TPS9616 Background: Tumor infiltrating lymphocyte (TIL) therapy is an autologous cellular therapy isolated from a patient's tumor. Clinical data demonstrated that TIL therapy in heavily pretreated melanoma patients yielded an objective response rate (ORR) in 30% of patients with 5-year overall survival (OS) of approximately 20%. TILs were approved by the FDA in February 2024 for the treatment of metastatic or unresectable melanoma that have progressed on anti-PD-1 therapy and BRAF+MEK inhibitors (in patients with BRAFV600 mutation). Non-melanoma skin cancers, including cutaneous squamous cell cancer (CSCC) and Merkel cell cancer (MCC), share biologic features with melanoma, including an “inflamed” tumor microenvironment and high responsiveness to anti-PD-1 therapy. However, approximately, a third of patients will experience disease progression after anti-PD-1 therapy or will discontinue treatment due to toxicity and subsequently experience disease progression. Given the overlap between melanoma and CSCC/MCC, we hypothesize that TIL therapy may induce immune response against non-melanoma skin cancers. Because some of these tumors are superficial and are often ulcerated and can contain polymicrobial contamination, this trial will also assess the feasibility of TIL production in this unique patient population, alongside its safety and efficacy. Methods: Ten patients with CSCC (Cohort A) and 4 patients with MCC (Cohort B) adults will be eligible for TIL if they have disease progression after anti-PD-1 therapy. Eligible patients should have adequate organ function and be able to receive dose-reduced non-myeloablative lymphodepletion (NMA-LD) and interlukin-2 (IL-2). Following tumor harvest and TIL manufacturing, patients will receive NMA-LD including cyclophosphamide (30 mg/kg on days –5 and –4), and fludarabine (25 mg/m2 on days –5 to –1). Subsequently, patients will receive TIL on day 0 followed by IL-2 (600,000 IU/kg) for up to 6 doses. The primary objective is to evaluate feasibility and safety of TIL production and administration in cohorts A and B (defined by successful tumor harvest that leads to a TIL product that contains ≥ 1 x 10^9 cells, administration of NMA-LD, infusion of TIL therapy and complete at least 1 dose administered of IL-2). Secondary objectives include ORR, progression-free survival, duration of response, OS and correlative studies. Within Cohort A, efforts will be made to achieve an approximately equal distribution of ulcerated and non-ulcerated lesions. To maintain this balance, ulceration status will be monitored throughout the enrollment process, and eligibility criteria may be modified as necessary to ensure proportional representation of each subtype. Clinical trial information: NCT07288073 .

Introduction:Immune checkpoint inhibitors (ICIs) have significantly improved survival for patients with metastatic melanoma, yet many experienceresistance due to immunosuppressive mechanisms within the tumor immune microenvironment (TIME). Understanding how the spatial architecture of immune and inflammatory components changes across disease stages may reveal novel prognostic biomarkers and therapeutic targets. Methods:We performed high-dimensional spatial profiling of two melanoma tissue microarrays (TMAs), representing Stage III (n = 157) and Stage IV (n = 248) metastatic tumors. Using imaging mass cytometry (IMC) and multiplex immunofluorescence (mIF), we characterized the phenotypic, functional, and spatial properties of the TIME. Cellular neighborhoods were defined by inflammatory marker expression, and spatial interactions between immune and tumor cells were quantified using nearest-neighbor functions (G-cross). Associations with survival were assessed using Cox proportional hazards models with robust variance estimation. Results:Stage IV tumors exhibited a distinct immune landscape, with increased CD74- and MIF-enriched inflammatory neighborhoods and reduced iNOS-associated regions compared to Stage III. Cytotoxic T lymphocytes (CTLs) and tumor cells were more prevalent in Stage IV TIME, while B cells and NK cells were depleted. Spatial analysis revealed that CTL-Th cell, NK-T cell, and B-NK cell interactions were linked to improved survival, whereas macrophage aggregation and excessive B-Th cell clustering in inflammatory regions correlated with worse outcomes. Organ-specific analyses showed that CTL infiltration near tumor cells predicted survival in gastrointestinal metastases, while NK-T cell interactions were prognostic in lymph node and skin metastases. Discussion:Our results reveal stage-specific shifts in immune composition and spatial organization within the melanoma TIME. In advanced disease, immunosuppressive neighborhoods emerge alongside changes in immune cell localization, with spatial patterns of immune coordination-particularly involving CTLs, NK cells, and B cells-strongly predicting survival. These findings highlight spatial biomarkers that may refine patient stratification and guide combination immunotherapy strategies targeting the inflammatory architecture of the TIME.

Introduction:Inducible nitric oxide synthase (iNOS) plays a critical role in inflammatory signaling and tumor immunology, contributing to both pro- and anti-tumor effects depending on the cellular context. While iNOS induction has been linked to immune activation and tumor progression, its expression in cancer cells is highly variable and often inconsistently reported across different tumor models. To address this gap, we developed a well-defined in vitro platform using the human colorectal adenocarcinoma cell line DLD-1 to model stimulus-dependent iNOS expression and nitric oxide (NO) signaling. Methods:DLD-1 cells were stimulated with a pro-inflammatory cytokine cocktail (lipopolysaccharide [LPS], interleukin-1β [IL-1β], and interferon-γ [IFN-γ]), resulting in marked upregulation of iNOS at both the mRNA and protein levels. iNOS specificity was confirmed using targeted siRNA knockdown. Functional assessment of NO production was performed using the Nitrate/Nitrite Colorimetric Assay Kit and the ENO-30 NOx Analyzer. Induction of iNOS was further associated with elevated levels of reactive nitrogen species (RNS), reactive oxygen species (ROS), and protein nitration, including 3-nitrotyrosine, detected by immunohistochemistry and Western blot. Results:Upon stimulation, DLD-1 cells consistently expressed enzymatically active, full-length human iNOS and produced biologically relevant levels of NO and downstream nitrosative stress markers. Treatment with selective iNOS inhibitors significantly reduced nitrite accumulation, confirming the functional activity of iNOS and the model's applicability for pharmacologic evaluation of NO-modulatory compounds. Discussion:Our findings establish the DLD-1 cell line as a reproducible and well-controlled in vitro system for studying inducible iNOS expression and downstream NO/RNS signaling in human epithelial cancer cells. This platform provides a valuable tool for mechanistic studies, screening of iNOS-targeted agents, and resolving discrepancies in iNOS detection across experimental models in cancer biology.

Supplementary Figure 1. Western blots. Supplementary Figure 2. Graphs showing tumor volume from mice following intradermal injection of (A) HMEL-BRAF^V600E cells, (B) WM115 cells, (C) 1205 Lu cells, and (D) pMEL-NRAS^G12D overexpressing GFP, RNF2 wild-type or catalytic mutant derivatives (R70C or I53S). Supplementary Figure 3. (A) RNF expression in the melanoma expression data for normal skin, nevi, and primary tumors. Supplementary Figure 4. (A) Heat map showing clustering of top 10 percent deregulated genes in duplicates of expression data from RNF2^WT overexpressing compared to GFP overexpressing HMEL-BRAF^V600E cells. Supplementary Figure 5. Chromatin state analysis on HMEL-BRAF^V600E cells.

PDF file, 212K, (A) H&E staining of representative, A375 human xenograft tumor sections excised from NOD/SCID mice. Prior to excision, mice with established tumors were treated for 3 days with either PLX4720 or DMSO vehicle control. Magnification: 4x. (B) RT-PCR analysis of human IL1α, IL1β, IL-8, GRO-α, PD-L1, and PD-L2 transcripts derived from A375 xenografts shown in (A). Data show the average of 3 mice per group, and are representative of 3 separate experiments.

Genes that are occupied by RNF2 and show changed expression in HMEL-RNF2WT cells. This file provides the gene names of those genes that show change in expression in HMEL-RNF2WT cells compared to HMEL-GFP cells and contain RNF2 binding site in their promoter.

This file contains supplementary methods, supplementary figure legends, supplementary table legends and supplementary references.

PDF file, 188K, Tumor-associated fibroblasts (TAF) cultures were derived from three separate metastatic melanoma biopsies from three distinct anatomical sites: lymph node, lung and soft tissue. Cultured TAFs were exposed overnight to IL-1α, and treated and untreated cells were subjected to Affymetrix gene expression analysis. (A) Heatmap displaying the 197 most differentially expressed genes selected at FDR 0.01. Relative expression values are indicated by the color bar. (B) Gene set enrichment analysis of IL-1α-treated TAFs demonstrates a strong correlation with gene sets previously identified to be associated with NF-kappaB upregulation and the interferon response. (C) Heatmap showing IL-1α induced expression of nine genes previously reported to be associated with T-cell suppression.

Figure S1. Chemical Structure of MSC2156119J (Tepotinib, EMD 1214063); Figure S2. Morphology of melanoma cell 2D ambient air cultures and spheroids; Figure S3. The immunofluorescent staining of DAPI and LOX-1 in A375 2D ambient air cultures; Figure S4. The immunofluorescent staining of LOX-1 in MEL1617 3D spheroids on day 3 and 8 of cultures; Figure S5. The human phospho-kinase antibody array of 451Lu spheroids and 2D ambient air cultures; Figure S6. The human phospho-kinase antibody array of A375 spheroids and 2D ambient air cultures; Figure S7. Effect of PLX4032 on BRAF-ERK pathway in melanoma cells under hypoxic conditions. Supplementary Table S1: Primers for RT-PCR and Real-time PCR; Supplementary Table S2: Patient Characteristics; Supplementary Table S3: List of cutaneous melanoma cell lines in CCLE project

PDF file - 67K, A375 cells were treated with L-NIL (300 μM and 1000 μM) or the NO scavenger PTIO (300 μM) for 24 hours prior to harvest and preparation of protein lysates. Western Blot analyses of AKT phosphorylation at serine 473 and total AKT demonstrated lack of downregulation of AKT phosphorylation under conditions which strongly reduce downstream mTOR pathway activation.

PDF file - 132K, Mel 624 (A) and MeWo cells (B) were transfected with iNOS-specific or scrambled control siRNA and inoculated on the CAM for 5 days prior to photography of macroscopic tumors, histological processing, and trypan exclusion viability assay as described in Figure 1. Each graph represents data derived from 2 independent experiments, and error bars indicate standard error of the mean.

Aberrant expression of inducible nitric oxide synthase (iNOS) and chronic nitrosative stress is correlated with growth, metastasis, resistance to therapy, and poor patient survival of metastatic melanoma. One physiological influence of nitric oxide (NO) is exerted directly through the post-translational modification of many proteins, including the unique S-nitrosylation (SNO). To date, the role of SNO in melanoma development has not been fully characterized. Herein, we focus on the mechanism of how nitrosative stress regulates the tumor suppressor p53 in melanoma cells. We first examined the effects of different levels of NO donors, S-nitrosoglutathione (GSNO) and diethylenetriamine (DETA) NONOate, on the growth of melanoma cells by the MTT assay. Low levels of nitrosative stress (NO donors ≤ 20 µM) did not suppress the growth of human melanoma A375 and SB2 cells, but high levels of nitrosative stress (NO donors ≥ 50 µM) inhibited melanoma cell growth. Our biotin switch assays (BSA) showed that the treatment of NO donors significantly increased total S-nitrosylated proteins in A375 and SB2 cells. Markedly, p53 is confirmed to be one of the S-nitrosylated proteins derived from BSA. Our mass spectrometry analysis of SNO proteins in melanoma cells confirms that p53 is S-nitrosylated in A375 cells under nitrosative stress. Further proteomic characterization identifies Cys242, Cys275, and Cys277 as the SNO sites of p53. We conducted a molecular dynamics (MD) simulation of p53-DNA binding under SNO modification. The structural analysis shows that the SNO of Cys277 reduces the hydrogen bonds between p53 and the targeted DNA, interfering with the recognition and binding of p53 to DNA. Moreover, the chelating ability of Cys242 for Zn2+ is weakened after SNO, indicating the SNO of p53 reduces p53 binding to Zn2+ and affects its active form. The electrophoretic mobility shift assay also confirmed that the treatment of 100 µM of DETA NONOate, significantly decreased the nuclear p53 of A375 and SB2 cells binding to the consensus p53-binding DNA. The induction of nitrosative stress by NO donors also significantly increased mouse double minute 2 homolog (MDM2), p21, and p53 expression but decreased p53 upregulated modulator of apoptosis (PUMA) expression.This study confirms that the crucial tumor suppressor, p53, is S-nitrosylated in melanoma cells under nitrosative stress. Notably, for the first time, the SNO sites of p53 and the effects of SNO on p53 functions were characterized. Our results describe an important mechanism of how nitrosative stress modifies crucial cysteines of p53, alters p53 DNA-binding activities, and regulates downstream gene expression through SNO. Our study provides much-needed insights into identifying novel NO-driven SNO proteins in melanoma as new biomarkers and targets for drug development. Citation Format: Jordan Winfield, Mariana Grigoruta, Yiliang Li, Fancui Meng, Yong Qin, Elizabeth Grimm, Leyuan Chen, Kevin Rosenblatt, Li Li. S-nitrosylation of p53 in melanoma alters p53-DNA binding and downstream gene expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4785.

Clinical outcomes by CD74 categorical expression (both number and intensity) in tumor cells (A) in discovery and (B) in validation datasets of stage III melanoma patients. Kaplan-Meier analyses are shown for OS and RFS.

Binding sites for RNF2 in HMEL-RNF2WT cells. This file contains list of all chromosomal locations that show enrichment by MACS for RNF2 binding by V5 ChIP-Seq in HMEL-RNF2 cells.

The influence of ptgs1, ptgs2, and ptges KO on the production of arachidonic acid metabolites from murine BrafV600E melanoma cells. A, Schematic diagram of the arachidonic acid metabolic cascade. PGG2, prostaglandin G2; PGH2, prostaglandin H2; TxAS, thromboxane-A synthase; TxA2, thromboxane A2; PGDS, prostaglandin D synthase; cPGES, cytosolic prostaglandin E synthase; PGFS, prostaglandin F synthase; PGIS, prostaglandin I synthase. B and C,ptgs1, ptgs2, and ptges genes in murine BrafV600E melanoma cells were knocked out via CRISPR/CAS9. The mRNA and protein levels were analyzed by qRT-PCR (B) and Western blotting (C). Bar graphs show the fold change relative to mRNA levels of scramble control for each gene (B, n = 4). GAPDH was used as a loading control (C). D–H, Bar plot showing the concentrations of arachidonic acid metabolites released in supernatants obtained from scramble control, ptgs1-KO, ptgs2-KO, and ptges-KO cells. All prostanoids were measured by ELISA (n = 4): PGE2 (D), TxB2 (E), PGD2 (F), PGF2α (G), and 6-keto PGF1α (H). Graph values represent mean ± SD. Significance in difference between two groups was determined by Student t test. **, P < 0.01; *, P < 0.05.

This file contains distribution of the markers in discovery and validation set samples

Supplementary Figure 1. Gating strategies for flow cytometry data analysis. Supplementary Figure 2. Co-staining of mPGES1 and S100 in human melanoma tissues. Supplementary Figure 3. Chemokines/cytokines related to COX2 or mPGES1 in melanoma. Supplementary Figure 4. Effect of COX2 or mPGES1 inhibitors on surface expression of PD-L1. Supplementary Figure 5. Comparison of PGE2 level and cell viability in scramble, PTGES KO and PTGS1/PTGS2 double KO mouse melanoma cell lines. Supplementary Figure 6. Prognostic effect of mPGES1 in Stage IV melanoma. Supplementary Figure 7. Association between mPGES1 levels and response of anti-PD-1 therapy.

Supplementary Figure 1. Dose response in Treg and NK cell subsets upon WT IL-2 or F42K stimulation Supplementary Figure 2. Gating strategy for Tregs upon WT IL-2 or F42K stimulation Supplementary Figure 3. CD127 expression by ICOS+ Tregs and CD4+Foxp3- effector T cells Supplementary Figure 4. KI67 expression by NK cells after WT IL-2 or F42K stimulation Supplementary Figure 5. Effect of IL-2 mutant on purified cell populations Supplementary Figure 6. NK cell phenotype 3 days after WT IL-2 or F42K stimulation Supplementary Figure 7. Expression of activation markers by NK cells 6 days after WT IL-2 or F42K stimulation Supplementary Figure 8. Expression of inhibitory molecules by NK cell after WT IL-2 or F42K stimulation Supplementary Figure 9. Expression of cytolytic mediators by NK cells 6 days after WT IL-2 or F42K stimulation Supplementary Figure 10. IL-2 serum level post hydrodynamic gene transfer in mouse model Supplementary Figure 11. Changes of ICOS+ Tregs and NK cells in mouse model Supplementary Table 1. Immune response-related genes differentially regulated in F42K to WT IL-2

Additional information for materials and methods