Cytotoxic Gene Research Articles

LFA-1/ ICAM-1 promotes NK cell cytotoxicity associated with the pathogenesis of ocular toxoplasmosis in murine model.

Ocular toxoplasmosis (OT) is one of the most common causes of posterior uveitis. However, the pathogenic mechanisms of OT have not been well elucidated. Here, we used C57BL/6 (B6) mice to establish OT by peroral infection with 20 cysts of the TgCtWh6 strain, and severe ocular damage was observed by histopathological analysis in the eyes of infected mice. RNA-sequencing results showed that infection with T. gondii increased the expression of the NK-mediated cytotoxicity gene pathway at Day 30 after ocular T. gondii infection. Both NK-cell and CD49a+ NK-cell subsets are increased in ocular tissues, and the expression levels of LFA-1 in NK cells and ICAM-1 in the OT murine model were upregulated upon infection. Furthermore, inhibition of the interaction between LFA-1 and ICAM-1 with lifitegrast, a novel small molecule integrin antagonist, inhibited the protein expression of LFA-1 and ICAM-1 in murine OT and NK cells, improved the pathology of murine OT and influenced the secretion of cytokines in the OT murine model. In conclusion, the interaction between LFA-1 and ICAM-1 plays a role in the early regulation of the CD49a+ NK-cell proportion in an OT murine model. LFA-1/ ICAM-1 may be a key molecule in the pathogenesis of OT, and may provide new insights for potential immunotherapy.

CD8A as a Prognostic and Immunotherapy Predictive Biomarker Can Be Evaluated by MRI Radiomics Features in Bladder Cancer.

Simple SummaryIt is necessary to explore a reliable predictive biomarker for immunotherapeutic strategies in bladder cancer. As an important member of tumor-infiltrating immune cells, cytotoxic T cells are the ultimate effectors of cancer rejection because of their specificity against cancer cells and cytolytic capabilities. Based on RNA expression data, we found that among T cytotoxic pathway-related genes, CD8A is a novel protective gene in bladder cancer. CD8A expression is highly associated with tumor microenvironment characteristics and tumor mutation burden, which partially explain CD8A as a prognostic and immunotherapy predictive biomarker in bladder cancer. In addition, we performed the radiogenomics in bladder cancer based on the RNA-sequence data and MRI-based radiomics features in our center. In this way, an MRI-based radiomics signature has the potential to preoperatively predict the expression of CD8A, thereby contributing to the preoperative prediction of prognosis and immunotherapeutic susceptibility in bladder cancer.As an important member of T cytotoxic pathway-related genes, CD8a molecule (CD8A) may be a useful biomarker of immunotherapeutic response and immune cell infiltration. We aimed to investigate the clinical predictive value of CD8A in prognosis and tumor microenvironment (TME) and preoperatively predict the expression of CD8A using radiogenomics in bladder cancer (BCa). Among 12 T cytotoxic pathway-related genes, CD8A was a novel protective gene and had the highest correlations with T cells and Macrophages M1 in BCa. In advanced cancer patients treated with immunotherapy, low CD8A expression was associated with immunotherapeutic failure and poor survival outcomes. CD8A expression was highly related to tumor mutation burden, critical immune checkpoint genes and several types of tumor-infiltrating immune cells, predicting effective response to immunotherapy. The preoperative MRI radiomics features and RNA-sequence data of 111 BCa samples were used to develop a radiomics signature that achieved good performance in the prediction of CD8A expression in both the training (area under curve (AUC): 0.857) and validation sets (AUC: 0.844). CD8A is a novel indicator for predicting the prognosis and immunotherapeutic response in BCa. A radiomics signature has the potential to preoperatively predict the expression of CD8A in BCa patients.

Network Pharmacological Analysis of the Red Sea Sponge Hyrtios erectus Extract to Reveal Anticancer Efficacy of Corresponding Loaded Niosomes.

In this study, the LC-HRMS-assisted chemical profiling of Hyrtios erectus sponge led to the annotation of eleven major compounds (1–11). H. erectus-derived crude extract (HE) was tested in vitro for its antiproliferative activity against three human cancer cell lines, Hep-G2 (human liver cancer cell line), MCF-7 (breast cancer cell line), and Caco-2 (colon cancer cell line), before and after encapsulation within niosomes. Hyrtios erectus extract showed moderate in vitro antiproliferative activities towards the studied cell lines with IC50 values 18.5 ± 0.08, 15.2 ± 0.11, and 13.4 ± 0.12, respectively. The formulated extract-containing niosomes (size 142.3 ± 10.3 nm, PDI 0.279, and zeta potential 22.8 ± 1.6) increased the in vitro antiproliferative activity of the entrapped extract significantly (IC50 8.5 ± 0.04, 4.1 ± 0.07, and 3.4 ± 0.05, respectively). A subsequent computational chemical study was performed to build a sponge–metabolite–targets–cancer diseases network, by focusing on targets that possess anticancer activity toward the three cancer types: breast, colon, and liver. Pubchem, BindingDB, and DisGenet databases were used to build the network. Shinygo and KEGG databases in addition to FunRich software were used for gene ontology and functional analysis. The computational analysis linked the metabolites to 200 genes among which 147 genes related to cancer and only 64 genes are intersected in the three cancer types. The study proved that the co-occurrence of compounds 1, 2, 3, 7, 8, and 10 are the most probable compounds possessing cytotoxic activity due to large number of connections to the intersected cytotoxic genes with edges range from 9-14. The targets possess the anticancer effect through Pathways in cancer, Endocrine resistance and Proteoglycans in cancer as mentioned by KEGG and ShinyGo 7.1 databases. This study introduces niosomes as a promising strategy to promote the cytotoxic potential of H. erectus extract.

Alphaviruses in Immunotherapy and Anticancer Therapy.

Alphaviruses have been engineered as expression vectors for vaccine development and gene therapy. Due to the feature of RNA self-replication, alphaviruses can provide exceptional direct cytoplasmic expression of transgenes based on the delivery of recombinant particles, naked or nanoparticle-encapsulated RNA or plasmid-based DNA replicons. Alphavirus vectors have been utilized for the expression of various antigens targeting different types of cancers, and cytotoxic and antitumor genes. The most common alphavirus vectors are based on the Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus, but the oncolytic M1 alphavirus has also been used. Delivery of immunostimulatory cytokine genes has been the basis for immunotherapy demonstrating efficacy in different animal tumor models for brain, breast, cervical, colon, lung, ovarian, pancreatic, prostate and skin cancers. Typically, therapeutic effects including tumor regression, tumor eradication and complete cure as well as protection against tumor challenges have been observed. Alphavirus vectors have also been subjected to clinical evaluations. For example, therapeutic responses in all cervical cancer patients treated with an alphavirus vector expressing the human papilloma virus E6 and E7 envelope proteins have been achieved.

Balancing Affinity, Selectivity, and Cytotoxicity of Hydrazone-Based G-Quadruplex Ligands for Activation of Interferon β Genes in Cancer Cells.

G-quadruplex (G4) ligands are investigated to discover new anticancer drugs with increased cell-killing potency. These ligands can induce genome instability and activate innate immune genes at non-cytotoxic doses, opening the discovery of cytostatic immune-stimulating ligands. However, the interplay of G4 affinity/selectivity with cytotoxicity and immune gene activation is not well-understood. We investigated a series of closely related hydrazone derivatives to define the molecular bases of immune-stimulation activity. Although they are closely related to each other, such derivatives differ in G4 affinity, cytotoxicity, genome instability, and immune gene activation. Our findings show that G4 affinity of ligands is a critical feature for immune gene activation, whereas a high cytotoxic potency interferes with it. The balance of G4 stabilization versus cytotoxicity can determine the level of immune gene activation in cancer cells. Thus, we propose a new rationale based on low cell-killing potency and high immune stimulation to discover effective anticancer G4 ligands.

Single-cell transcriptome landscape and antigen receptor dynamic during SARS-CoV-2 vaccination

Vaccination by inactivated vaccine is an effective strategy to prevent the COVID-19 pandemic. However, the detailed molecular immune response at single-cell level is poorly understood. In this study, we systematically delineated the landscape of the pre- and post-vaccination single-cell transcriptome, TCR (T cell antigen receptor) and BCR (B cell antigen receptor) expression profile of vaccinated candidates. The bulk TCR sequencing analysis of COVID-19 patients was also performed. Enrichment of a clonal CD8+ T cell cluster expressing specific TCR was identified in both vaccination candidates and COVID-19 patients. These clonal CD8+ T cells showed high expression of cytotoxicity, phagosome and antigen presentation related genes. The cell–cell interaction analysis revealed that monocytes and dendritic cells could interact with these cells and initiate phagocytosis via ICAM1-ITGAM and ITGB2 signaling. Together, our study systematically deciphered the detailed immunological response during SARS-CoV-2 vaccination and infection. It may facilitate understanding the immune response and the T-cell therapy against COVID-19.

Antioxidant properties and cytotoxic effects of Alternanthera sissoo and Alternanthera bettzickiana extracts against cancer cells

This work aimed to evaluate the antioxidant capacities and cytotoxic attributes of Alternanthera sissoo (AS) and Alternanthera bettzickiana Green (AB) which are commonly grown in Thailand, yet not extensively-studied. Dried leaves were extracted by maceration using ethanol for 48 h and ethanolic extract solution was used. Cytotoxic, anti-proliferative, anti-migratory capacities and apoptosis-related gene expressions of AS and AB extracts on MCF-7, HepG2, and HeLa cell lines were investigated using 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetra zolium bromide (MTT) assay, anti-colony formation and wound healing test, respectively. The antioxidant activities by DPPH assay and FRAP assay of AS (130.62±1.61 µg TE/g extract and 24.76 ± 0.54 mg Fe(II)/g extract, respectively) were significantly higher than those of AB (104.34±3.68 µg TE/g extract and 23.13± 0.90 mg Fe(II)/g extract, respectively). Likewise, total phenolic content and the total flavonoid content of AS (7.43±0.39 mg GAE/g and 184.22±3.20 mg RE/g extract, respectively) were significantly higher than AB (6.10±0.29 mg GAE/g extract and 168.07±7.90 mg RE/g extract, respectively). HPLC analysis showed the predominance of myricetin, rutin and ferulic acid. For MCF-7, HepG2 and HeLa, the lowest IC50 values of 48.53 µg/mL, 69.94 µg/mL and 43.98 µg/mL, respectively were found in AS extract at 72 h exposure using MTT assay. Apoptotic bodies, gene expression and protein expressions related to apoptosis were more pronounced in AS treatment. Both AS and AB can be served as local foods with chemopreventive effects against breast, liver and cervical cancers. This work provided a foundation for future pharmacological research on Alternanthera plant extracts.

Single-cell transcriptomics identifies pathogenic T-helper 17.1 cells and pro-inflammatory monocytes in immune checkpoint inhibitor-related pneumonitis

BackgroundImmune checkpoint inhibitor (ICI)-related pneumonitis is the most frequent fatal immune-related adverse event associated with programmed cell death protein-1/programmed death ligand-1 blockade. The pathophysiology however remains largely unknown, owing to...

Abstract PO015: Intrinsic and extrinsic role of Axl in models of hepatocellular carcinoma

Abstract The activation of the receptor tyrosine kinase Axl by binding to its high affinity ligand Gas6 is a major driver of tumor progression and metastasis in various cancer entities. In hepatocellular carcinoma (HCC), about 40% of patients show upregulation of Axl expression correlating with vascular invasion and poor survival of patients. In pre-malignant stages of HCC, Axl is essentially involved in developing hepatic fibrosis and cirrhosis by activation of hepatic stellate cells. Despite recent insights, the dichotomy of intrinsic and extrinsic Axl functions in HCC is poorly understood. Therefore, we aimed at analyzing cell-specific aspects of Axl in liver tumorigenesis using advanced mouse tumor models. To particularly address the tumor-cell specific role of Axl, we employed a transplantation-based mouse models. In order to investigate both the tumor-intrinsic and extrinsic impact of Axl, we employed an autochthonous model of diethylnitrosamine (DEN)-induced liver tumor formation in systemic Axl-deficient mice. We found that tumor-intrinsic Axl expression in HCC cells did not affect proliferation in vitro and in vivo, however increased the number of pulmonary metastatic colonies and reduced overall survival of mice. In line, Gas6/Axl signaling enhanced the invasive abilities of tumor cells in vitro as observed in 3-dimensional cell culture settings. The Axl-driven invasive phenotype of HCC cells associated with the upregulated expression of transcription factors Snai1/Snai2 and downregulation of E-Cadherin indicating changes in epithelial plasticity. Intriguingly, the tumor burden was enhanced in DEN-treated Axl−/− mice in the background of CCl4-induced inflammation by showing higher proliferative rates of malignant hepatocytes. Interestingly, these tumors were less infiltrated with cytotoxic CD8+ T cells and granzyme B+ cells. Additionally, we observed an increased infiltration of FoxP3+ regulatory T cells and F4/80+ macrophages as well as elevated expression of M2-macrophage-specific Mrc-1, Fizz-1 and TGF-β1 suggesting that Axl−/− tumors shift towards an immunosuppressive environment. Remarkably, PD-L1 levels did not vary between Axl+/+ and Axl−/− tumors, suggesting alternative immune escape mechanisms. In accordance, analysis of HCC patient datasets showed a positive correlation of Axl expression with cytotoxic immune gene signatures. From these data we conclude that tumor intrinsic Gas6/Axl signaling triggers the metastatic phenotype of HCC cells while Axl deficiency escapes immune surveillance and therefore promotes liver tumorigenesis. Together, our findings show that Axl plays a prominent role in HCC dissemination and highlight the importance of Axl expression in the tumor microenvironment. Citation Format: Kristina Breitenecker, Denise Heiden, Gerhard Weber, Wolfgang Mikulits. Intrinsic and extrinsic role of Axl in models of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO015.

Association of Cytotoxic T-Lymphocyte Antigen-4 Gene Polymorphism with Type 1 Diabetes Mellitus: In silico Analysis of Biological Features of CTLA-4 Protein on Ethiopian Population.

BackgroundT1DM is a chronic organ-specific T-cell-mediated autoimmune disease characterized by the selective destruction of β-cells in the islets of Langerhans, resulting in insulin deficiency and hyperglycemia. Genes for cytotoxic T lymphocyte-associated antigen 4 have been hypothesized as possible contender genes for T1DM vulnerability. However, it has not been studied in the Ethiopian population yet.ObjectiveThe aim of the study was to investigate CTLA-4 exon 1 was linked to A49G polymorphism with T1DM and its biological features of CTLA-4 among T1DM patients, in Ethiopia.MethodsA case–control study was done from December 2019 to March 2020 on 210 study participants (105 T1DM patients and 105 healthy controls). Polymerase Chain Reaction amplification with forward and reverse primers was followed by restriction fragment length polymorphism and gel electrophoresis to determine gene polymorphism. Bioinformatics data of SNP was retrieved from National Centers for Biotechnology Information databases. The chi-square test and logistic regression were used. Statistical significance was defined as a P-value of less than 0.05.ResultsThe CTLA-4 (+A49G) gene polymorphism was observed on 56 (26.7%) study participants, 39 (18.57%) of T1DM patients, and 17 (0.08%) were controls. In T1DM and controls, the frequency of the A allele was 73.3% and 89.5%, while the G allele was 26.7% and 10.5%, respectively. The G allele was found to be associated with T1DM (OR=3.1; 95% CI, 1.82 −5.32; P=0.001). Statistical analysis revealed an association between the likelihood of T1DM and GG genotype of the CTLA-4 (+A49G) gene polymorphism (OR=3.11; 95% CI, 1.37–10.90; P=0.01). Further in silico analyzed the SNP to assess its biological features.ConclusionThe study showed as CTLA-4 (+A49G) gene polymorphism is linked with T1DM in the Ethiopian population.

Distinct patterns of the natural evolution of soft tissue sarcomas on pre-treatment MRIs captured with delta-radiomics correlate with gene expression profiles.

Radiomics of soft tissue sarcomas (STS) is assumed to correlate with histologic and molecular tumor features, but radiogenomics analyses are lacking. Our aim was to identify if distinct patterns of natural evolution of STS obtained from consecutive pre-treatment MRIs are associated with differential gene expression (DGE) profiling in a pathway analysis. All patients with newly diagnosed STS treated in a curative intent in our sarcoma reference center between 2008 and 2019 and with two available pre-treatment contrast-enhanced MRIs were included in this retrospective study. Radiomics features (RFs) were extracted from fat-sat contrast-enhanced T1-weighted imaging. Log ratio and relative change in RFs were calculated and used to determine grouping of samples based on a consensus hierarchical clustering. DGE and oncogenesis pathway analysis were performed in the delta-radiomics groups identified in order to detect associations between delta-radiomics patterns and transcriptomics features of STS. Secondarily, the prognostic value of the delta-radiomics groups was investigated. Sixty-three patients were included (median age: 63 years, interquartile range: 52.5-70). The consensus clustering identified 3 reliable delta-radiomics patient groups (A, B, and C). On imaging, group B patients were characterized by increase in tumor heterogeneity, necrotic signal, infiltrative margins, peritumoral edema, and peritumoral enhancement before the treatment start (p value range: 0.0019-0.0244), and, molecularly, by downregulation of natural killer cell-mediated cytotoxicity genes and upregulation of Hedgehog and Hippo signaling pathways. Group A patients were characterized by morphological stability of pre-treatment MRI traits and no local relapse (log-rank p = 0.0277). This study highlights radiomics and transcriptomics convergence in STS. Proliferation and immune response inhibition were hyper-activated in the STS that were the most evolving on consecutive imaging. • Three consensual and stable delta-radiomics clusters were identified and captured the natural patterns of morphological evolution of STS on pre-treatment MRIs. • These 3 patterns were explainable and correlated with different well-known semantic radiological features with an ascending gradient of pejorative characteristics from the A group to C group to B group. • Gene expression profiling stressed distinct patterns of up/downregulated oncogenetic pathways in STS from B group in keeping with its most aggressive radiological evolution.

KIR2DS2 Expression Identifies NK Cells With Enhanced Anticancer Activity.

NK cells are promising cellular therapeutics against hematological and solid malignancies. Immunogenetic studies have identified that various activating killer cell Ig-like receptors (KIRs) are associated with cancer outcomes. Specifically, KIR2DS2 has been associated with reduced incidence of relapse following transplant in hematological malignancies and improved outcomes in solid tumors, but the mechanism remains obscure. Therefore, we investigated how KIR2DS2 expression impacts NK cell function. Using a novel flow cytometry panel, we show that human NK cells with high KIR2DS2 expression have enhanced spontaneous activation against malignant B cell lines, liver cancer cell lines, and primary chronic lymphocytic leukemia cells. Surface expression of CD16 was increased on KIR2DS2high NK cells, and, accordingly, KIR2DS2high NK cells had increased activation against lymphoma cells coated with the clinically relevant anti-CD20 Abs rituximab and obinutuzumab. Bulk RNA sequencing revealed that KIR2DS2high NK cells have upregulation of NK-mediated cytotoxicity, translation, and FCGR gene pathways. We developed a novel single-cell RNA-sequencing technique to identify KIR2DS2+ NK cells, and this confirmed that KIR2DS2 is associated with enhanced NK cell-mediated cytotoxicity. This study provides evidence that KIR2DS2 marks a population of NK cells primed for anticancer activity and indicates that KIR2DS2 is an attractive target for NK-based therapeutic strategies.

Integration of tumor extrinsic and intrinsic features associates with immunotherapy response in non-small cell lung cancer

The efficacy of immune checkpoint blockade (ICB) varies greatly among metastatic non-small cell lung cancer (NSCLC) patients. Loss of heterozygosity at the HLA-I locus (HLA-LOH) has been identified as an important immune escape mechanism. However, despite HLA-I disruptions in their tumor, many patients have durable ICB responses. Here we seek to identify HLA-I-independent features associated with ICB response in NSCLC. We use single-cell profiling to identify tumor-infiltrating, clonally expanded CD4+ T cells that express a canonical cytotoxic gene program and NSCLC cells with elevated HLA-II expression. We postulate cytotoxic CD4+ T cells mediate anti-tumor activity via HLA-II on tumor cells and augment HLA-I-dependent cytotoxic CD8+ T cell interactions to drive ICB response in NSCLC. We show that integrating tumor extrinsic cytotoxic gene expression with tumor mutational burden is associated with longer time to progression in a real-world cohort of 123 NSCLC patients treated with ICB regimens, including those with HLA-LOH.

Lymphocytic Airway Inflammation in Lung Allografts.

Lung transplant remains a key therapeutic option for patients with end stage lung disease but short- and long-term survival lag other solid organ transplants. Early ischemia-reperfusion injury in the form of primary graft dysfunction (PGD) and acute cellular rejection are risk factors for chronic lung allograft dysfunction (CLAD), a syndrome of airway and parenchymal fibrosis that is the major barrier to long term survival. An increasing body of research suggests lymphocytic airway inflammation plays a significant role in these important clinical syndromes. Cytotoxic T cells are observed in airway rejection, and transcriptional analysis of airways reveal common cytotoxic gene patterns across solid organ transplant rejection. Natural killer (NK) cells have also been implicated in the early allograft damage response to PGD, acute rejection, cytomegalovirus, and CLAD. This review will examine the roles of lymphocytic airway inflammation across the lifespan of the allograft, including: 1) The contribution of innate lymphocytes to PGD and the impact of PGD on the adaptive immune response. 2) Acute cellular rejection pathologies and the limitations in identifying airway inflammation by transbronchial biopsy. 3) Potentiators of airway inflammation and heterologous immunity, such as respiratory infections, aspiration, and the airway microbiome. 4) Airway contributions to CLAD pathogenesis, including epithelial to mesenchymal transition (EMT), club cell loss, and the evolution from constrictive bronchiolitis to parenchymal fibrosis. 5) Protective mechanisms of fibrosis involving regulatory T cells. In summary, this review will examine our current understanding of the complex interplay between the transplanted airway epithelium, lymphocytic airway infiltration, and rejection pathologies.

Transcriptome Analysis of Tumor-Infiltrating Lymphocytes Identifies NK Cell Gene Signatures Associated With Lymphocyte Infiltration and Survival in Soft Tissue Sarcomas.

PurposeClinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS.Experimental DesignUsing fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3-CD56+ NK and CD3+ T cells and CD45- viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression.ResultsSorted intra-tumoral CD3+ T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45- tumor cells. TCGA analysis revealed that tumors with high TLR4 expression (P = 0.03) and low expression of STMN1 involved in microtubule polymerization (P < 0.001) were associated with significantly improved survival.ConclusionsUnlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS.

Three residues in the BTB domain promote a good partnership between NuRD and Thpok.

Among the BTB-ZF transcription factor family, three amino acids in the BTB domain make Thpok unique in repressing cytotoxic lineage-related genes via recruitment of the NuRD chormatin-remodeling complex (see the related Research Article by Gao et al.).

Abstract 1983: Modulation of immune gene expression by intra-tumoral oncolytic adenovirus ONCOS-102 is associated with clinical response in anti-PD-1 refractory/resistant melanoma

Abstract Background: Defining molecular parameters of clinical response to immune modulators may lead to development of novel biomarkers, new therapeutic targets and drug combinations in cancer treatment. We recently completed phase I/II testing of ONCOS-102, a GM-CSF-encoding oncolytic adenovirus (Ad5/3-D24-GMCSF), for therapeutic efficacy and capacity to remodel tumor micro-environment (TME) in combination with pembrolizumab (pem) in patients (pts) with non-resectable, stage III-IV, anti-PD-1 resistant/refractory (R/R) melanoma (NCT03003676). Here we report comparative, longitudinal gene expression analysis of tumor samples from pts with complete/partial response (CR/PR), stable (SD) or progressive (PD) disease by RECIST1.0. Methods: In study part 1, n=9 pts received three intra-tumoral (i.t.) injections of ONCOS-102 every 3 days during the first week of treatment. Pem was administered i.v. at 10 mg/kg from day 22 every 3 weeks for 6 months. In study part 2, n=12 pts received additional injections of ONCOS-102 at day 15 and then every 3 weeks along with pem. On-treatment tumor biopsies were collected at baseline, day 22 and 64. Total RNA sequencing followed by differential gene expression analysis using DESeq1 was conducted on n=17 pts to disclose dynamic transcriptome changes associated with ONCOS-102 administration and clinical response. Results: Baseline biopsies of CR/PR pts showed 1,5-2 fold higher levels of expression of innate and adaptive immune response-related genes compared to PD pts. Treatment with ONCOS-102 upregulated immune related genes at day 22 across most tumors with no clear PR/PD differentiation. However, at day 64 a striking distinction between pts with CR/PR compared to PD emerged, showing 4-5 fold increase in expression levels for several immune-related gene categories including check-points, co-stimulatory and cellular cytotoxicity genes. TME remodeling in CR/PR pts was sustained at least until day 64. On individual genes,higher and more sustained upregulation of several co-stimulatory molecules (4-1BB, OX40, CD28) and checkpoints (LAG-3, TIGID, TIM-3) was seen in responders. Expression of adenovirus genes was more prevalent in CR/PR (2 of 3 pts) than PD (0 of 2 pts) on day 64, suggesting that tumor susceptibility to virus entry and replication drives clinical benefit of ONCOS-102. Our findings were supported by analysis of other gene subsets and comparison of different schedules of ONCOS-102 administration. Conclusions: ONCOS-102 drives pro-inflammatory modulation of the TME in PD-1 R/R melanoma tumors. Clinical response ONCOS-102 is associated with efficient viral gene expression and strong, sustained activation of immune-related genes, which may be targets for future combinations of ONCOS-102 and immune-modulators beyond PD-1/PD-L1 inhibitors. Citation Format: Erik Digman Wiklund, Lukasz Kuryk, Sandeep Kumar, Magnus Jaderberg, Victor levitsky, Thomas Birkballe Hansen, Anne-Sophie Moller, Alexander Shoushtari. Modulation of immune gene expression by intra-tumoral oncolytic adenovirus ONCOS-102 is associated with clinical response in anti-PD-1 refractory/resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1983.

SiRNA Targeting Mcl-1 Potentiates the Anticancer Activity of Andrographolide Nanosuspensions via Apoptosis in Breast Cancer Cells.

Breast cancer is the second leading cause of cancer-related death in the US. However, recurrence is frequently found despite adjuvant therapy being available. Combination therapy with cytotoxic drugs and gene therapy is being developed to be a new promising cancer treatment strategy. Introducing substituted dithiocarbamate moieties at the C12 position of andrographolide (3nAG) could improve its anticancer selectivity in the MCF-7 breast cancer cell line. However, its hydrophobicity is one of its main drawbacks. This work successfully prepared 3nAG nanosuspension stabilized with the chitosan derivative NSC (3nAGN-NSC) to increase solubility and pharmacological effectiveness. siRNAs have emerged as a promising therapeutic alternative for interfering with particular mRNA. The 3nAGN-NSC had also induced Mcl-1 mRNA expression in MCF-7 human breast cancer cells at 8, 12, and 24 h. This indicates that, in addition to Mcl-1 silencing by siRNA (siMcl-1) in MCF-7 with substantial Mcl-1 reliance, rationally devised combination treatment may cause the death of cancer cells in breast cancer. The Fa-CI analysis showed that the combination of 3nAGN-NSC and siMcl-1 had a synergistic effect with a combination index (CI) value of 0.75 (CI < 1 indicating synergistic effects) at the fractional inhibition of Fa 0.7. The synergistic effect was validated by flow cytometry, with the induction of apoptosis as the mechanism of reduced cell viability. Our findings suggested the rational use of 3nAGN-NSC in combination with siMcl-1 to kill breast cancer cells.

DNMT3A mutations define a unique biological and prognostic subgroup associated with cytotoxic T cells in PTCL-NOS

AbstractPeripheral T-cell lymphomas (PTCLs) are heterogenous T-cell neoplasms often associated with epigenetic dysregulation. We investigated de novo DNA methyltransferase 3A (DNMT3A) mutations in common PTCL entities, including angioimmunoblastic T-cell lymphoma and novel molecular subtypes identified within PTCL–not otherwise specified (PTCL-NOS) designated as PTCL-GATA3 and PTCL-TBX21. DNMT3A-mutated PTCL-TBX21 cases showed inferior overall survival (OS), with DNMT3A-mutated residues skewed toward the methyltransferase domain and dimerization motif (S881–R887). Transcriptional profiling demonstrated significant enrichment of activated CD8+ T-cell cytotoxic gene signatures in the DNMT3A-mutant PTCL-TBX21 cases, which was further validated using immunohistochemistry. Genomewide methylation analysis of DNMT3A-mutant vs wild-type (WT) PTCL-TBX21 cases demonstrated hypomethylation in target genes regulating interferon-γ (IFN-γ), T-cell receptor signaling, and EOMES (eomesodermin), a master transcriptional regulator of cytotoxic effector cells. Similar findings were observed in a murine model of PTCL with Dnmt3a loss (in vivo) and further validated in vitro by ectopic expression of DNMT3A mutants (DNMT3A-R882, -Q886, and -V716, vs WT) in CD8+ T-cell line, resulting in T-cell activation and EOMES upregulation. Furthermore, stable, ectopic expression of the DNMT3A mutants in primary CD3+ T-cell cultures resulted in the preferential outgrowth of CD8+ T cells with DNMT3AR882H mutation. Single-cell RNA sequencing(RNA-seq) analysis of CD3+ T cells revealed differential CD8+ T-cell subset polarization, mirroring findings in DNMT3A-mutated PTCL-TBX21 and validating the cytotoxic and T-cell memory transcriptional programs associated with the DNMT3AR882H mutation. Our findings indicate that DNMT3A mutations define a cytotoxic subset in PTCL-TBX21 with prognostic significance and thus may further refine pathological heterogeneity in PTCL-NOS and suggest alternative treatment strategies for this subset.

Multiomics profiling and association with molecular and immune features in association with benefits from immunotherapy for patients with previously treated stage IV or recurrent squamous cell lung cancer from the phase III SWOG LungMAP S1400I trial.

9046 Background: Immune checkpoint blockade (ICB) has become a standard pillar of treatment for lung cancer. However, only ̃20% of unselected patients can achieve durable clinical benefits. We performed immunogenomic profiling of tissue specimens from a randomized Phase III trial S1400I on metastatic lung squamous cell carcinoma (SCC) to evaluate if there were factors associated with better prognoses with ICB from single-agent versus combined targeting PD-1/CTLA-4 and evaluate if any differentiated between the treatments. Methods: We utilized FFPE tumor tissue submitted for Lung-MAP screening provided by the SWOG bank. SCC samples from 82 eligible patients treated with combined nivolumab+ipilimumab (N+I) or single agent nivolumab (N) were subjected to multiplex immunofluorescence (mIF, n = 82) and NanoString (ncounter PanCancer Immune Profiling Panel, n = 32). Cell density phenotypes (cells/mm2) were defined using image analysis of staining for cytokeratin, CD3, CD8, granzyme B, CD45RO, FOXP3, PD1, PD-L1, and CD68. Immunogenomic features were associated with response, PFS, and OS derived from data provided by the LungMap team to the CIDC portal. For statistical analyses, non-parametric tests were utilized to assess associations of cell phenotypes versus continuous or categorical variables, and log-rank test analysis was performed to identify cell phenotypes or genes correlated with survival. Results: In both arms higher densities of total CD3+CD45RO+ T cells ( P= 0.041), CD3+PD-1+ T cells ( P= 0.024) and CD3+CD8+PD-1 T cells in stroma ( P= 0.042) and CD3+CD8+GZMB+ T cells in the tumor compartment ( P= 0.011) were positively associated with PFS. In the N+I arm but not in the N arm, higher densities of CD3+CD8+GZMB+ T cells in the tumor compartment were associated with better PFS ( P= 0.015) and higher densities of stroma CD3+CD8-FOXP3+ T cells with worse OS. Spatial analysis showed that the presence of CD8+GZMB+ T cells close to malignant cells (median, ≤19.27 µm) was associated with better PFS ( P= 0.037) in N+I arm and cluster analysis showed low clustering of cells in TMB-high vs. TMB-low tumors (P &lt; 0.01). Gene expression profiling demonstrated that myeloid infiltration, immune recruitment, and inflammation genes were associated with a positive clinical outcome ( P&lt; 0.05). In both arms, BLNK, CD163, FCGR2A were associated with better OS ( P&lt; 0.01), IRF1 and BLNK were associated with increased PFS ( P&lt; 0.01). In the N+I arm but not in the N arm, we observed significantly higher CD45 immune cell scores, including CD8 T cells and neutrophils, in responders versus non-responders. Conclusions: Our findings suggest a potential advantage in PFS and OS with an increased presence of cytotoxic immune cells and genes associated with the recruitment and proliferation of these cell types before therapy.