Abstract Checkpoint-inhibitors (CPIs) improve outcomes in metastatic melanoma (MM), but resistance limits benefit. This phase I/II (NCT02706353) study evaluated intratumoral sotigalimab (an anti-CD40 agonistic antibody) with pembrolizumab in 32 CPI-naïve MM patients. Here, we report the final safety outcomes, clinical efficacy results, and comprehensive biomarker analyses from this trial. Primary endpoints included determining safety and tolerability, the recommended phase 2 dose (RP2D) of sotigalimab, and the objective response rate (ORR). The most common adverse events (AEs) related to sotigalimab were injection-site reactions, pruritus, and fatigue. Sotigalimab was well tolerated; common adverse events were injection-site reactions, and fatigue. At the RP2D, the ORR was 50% and the disease control rate (DCR) was 92%, with ORR of 67% in injected tumors and 50% in non-injected tumors. Multiomic biomarker analyses of tumor and blood samples collected before and during treatment demonstrated that sotigalimab effectively engaged the CD40 pathway, boosting the infiltration and activation of myeloid cells, including CD11c+DC-LAMP+ dendritic cells (DCs) and macrophages. The combination therapy activated innate and adaptive immunity in injected tumors and cytotoxic responses in non-injected tumors. TCR sequencing analysis showed increased T-cell clonality with expanded new clones shared across tumors. Clinical responses correlated with these immunologic changes, but not with baseline immunological features associated with response to anti-PD1 monotherapy. These findings suggest that intratumoral sotigalimab may enhance anti-PD1 therapy, supporting the need for further randomized phase 2 trials to better evaluate its therapeutic potential in the 'in situ' immunization approach. Citation Format: Salah-Eddine Bentebibel, Daniel J McGrail, Veena Kochat, Emre Arslan, Reham Abdel-Wahab, Sung-Nam Cho, Xiaodong Yang, Daniel H Johnson, Rodabe N Amaria, Isabella C Glitza, Patrick Hwu, Hussein A Tawbi, Jared K Burks, Michael A Davies, Kunal Rai, Suhendan Ekmekcioglu, Adi Diab. Intratumoral sotigalimab with pembrolizumab induces rapid activation of antigen presenting cells and drives anti-tumor responses in non-injected tumors in metastatic melanoma: A phase I/II study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT238.

Abstract Background: Mucosal melanoma (MuM) is a rare melanoma subtype, accounting for only 1-2% of all melanoma cases, yet is highly aggressive, demonstrating poorer responsiveness to immune checkpoint blockade than the more common cutaneous melanoma. However, the biological mechanisms driving therapeutic resistance in MuM remain poorly understood. This study aims to uncover spatial and molecular mechanisms underlying response and resistance of MuM to immune checkpoint blockade therapies. Study Design and Methods: An integrative spatial multi-omics framework was applied, which combined single-cell spatial proteomics using COMET platform with high mass resolution spatial metabolomics using imaging mass spectrometry (MALDI-IMS). We comprehensively profiled cellular compositions and spatially defined cellular neighborhoods (CNs) across 97 FFPE tissue cores from 26 MuM patients treated with PD-1/PD-L1 or CTLA-4 inhibitors. Spatial organization, cell-cell interactions, proteomic profiles and metabolomic features of CNs were further compared across responders and non-responders. Results: The comprehensive approach enabled spatially resolved profiling of 695,444 single cells, which were categorized into 25 cellular phenotypes spanning 9 major cell lineages. Spatial cellar neighborhood analysis revealed 15 biologically distinct CNs that differed in their composition and spatial distribution of tumor, immune, and stromal cell compartments. In patients who responded to immunotherapy, three tumor-associated CNs—the central tumor, invasive tumor, and tumor boundary CNs—were significantly enriched and collectively formed unique spatial organization patterns. Notably, the invasive tumor CN and tumor boundary CN were characterized by spatial proximity among Ki67+ tumor cells, CD163+ macrophages, and CD11c+ dendritic cells. These CD163+ macrophages exhibited reduced expression of IRF4 and Arg1, consistent with lower immunosuppressive activity. Conversely, non-responders exhibited a stromal-dominant CN composed primarily of SMA- stromal cells and demonstrated reduced immune infiltration in both pre-treatment and post-treatment samples. Spatial metabolomic profiling further revealed a pronounced reduction of tryptophan-derived indole metabolites in responders, which significantly correlated with CD11c and CD163 expression, indicating coordinated immunometabolic remodeling within the tumor microenvironment. Conclusions: These findings highlight that spatial tumor-immune architecture, stromal exclusion, and metabolic rewiring collectively shape immunotherapy response in MuM. The identified spatially resolved tryptophan-derived metabolite signatures offer promising biomarkers and potential therapeutic targets to improve treatment outcomes in this clinically challenging melanoma subtype. Citation Format: Jun Wang, Priyadharsini Nagarajan, Sungnam Cho, Yunhe Liu, Erin H. Seeley, Yibo Dai, Yang Liu, Jared K. Burks, Jennifer L. McQuade, Adi Diab, Linghua Wang, Suhendan Ekmekcioglu. Integration of spatial single cell proteomics and spatial metabolomics reveals tumor microenvironment predictive of immunotherapy response in mucosal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7750.

Mucosal melanoma (MuM) is a rare but aggressive malignancy with limited benefit from immune checkpoint inhibition and few predictive biomarkers. We integrated single-cell spatial proteomics (COMET) and spatial metabolomics (MALDI-IMS) to profile 97 tissue cores from 26 patients treated with PD-1/PD-L1 and/or CTLA-4 inhibitors. We profiled 695,444 cells and resolved 25 cell states across eight major cell types. Cellular neighborhood (CN) analysis revealed distinct tumor- and stromal-associated spatial architectures. Responders were enriched for tumor-associated CNs (invasive tumor and tumor boundary) with close spatial proximity among Ki67+ tumor cells, CD163+ macrophages, and CD11c+ dendritic cells (DCs), and increased proliferating/cytotoxic CD8+ T-cell subsets. Non-responders showed stromal CN dominance with reduced immune infiltration. Spatial metabolomics identified lower abundance of indole-derived metabolites and reduced indole/tryptophan pathway activity in responders within tumor and TME regions that tracked with DC/macrophage-enriched spatial contexts. This study advances MuM spatial biology and provides a framework for biomarker-driven immunotherapy strategies.

Immune checkpoint blockers (ICB) improve outcomes in metastatic melanoma (MM), but resistance limits benefit. This phase I/II (NCT02706353) study evaluated intratumoral sotigalimab (anti-CD40 agonist) with pembrolizumab in 32 patients with ICB-naïve MM. Primary endpoints were safety and objective response rate (ORR). Sotigalimab was well tolerated. At the recommended phase II dose, the ORR was 50%, and the disease control rate was 92%, with ORRs of 67% in injected and 50% in non-injected tumors. Multiomic analyses of tumor and blood showed that sotigalimab effectively engaged the CD40 pathway, boosting infiltration and activation of myeloid cells, including CD11c+DC-LAMP+ dendritic cells and macrophages. The combination therapy activated innate and adaptive immunity in injected tumors and cytotoxic responses in non-injected tumors. T-cell receptor sequencing showed increased T-cell clonality with expanded new clones shared across tumors. Clinical responses correlated with these immunologic changes but not with baseline features associated with response to anti-PD-1 monotherapy. SIGNIFICANCE:In this study, we provide compelling data that intratumoral sotigalimab combined with pembrolizumab is safe, activates antigen-presenting cells, and elicits broad innate and adaptive immune responses in both injected and non-injected tumors, supporting further randomized phase II trials to evaluate sotigalimab's potential to enhance anti-PD-1 therapy through "in situ" immunization.

Mucosal melanoma (MuM) is a rare and aggressive cancer with limited response to immunotherapy. Despite its clinical severity, the mechanisms that drive therapeutic resistance in MuM are still largely unclear. To elucidate the spatial and molecular determinants of therapeutic outcomes, we designed this study to identify the spatial and molecular factors that shape both responsiveness and resistance of MuM to immune checkpoint blockade therapies. We employed a comprehensive spatial multi-omics strategy that integrated COMET-based single-cell spatial proteomics with high-resolution metabolite imaging via MALDI-IMS. Using this platform, we analyzed the cellular composition and mapped spatially organized cellular neighborhoods (CNs) across 97 FFPE tissue cores obtained from 26 MuM patients who received PD-1/PD-L1 or CTLA-4 blockade. We further examined how spatial architecture, cell–cell interactions, proteomic signatures, and metabolomic profiles of CNs varied between treatment responders and non-responders. This integrative strategy enabled spatially resolved characterization of about 700K single cells, which were classified into 25 phenotypic clusters across nine major cellular lineages. Spatial neighborhood analysis identified 15 biologically distinct CNs, each exhibiting unique compositions and spatial arrangements of tumor, immune, and stromal components. Among patients who responded to immunotherapy, three tumor-associated CNs—the central tumor, invasive tumor, and tumor boundary CNs—were markedly enriched, displaying distinctive spatial architecture. Notably, the invasive and boundary CNs showed close spatial proximity among proliferating tumor cells, macrophages, and dendritic cells. These CD163+ macrophages demonstrated decreased IRF4 and Arg1 expression, indicative of reduced immunosuppressive function. In contrast, non-responders showed a predominance of a stromal-rich CN characterized mainly by SMA- stromal cells with limited immune cell infiltration, observed in both baseline and post-treatment samples. Spatial metabolomic analysis further revealed a substantial decrease in tryptophan-derived indole metabolites in responders, which strongly correlated with CD163 and CD11c expression, suggesting a coordinated immunometabolic reprogramming within the tumor microenvironment. Collectively, these insights underscore that immunotherapy response in MuM is orchestrated by an interplay of spatial tumor–immune architecture, stromal insulation, and metabolomic reprogramming collectively underpin differential immunotherapy responses in MuM. The spatially resolved signatures of tryptophan-derived metabolites not only emerge as promising biomarkers but also present tractable therapeutic targets to potentially overcome resistance in this aggressive melanoma subtype. Suhendan Ekmekcioglu, Jun Wang, Priyadharsini Nagarajan, SungNam Cho, Yunhe Liu, Erin H. Seeley, Jared K. Burks, Adi Diab, Linghua Wang. Spatial multi-omics identifies a tumor microenvironment signature predictive of immunotherapy response in mucosal melanomas [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A036.

12134 Background: Immune checkpoint inhibitors (ICIs) have transformed cancer therapy, but grade ≥ 3 immune-related adverse events (irAEs) affect 60% of patients. The mechanisms driving irAEs remain poorly understood, hindering the development of strategies to mitigate toxicity without compromising efficacy. Methods: We analyzed biomarkers from an ongoing phase I/II trial (NCT04940299) evaluating tocilizumab (IL-6R blockade) in two regimens; 162 mg subcutaneously weekly (regular dose, RD) or bi-weekly (dose-dense, DD), combined with ipilimumab (3 mg/kg) and nivolumab (1 mg/kg) up to 12 weeks as front-line therapy for advanced melanoma. Longitudinal analyses of blood, tumor, and inflamed tissue biopsies were performed to identify biomarkers for risk stratification and to elucidate the immunobiology of irAEs and antitumor responses across four patient subgroups, categorized by tumor response and the presence or absence of grade ≥ 3 irAEs. Results: A total of 35 patients were treated with the triplet and followed for up to 15 months. By week 12 after treatment initiation, grade ≥ 3 irAEs occurred in 15 patients (43%), with similar frequencies in the RD (44%) and the DD (40%) cohorts. Within 90 days after last tocilizumab dose, the incidence of grade ≥ 3 irAEs increased to 56% in the RD cohort but remained unchanged in the DD cohort, resulting in an overall incidence of 51%. The best overall response rate (ORR) was 66%, with 64% in the RD cohort and 70% in the DD cohort. NanoString analysis of tumor biopsies identified 31 upregulated genes in patients with grade ≥ 3 irAEs compared to those without, including RORC, a key regulator of Th17 differentiation, which was significantly elevated in longitudinal biopsies (pre: n = 8; post: n = 6) of patients with high-grade irAEs. IL-17, IL-1, and TNF signaling pathways were significantly decreased after tocilizumab treatment in the subgroup of patients without the grade ≥ 3 irAEs (pre: n = 14, post: n = 9). LunaPhore-COMET analysis showed elevated Th17 and γδ T-cell subsets (CD4+ and CD8+) in inflamed tissues compared to matched healthy or tumor tissues. CyTOF profiling of blood showed higher γδ T-cell levels at baseline in patients with grade ≥ 3 irAEs (n = 6), which remained elevated following tocilizumab treatment. Conclusions: These findings underscore the pivotal role of Th17 cells in the development of irAEs and suggest that current tocilizumab regimens may provide insufficient IL-6 blockade or require combination strategies, such as concurrent IL-23 inhibition, to more effectively target Th17 cells expansion and maintenance. Notably, this study is the first to identify γδ T-cells as potential predictive biomarkers for irAEs, yet their utility requires further validation. A randomized phase II trial exploring these mechanisms is underway. Clinical trial information: NCT04940299 .

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.

The Immune Checkpoint Inhibitors (ICIs) have significantly changed treatment approaches for patients with various cancers, including melanoma, in advanced and/or adjuvant settings. Acral melanomas are rare but aggressive clinical subtype of melanomas and there is an urgent need to improve care for acral melanomas aimed at the treatment with ICIs. While biomarker studies and drug discoveries in cutaneous melanoma have achieved substantial progress, little is known about either what are the prognostic markers for acral melanomas and how to predict response to ICI treatment modalities. Here, we target to understand prognostic significance of Tumor Immune Micro Environment (TIME) characteristics and progression of acral melanomas as well as the predictive factors for the ICI-treatment options, with a specific focus on complex immune-biology between white and non-white patients with acral melanomas. In our current study, we hypothesized that innate inflammatory enzymes and their mediators in acral melanoma TIME causes “immune exclusion” by altering the tissue structure that could be otherwise “immune inflamed” and incline the tumor responsiveness. To understand the complexity of the TIME in these patients, we visualized this highly complex cellular structure and studied it in its intact form for many protein biomarkers. We performed a retrospective analysis of 40 tumor tissue samples from patients with advanced or metastatic acral melanoma, treated with anti-CTLA4 or anti-PD1 immunotherapy to characterize the immune signatures and perform and quantify immune cell profiling in pre-treatment and on-treatment tumor tissue samples. For detailed immunophenotyping of TIME, we used cycling IHC (by Lunaphore-Comet) for 40 individual cell surface and intracellular markers per cell, as well as additional structural and functional markers. We further performed inflammatory markers by multiplex IHC (by Opal system) to align our inflammatory cores with immune cell neighborhoods. We have also performed imaging of metabolites, glycans, and peptides MALDI-TOF mass spectrometer in the same cohort and encouraged by our preliminary results on differential characteristics on tumor areas with various immune infiltration, such as no immune cells, versus peripheral or intratumoral localizations. Our initial analyses revealed that the differences in immune cell distribution inside tumor areas (peri- versus intra-tumoral areas), as well as patient ethnicities (Hispanic or Latino versus non-Hispanic white), specifically on NK cells and macrophage subtypes, which supports our original hypothesis on differential and complex immune-biology on white and non-white patients with acral melanomas. Our ongoing proteomics and metabolomics analyses of TIME in this unique cohort of acral melanomas, as one of the rarest and relatively under explored group of patients, will further guide us in stratifying patient selections and personalize the future treatment decisions. Citation Format: Suhendan Ekmekcioglu, Kaysia Ludford, SungNam Cho, Salah Eddine-Bentebibel, Akshay V Basi, Reham Abdel-Wahab, Khalida M Wani, Noha Abdel-Wahab, Jared K Burks, Adi Diab. Immune and metabolic architecture of TIME in metastatic acral melanoma patients treated with checkpoint inhibitors [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A070.

Introduction:K-ras mutant lung adenocarcinoma (KM-LUAD) is a difficult-to-treat cancer subtype in which chronic inflammation pervades the tumor immune microenvironment (TIME). Pro-inflammatory pathways dampen the response to treatments, including immune checkpoint inhibitors, necessitating therapies that target this inflammatory signaling network in the TIME. One of the lynchpins of chronic inflammation in KM-LUAD is signal transducer and activator of transcription 3 (STAT3). Methods:Here, we tested the anti-tumor and early immunotherapeutic efficacy of TTI-101, a selective small-molecule inhibitor of canonical STAT3 signaling, in a K-rasG12D mutant lung cancer mouse model (CC-LR). Results:Treatment of CC-LR mice with TTI-101 resulted in reduced tumor burden while increasing dendritic cell (DC) and T helper 1 (Th1) infiltration into the TIME. TTI-101 treatment decreased pY-STAT3 expression in tumors with accompanying increases in several NF-κB anti-tumor target genes including CXCL9, a chemokine for primed T cells. Transcriptional profiling of the TIME revealed improved immune activation and anti-tumor skewing, as well as B cell signaling enrichment. Analysis of human LUAD data demonstrated negative correlations between STAT3 and Th1/DC infiltration, with DC infiltration also conferring improved survival in LUAD patients with low STAT3. Discussion:Our results highlight the importance of STAT3 in driving early tumorigenesis and offer a preventative treatment window for high-risk individuals and patients with early-stage KM-LUAD.

Interferon gamma (IFNγ) is a critical cytokine known for its diverse roles in immune regulation, inflammation, and tumor surveillance. However, while IFNγ levels were elevated in sera of most newly diagnosed acute myeloid leukemia (AML) patients, its complex interplay in AML remains insufficiently understood. We aim to characterize these complex interactions through comprehensive bulk and single-cell approaches in bone marrow of newly diagnosed AML patients. We identify monocytic AML as having a unique microenvironment characterized by IFNγ producing T and NK cells, high IFNγ signaling, and immunosuppressive features. IFNγ signaling score strongly correlates with venetoclax resistance in primary AML patient cells. Additionally, IFNγ treatment of primary AML patient cells increased venetoclax resistance. Lastly, a parsimonious 47-gene IFNγ score demonstrates robust prognostic value. In summary, our findings suggest that inhibiting IFNγ is a potential treatment strategy to overcoming venetoclax resistance and immune evasion in AML patients.

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.

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.

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.

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.

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.