance to targeted therapy in HER2-positive breast cancer remains a clinical challenge, especially for patients with relapsed or metastatic disease. Particularly, persistent activation of hypoxia-inducible factor 1 (HIF-1) signalling is well documented in the context of trastuzumab and trastuzumab emtansine resistance. To achieve a deeper understanding of how HIF-1 activity modulates the response to anti-HER2 treatment, we functionally characterized a cellular model of hypoxia-induced drug resistance for HER2-positive breast cancer using shotgun proteomics. By global phosphoproteomics profiling, the Rac1 pathway was identified as one of the most enriched signalling networks under hypoxia. Furthermore, the selective Rac1 blockade with the 1A-116 small-molecule inhibitor sensitised HER2-positive cells to trastuzumab in both 2D and 3D culture systems. Altogether, our findings demonstrate that hypoxic conditions induce the resistance of HER2-positive breast cancer cells to targeted therapy and suggest the therapeutic potential of Rac1 inhibition to enhance trastuzumab efficacy. HighlightsO_LIHypoxic conditions induce trastuzumab resistance in HER2-positive breast cancer. C_LIO_LIRac1 signalling was mapped under hypoxia by phosphoproteomics profiling. C_LIO_LIRac1 inhibition sensitises HER2-positive cells to trastuzumab. C_LI

Head and neck squamous cell carcinoma (HNSCC) ranks as the seventh most common cancer, with increasing incidence and mortality rates and limited therapeutic progress. The heterohexameric prefoldin complex, a highly conserved co-chaperone assembly composed of six PFDN subunits, exhibits expression levels strongly correlated with cancer progression. Among these subunits, the PFDN5 gene presents a paradoxical role in cancer biology, demonstrating both tumor-promoting and tumor-suppressive activities. Notably, the PFDN5 gene generates two distinct protein isoforms through alternative splicing, yet their individual contributions to cancer remain unexplored. In this study, we reveal that an elevated short-to-long PFDN5 alternative splice variants ratio is significantly associated with improved overall survival in HNSCC patients. Using proximity-dependent biotin identification (BioID), we mapped shared and isoform-specific protein-protein interaction networks for PFDN5. Our analysis uncovered novel proximal interactors, implicating PFDN5 isoforms in unexpected functions, including spindle organization, transcriptional complexes, and NF-{kappa}B signaling. These results provide a foundation for exploring PFDN5 isoforms as potential therapeutic targets in HNSCC.

BackgroundLung adenocarcinoma (LUAD) is clinically and molecularly defined by oncogenic driver mutations, identification of which has led to the development of driver-targeted therapies and substantial improvements in prognosis for subsets of LUAD patients. Recent studies assessing clinical outcomes in the context of multigenic alterations have identified secondary mutations that might explain differential responses to targeted therapies, chemotherapies and immunotherapies. Genetic inactivation or loss of SMARCA4, which frequently co-occurs with loss-of-function mutations in STK11 and KEAP1, is especially predictive of poor prognosis and shorter overall survival in LUAD patients, regardless of driver status. We sought to examine the clinical and functional associations of SMARCA4 deficiency in LUAD, with or without co-associated STK11/KEAP1 loss-of-function. MethodsWe examined correlation between SMARCA4 loss, gene expression and prognosis through genomic and transcriptomic profiling of clinically annotated LUAD samples. We generated isogenic cell line models with genetic knockouts of SMARCA4 with or without concomitant STK11 and KEAP1 to profile mutationally-defined genotypes of interest in vitro and in vivo. Lastly, we interrogated the functional dependency of SMARCA4/STK11/KEAP1 triple mutant models on TGF-{beta} signaling to assess its potential as a therapeutic target. ResultsSMARCA4/STK11/KEAP1 triple mutant LUAD is associated with poor survival and high frequency of multisite metastasis. SMARCA4/STK11/KEAP1 triple knockout models showed enhanced migration and invasion in vitro, and diversified organotropism in an in vivo intracardiac xenograft metastasis assay. RNA-Seq and DNaseI-Seq of these in vitro models and clinical samples identified upregulation of TGF-{beta} signaling and EMT gene expression signatures, and corresponding changes in chromatin accessibility, in SMARCA4/STK11/KEAP1 triple mutant LUAD. ConclusionsWe identify SMARCA4/STK11/KEAP1 triple mutant LUAD as a prognostically significant disease subset and nominate TGF-{beta} signaling as a potential therapeutic target.

CD47 is a "dont eat me" signal that suppresses macrophage-mediated phagocytosis. Its upregulation in lung and other cancers facilitates tumour immune escape, making CD47 a promising immunotherapeutic target. Studies have demonstrated anti-tumour efficacy of CD47 blockade in preclinical lung cancer models, but monoclonal antibodies targeting CD47 have had limited efficacy as monotherapy in solid tumour patients to date. This discrepancy may in part reflect the use of human tumour xenografts in mice that do not have fully-functioning immune systems in preclinical efficacy studies. Thus, understanding tumour responses to CD47 inhibition using immune competent lung cancer models is needed to inform strategies to harness its therapeutic potential. Here, we characterized the effects of CD47 knockout (KO) on tumour growth and immune responses in two syngeneic, orthotopic murine lung cancer models, LLC-Luc (LLC) and CMT167 (CMT). As expected, CD47 KO impaired the fitness of LLC and CMT cells in vivo. Mice with CD47-deficient tumours exhibited prolonged survival and increased infiltration of anti-tumour leukocytes. However, although CD47 KO impaired lung tumour growth in syngeneic mice, KO tumours were ultimately lethal. Immunophenotyping revealed an increased prevalence of PD-L1+ cells in CD47-deficient tumours, nominating PD-L1-mediated suppression of tumour immunity as an acquired mechanism of resistance to CD47 blockade. Concordantly, dual inhibition of CD47 and PD-L1 extended the survival of CMT tumour-bearing mice compared to inhibition of either alone. These findings suggest that PD-L1 blockade could be leveraged to overcome resistance and potentiate the efficacy of CD47-targeted immunotherapy in lung cancer.

TP53 (also called TRP53 or p53) is a critical tumour suppressor that prevents cancer development by inducing a transcriptional program which can lead to diverse cellular responses, most prominently, cell proliferation arrest/senescence with survival of cells or cell death by apoptosis. Why distinct cell types undergo different outcomes after p53 activation remains unclear. Using integrated RNA-sequencing, proteomic and functional analyses across a diverse range of murine primary cell types, we demonstrate that cell fate is governed by the balance between pro-survival BCL-2 and pro-apoptotic BH3-only proteins. Cells resistant to apoptosis displays a higher starting ratio of pro-survival BCL-2 to pro-apoptotic BH3-only proteins, along with transcriptional upregulation of the pro-survival gene Bcl2l1, encoding BCL-XL. This control of cell fate is also seen in human wild-type p53 cancer cell lines. These findings reveal the mechanism for understanding p53-driven cell fate decisions, suggest therapeutic strategies to shift p53-induced cell proliferation arrest/senescence toward apoptotic cell death and allowed generation of an RNAseq data-based predictor of outcome for cancer cells after p53 activation.

Reactive oxygen species (ROS) are central signaling molecules in many biological processes by inducing oxidative modifications of protein cysteine residues, including S-glutathionylation. Increasing evidence supports that ROS contribute to cancer progression via promoting cancer cell migration, invasion, and metastasis. Nevertheless, the protein targets of S-glutathionylation that regulate cancer cell motility remain ill-defined. In this study, we report on the redox regulation of ARHGEF7, a guanine nucleotide exchange factor highly expressed in metastatic cancer cells, that plays a major role in regulating cell migration. Our data demonstrates that ARHGEF7 is selectively glutathionylated at the highly conserved C312 residue in its PH domain, which is implicated in regulating its enzymatic activity. Breast cancer cell lines showed increased cell migration and invasion upon glutathionylation of ARHGEF7 at C312 in response to both oxidative stress and epidermal growth factor (EGF). Mechanistically, upon C312 glutathionylation, ARHGEF7 exhibited significantly enhanced binding to Rac1 and increased Rac1 recruitment to the cell membrane and lamellipodia. ARHGEF7 S-glutathionylation also increased its enzymatic rate of GDP-GTP nucleotide exchange, resulting in Rac1 activation. Consequently, ARHGEF7 C312 S-glutathionylation induced Rac1-PAK1 activation and their downstream pathways, including LIMK1 and MEK1, thereby enhancing migration and invasion. Our data reveal a new redox player in cell migration, with its potential implications for ROS-induced cancer progression.

Introduction Depression is highly prevalent among cancer survivors and may be biologically distinct, although clinical studies investigating these mechanisms remain limited. Thus, the aims of this study were to (1) identify perturbed biological pathways associated with depressive symptom severity in cancer survivors, and (2) investigate whether these pathways are common or distinct to those perturbed in an age-matched non-cancer cohort. Methods We analyzed cross-sectional self-reported and transcriptomic data from the Multi-Ethnic Study of Atherosclerosis (PHD #39341). Cancer survivors and an age-matched non-cancer cohort (target ratio 1:2) were identified. The 20-item Center for Epidemiologic Studies Depression Scale (CES-D) was used to split participants into low (CES-D<16) and high ([&ge;]16) depressive symptom groups. Analyses were conducted separately for survivor and non-cancer cohorts. Differential gene expression between depressive symptom groups was evaluated with adjustments for covariates significantly associated with depression (survivor cohort: BMI; non-cancer cohort: marital status), with pathway impact analysis identifying perturbed pathways (FDR < 0.025). Results Ninety-three cancer survivors (11.8% with high depressive symptoms) and 176 non-cancer participants (9.7% with high depressive symptoms) were included. Sixty-eight and 72 perturbed pathways were associated with depression among survivor and non-cancer cohorts, respectively. Twenty-one of these pathways were perturbed uniquely among cancer survivors, which were related to neurodegeneration, reward circuitry, proliferation, and secretion. Inflammatory pathways were consistently perturbed across both cohorts. Conclusions Distinct biological mechanisms related to neurodegeneration, reward circuitry, autonomic secretion, and proliferative signaling may underlie depression in cancer survivors. Inflammation was implicated as a shared mechanism of depression across cancer and non-cancer populations. This study identifies potential therapeutic targets and highlights the need for precision medicine in treating depression among cancer survivors.

IntroductionResistance to immunotherapy remains a major clinical challenge in TP53-mutant head and neck squamous cell carcinoma (HNSCC), a disease subset characterized by immune exclusion, high recurrence, and poor outcomes. Given the constitutive activation of PI3K/AKT/mTOR signaling in TP53-mutant HNSCC and its role in disease progression, we investigated whether the mTOR inhibition (mTORi) could overcome immune resistance and improve outcomes. MethodsWe evaluated the effects of mTOR inhibitor everolimus in TP53-mutant, anti-PD-1-resistant syngeneic HNSCC cell lines and syngeneic tumor models. Tumor microenvironment (TME) changes, including immune cell infiltration, immune checkpoint expression, and key pathways associated with immune suppression and angiogenesis were assessed to define the mechanisms underlying TME remodeling. ResultsEverolimus significantly suppressed tumor growth in syngeneic HNSCC models. At the cellular level, everolimus significantly increased intratumoral CD8+ T cell and dendritic cell (DC) infiltration while reducing the accumulation of regulatory T cells (Tregs). Mechanistically, everolimus induced a cytokine/chemokine response, marked by increased TNF-/CXCL10 expression, leading to enhanced immune infiltration. Everolimus also inhibited the HIF-1/VEGFA angiogenic axis, a central driver of immune exclusion and myeloid-derived suppressor cell (MDSC) recruitment. Furthermore, everolimus treatment attenuated PD-1/PD-L1 signaling by reducing PD-1 and PD-L1 expression, respectively, in T cells and tumor cells, thereby restoring T-cell cytotoxic competence. DiscussionThese findings demonstrate that mTORi with everolimus reverses multiple mechanisms of immune resistance in TP53-mutant HNSCC by promoting immune cell recruitment, suppressing immunosuppressive pathways, and enhancing anti-tumor T cell activity. Collectively, these results support mTORi as a mechanistically rational strategy for reprogramming immune resistance in TP53-mutant HNSCC and provide a strong preclinical rationale for combining everolimus with immune therapy in patients who are likely to fail immunotherapy.

BackgroundOpportunistic nutrient uptake is a hallmark of cancer metabolism. Cancer cells upregulate macropinocytosis to acquire extracellular nutrients to support growth and stress adaptation. We previously showed that extracellular ATP (eATP) is internalized by macropinocytosis and promotes multiple cancer phenotypes. Here, we tested whether eATP uptake is prevalent across cancers and whether eATP also induces senescence through purinergic receptor (PR) signaling. MethodsIntracellular ATP (iATP) levels were measured following eATP exposure across multiple cancer cell lines. eATP internalization was visualized in vitro and in vivo using a non-hydrolyzable fluorescent ATP analog together with high-molecular-weight dextran as a macropinocytosis marker. Senescence was quantified using three SA-{beta}-galactosidase assays and flow cytometry. Pharmacologic inhibitors of macropinocytosis and purinergic receptors were used to define pathway dependence. Combination treatments with the glucose transporter inhibitor DRB18 and the senolytic navitoclax were evaluated for antiproliferative effects. ResultseATP produced dose- and time-dependent increases in iATP across diverse cancer cell types. Imaging demonstrated widespread macropinocytic internalization of ATP in vitro and in tumor xenografts. eATP induced senescence in NSCLC cells, confirmed by multiple {beta}-gal assays and flow cytometry. PR inhibition significantly reduced senescence, whereas macropinocytosis inhibition had minimal effect on senescence induction. ConclusionseATP acts through dual pathways in cancer cells: macropinocytic internalization that elevates iATP and PR signaling that drives senescence. Targeting metabolic uptake together with senolytic therapy may offer a novel anticancer strategy.

BackgroundCodon usage bias, the non-random usage of synonymous codons in coding sequences, represents a fundamental feature of genomic organization that has been largely understudied in cancer biology. Pancreatic ductal adenocarcinoma (PDAC), the predominant subtype of pancreatic cancer, is characterized by aggressive disease progression and limited therapeutic options, necessitating novel approaches to understand its molecular pathogenesis. Leveraging publicly available single-cell RNA sequencing data, we performed comprehensive codon usage analyses across different cellular populations in PDAC. ResultsEmploying a variety of computational codon usage indices uncovered the connections between cancer-specific cellular state features and codon usage signatures. Our findings reveal that malignant pancreatic cells express genes with significantly higher GC content, demonstrate preferential usage of optimal codons through increased frequency of preferred synonymous codons, and exhibit a marked preference for more cost-effective amino acids. Analysis of transcript-level bulk RNA-seq data from PDAC tumors revealed that these codon optimization patterns extend to alternative isoform usage, with highly expressed isoforms displaying increased codon optimality and enhanced mRNA stability. ConclusionThese codon usage-dependent adaptations operating at both gene expression and transcript isoform levels may enable malignant cells to enhance gene expression rates, potentially leading to increased translational efficiency and protein production. These insights into the codon usage landscape of PDAC may provide potential biomarkers for disease monitoring and treatment response prediction.

MicroRNAs (miRNAs) have been widely implicated in cancer initiation and progression, yet examination of the effects of global miRNA disruption on these processes has been limited. We developed novel genetically engineered mouse models of Kras-driven pulmonary adenocarcinoma (LUAD) with cell-type-specific disruption of miRNA biosynthesis via Dicer1 allele deletion, which exhibit significant differences in tumor progression rates and expected survival. Dicer1 is an RNase III enzyme that is required for the biogenesis of mature, functional miRNAs. Lung tumor progression was accelerated, and expected survival was decreased only when we initiated tumors and deleted one allele of Dicer1 in club cells and mutated Dicer1 in alveolar type 2 (AT2) cells. Reversing the cell types by inducing tumorigenesis, deleting one Dicer1 allele in AT2 cells, and mutating Dicer1 in club cells modestly accelerated tumor progression and had no effect on expected survival. Collectively, our results demonstrate that Dicer1 disruption accelerates lung cancer progression in a cell-type-dependent and non-cell-autonomous manner, and our mice represent tools for investigating the roles of miRNAs and miRNA-mediated intercellular communication in tumor progression. SummaryKras-driven mouse models show that Dicer1 mutations accelerate lung adenocarcinoma (LUAD) progression in a cell-type-dependent manner and suggest that the influence of miRNA-mediated intercellular communication is unidirectional and non-cell-autonomous.

IntroductionMTAP emerges as potential predictive biomarker for MTA-cooperative PRMT5-inhibitors. Although MTAP attracts increasing attention in non-small cell lung cancer, its role in pulmonary neuroendocrine neoplasms (NENs) remains largely unexplored. MethodsHere, we assessed the prevalence of MTAP deficiency in 209 pulmonary NENs using immunohistochemistry (IHC). Additionally, we performed fluorescence in situ hybridization (FISH), whole exome sequencing (WES), deep proteomic profiling, transcriptomic, and methylation analyses of selected MTAP deficient and proficient carcinoids to further elucidate the underlying mechanisms of MTAP expression pattern. ResultsMTAP deficiency by IHC was detected in all neuroendocrine precursor lesions (n=17), 92% of typical carcinoids (n=51), 86% of atypical carcinoids (n=21), and 10% of large cell neuroendocrine carcinomas (LCNEC) (n=30). In contrast, all small cell lung cancers (SCLC) were MTAP proficient (n=90). In MTAP deficient carcinoids, FISH and WES did not detect homozygous 9p21 deletions, and methylation analysis showed no evidence of MTAP promoter hypermethylation. Comparing MTAP deficient and MTAP proficient carcinoids, proteomic data showed a clear separation between the two groups. Further, there was an inverse correlation between the expression of MTAP and OTP (orthopedia homeobox protein), which is known as a strong prognostic marker in pulmonary carcinoids. DiscussionMTAP deficiency is a novel hallmark of neuroendocrine precursors and most pulmonary carcinoids, clearly distinguishing the latter from SCLC and most LCNEC. It is neither caused by 9p21 deletion, nor by MTAP promoter hypermethylation. MTAP deficiency is a group-defining feature of carcinoids that might pave the way for new therapeutic approaches.

Melanoma remains one of the most treatment-refractory malignancies due to immune evasion, high mutational burden, and profound tumor heterogeneity. Although immune checkpoint inhibitors have transformed frontline management, a substantial proportion of patients develop resistance or experience relapse, underscoring the need for alternative and complementary immunotherapeutic strategies. Tumor-infiltrating lymphocyte (TIL) therapy and engineered viral vector-based immunotherapies represent mechanistically distinct yet clinically promising approaches for advanced melanoma. This systematic review and Bayesian meta-analysis evaluated the comparative efficacy of TIL therapy and engineered viral vector immunotherapies in advanced melanoma. A structured search of PubMed, Embase, Scopus, and Web of Science (2015-2025) identified 13 eligible studies, including four randomized controlled trials and nine prospective single-arm studies, reporting objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and treatment-related adverse events. Eight studies met criteria for inclusion in the Bayesian quantitative synthesis of ORR outcomes. Risk of bias and certainty of evidence were assessed using Cochrane and GRADE frameworks. TIL therapy demonstrated substantial standalone efficacy, particularly in PD-1-refractory populations, with reported ORRs reaching 49%, median PFS of 7.2 months, and OS extending to 25.8 months. Viral vector-based therapies, including talimogene laherparepvec (T-VEC) and RP1, showed more modest monotherapy activity but demonstrated improved responses when combined with immune checkpoint inhibitors. Among the studies included in the Bayesian quantitative synthesis, the pooled ORR estimate was 37.8% (95% highest density interval [HDI]: 30.6%-45.3%). Sensitivity analysis excluding the small-sample Cui et al. (2022) study yielded a similar pooled estimate of 38.3% (95% HDI: 30.4%-46.2%). Exploratory meta-regression supported the overall robustness of the findings. Certainty of evidence for ORR was moderate, whereas survival and safety outcomes were downgraded due to heterogeneity, sparse reporting, and inconsistent endpoint definitions. Collectively, these findings support complementary rather than competing roles for TIL and engineered viral vector immunotherapies within evolving melanoma treatment paradigms. The results further highlight the potential importance of biomarker-guided sequencing strategies, including viral immune priming followed by adoptive cellular therapy, as a framework for optimizing personalized immunotherapy in both refractory and earlier-line melanoma settings.

Squamous cell carcinoma of the lung is a difficult-to-treat cancer with high prevalence in the US, and particularly in Kentucky. The goals of this work were to test if the EZH1/2 inhibitor valemetostat improves anti-PD1 responses in squamous cell lung cancer models, and to develop ex vivo models to test immunotherapy drug combinations. We found that valemetostat produced augmented anti-tumor responses to anti-PD1 therapy through up-regulation of tumor cell specific Major Histocompatibility Complex Class II (MHC Class II), and a shift towards activated CD8+ T cells. Neutrophils predominated in these tumors regardless of therapy, but examination of bone marrow revealed that valemetostat treated mice and mice that rejected tumors both had more mature neutrophils. Likewise, Ezh2 knock-out mice produced neutrophils that were more apoptotic, less migratory, and less able to produce extracellular nets, but had similar ability to kill bacteria as Ezh2-WT neutrophils. To test tumor responses to differing neutrophil populations, we engineered three-dimensional air-liquid interface cultures with tumoroids, lung mesenchymal cells, and T cells, with and without bone marrow containing neutrophils and myeloid progenitors from distinct donors. Bone marrow from tumor-naive or mice with actively growing untreated tumors boosted tumoroid growth, while bone marrow from tumor-rejected or mice with tumors treated with valemetostat was anti-tumor. MHC Class II blockade lowered the ability of bone marrow to boost tumor growth, and reduced the ability of valemetostat with anti-PD1 to reduce tumoroid growth. Patient samples revealed a strong negative correlation between EZH2 and MHC Class II, suggesting that targeting EZH2 activity could lead to marked increase in MHC Class II and improve treatment responses in lung squamous cell carcinomas.

Abstract Introduction: IL-15 is one of the most promising candidate cytokines in cancer immunotherapy due to its ability to promote the activity of different cytotoxic innate immune cell subsets such as NK, ILC1 and gammadelta T cells. During biosynthesis, IL-15 associates with IL-15alpha and is transported to the cell surface where IL-15Ralpha trans-presents IL-15 to target neighboring cells expressing the beta chain (IL-2Rbeta) and the common gamma chain. Our group previously showed that in autoimmune type 1 diabetes and early innate immune responses to infections trans-presentation by IL-15Ralpha is dispensable. Here we addressed the relative roles of IL-15 and trans-presented IL-15 in the control of established tumors and spontaneous tumor development. Methodology: Growth kinetics of tumor cell lines were monitored in WT, Il15-/- and Il15ra-/- mice. Spontaneous fibrosarcoma was induced with Methylcholanthrene (MCA) in WT, Il15-/- and Il15ra-/- mice. Cell lines were established from MCA-induced tumors to characterize their immunogenicity. Results: Growth of established tumor cell lines were comparable in the three genotypes. MCA-induced tumor incidence was reduced in Il15ra-/- mice when compared to WT and Il15-/- mice. In vitro, MCA tumor-derived cell lines expressed MHC-I and PD-L1 and had comparable proliferation rates. In vivo, MCA tumor-derived cell lines established from the 3 genotypes showed comparative growth in WT mice suggesting that IL-15 does not impact immunoediting. Nonetheless, NLRC5 expressing B16-F10 tumors were contained in WT and Il15ra-/- mice but not in Il15-/- mice. Conclusions: Taken together, these results show that in the absence of trans-presentation by IL-15Ralpha, IL-15 can better control spontaneous tumor development and that IL-15 signaling plays a minor role in immunosurveillance in this model. IL-15 signaling, independent of IL-15Ralpha has a significant role in the control of solid tumors.