Differentially expressed genes within T1, comparing lineage 4 to all others and excluding significant DEGs from the T1 lineage 1 to the rest comparison (WM4007).

Ultraviolet (UV) radiation induces DNA damage and oxidative stress in melanocytes, shaping pigmentation phenotypes and elevating photocarcinogenesis risk. Human models that capture donor-linked genetic determinants of UV sensitivity remain limited. Here, we establish a genotype-informed UV response model using induced pluripotent stem cell (iPSC)-derived melanocytes from donors carrying defined MC1R variants. Differentiated cells recapitulated melanocytic morphology, marker expression, and pigmentation consistent with donor sun-sensitivity traits. Following narrowband UVB exposure, melanocyte lines with higher UV sensitivity showed reduced survival, prolonged checkpoint activation, and CPD-associated DNA damage signaling dynamics. Mechanistic analysis suggests that the interferon-regulated GTPase MX2 is associated with amplification of UV-induced p53 and p38 activation while promoting apoptosis independently of AKT. These findings support MX2 as a physiological enhancer of DNA damage signaling in normal melanocytes, distinct from its interferon-mediated role in melanoma. Our study provides a human-relevant platform linking pigmentation genotype to UV resilience and supports iPSC-derived systems as new approach methodologies (NAMs) for mechanistic and translational phototoxicology.

Abstract There are currently no effective targeted therapies for BRAF-mutant metastatic melanoma patients with acquired resistance to approved BRAF and MEK inhibitors (BRAFi and MEKi), and very few ongoing clinical trials. Anti-apoptotic BCL2 family proteins promote de novo resistance to several therapies, including single-agent BRAFi in BRAF-mutant melanomas. In this study, in vivo testing of a large collection of patient-derived xenograft (PDX) models from melanoma patients with acquired resistance to BRAFi or BRAFi+MEKi shows that combining BCL2 inhibitors (BCL2i; navitoclax or venetoclax) with BRAFi+MEKi induces tumor regressions in a subset of these PDXs. High basal BCL2 predicts response whereas high basal MCL1 predicts resistance to this strategy. MCL1 overexpression studies functionally validate its role in resistance. Further, combining BRAFi+MEKi with an MCL1 inhibitor (MCL1i) counteracts resistance and interestingly decreases MCL1i-associated markers of cardiotoxicity. Together these studies identify potential personalized strategies to improve outcomes in this challenging patient population.

Supplementary Figure 4: Effects of resiquimod on CD45-and CD45+ cell fractions in MPDOs.

Differentially expressed MAPK-pathway genes before and immediately after the initiation of treatment (T0 v T1) (WM4007).

Melanoma is a common and aggressive cancer, with rising incidence in most developed countries. Major discoveries in melanoma biology have been rapidly translated, allowing cures for patients in late-stage disease. Despite these advances, many tumors remain refractory, in part due to an incomplete understanding of the genes and pathways gained or lost during melanoma tumorigenesis. To address this gap and provide a broadly useful resource for the scientific community, we established melPDomiX, a multi-omics cohort of melanoma-patient-derived xenografts. By linking mutations with transcriptomic and proteomic features, melPDomiX enables systematic characterization of gain- and loss-of-function alterations in treatment-refractory melanoma. Using multi-omics integration and structural-context representation, we demonstrate how this resource distinguishes gain- from loss-of-function variants and uncovers new candidate melanoma drivers and therapeutic targets. Together, melPDomiX provides a comprehensive, deeply profiled set of tumor models that supports mechanistic discovery and facilitates the development of improved treatments for this devastating heterogeneous malignancy.

Differentially expressed genes between time point 1 and time point 0 with significant corrected p-values in the resistant lineages (1, 2) but not in the sensitive lineages (0, 5, 8) , in the sensitive but not resistant, and in both sensitive and resistant (WM4007).

Differentially expressed genes between pre-treatment (T0) and early-treatment (T1) that are significant in both models WM4007 and WM4237.

Background/Objectives: Uveal melanoma is the most common primary ocular cancer in adults. Patients with metastatic uveal melanoma (mUM) have limited treatment options and poor prognosis. mUM is characterized by high oxidative phosphorylation (OXPHOS), which may be a therapeutic vulnerability for this disease. ONC206 is an imipridone compound that can inhibit OXPHOS indirectly and is currently being evaluated in clinical trials. Thus, we tested the effects of ONC206 on human uveal melanoma cell lines and patient-derived xenografts (PDXs) in vitro and in vivo. Methods: The effects of ONC206 on cell survival, apoptosis, autophagy, oncogenic signaling pathways, and metabolic networks were assessed in vitro using human melanoma cell lines. ONC206 was then tested for safety and anti-tumor activity in vivo using two mUM PDX models. Results: ONC206 treatment produced dose-dependent inhibition of mUM cell growth in vitro, with induction of varying levels of apoptosis and autophagy. ONC206 treatment also downregulated OXPHOS effector proteins and metabolites, thereby impairing mitochondrial OXPHOS. Treatment with ONC206 significantly reduced tumor burden and improved survival in two UM PDX mouse models in vivo. Conclusions: Our findings position ONC206 as a mechanistically distinct agent to target mitochondrial metabolism and to inhibit mUM. As ONC206 is currently being evaluated in multiple clinical studies, our data support further evaluation as a potential new therapeutic strategy for mUM.

Despite advances in immune checkpoint blockade, resistance in metastatic melanoma remains a major challenge. To decode resistance mechanisms, we generate a comprehensive longitudinal, multi-omic, and spatial atlas of 45 tumor samples across 10 patients. Analysis reveals resistant tumors undergo convergent evolution toward a shared, spatially organized immunosuppressive ecosystem. We identify a structural mechanism characterized by spatial partitioning of immune checkpoints, where B7-H3 dominates MITF-high niches while IDO1 characterizes MITF-low zones. Furthermore, integrated single-cell and spatial analysis identifies a specific malignant subclone (c1) and a distinct architectural niche (RCN3), both exhibiting aberrant PI3K-mTOR signaling. Notably, c1 promotes the “ignored tumor” phenotype via FN1-ITGB1 and GDF15 signaling. Validated across independent cohorts, these spatial and molecular signatures predict poor survival and point to actionable targets. Ultimately, our study elucidates the spatial logic of resistance and provides a rationale for translating multi-omic discoveries into actionable, personalized therapeutic strategies.

Melanoma metastasis is driven by extensive intratumoral heterogeneity and phenotypic plasticity, yet how clonal identity relates to transcriptional programs during metastasis remains unclear. Here, we applied MeRLin, a single-cell lineage tracing platform, to dissect the clonal and transcriptional heterogeneity of metastatic melanoma in a patient-derived spontaneous metastasis model. Clonal analyses revealed hierarchical structures during tumor progression, with a subset of lineages from primary tumors consistently enriched across metastatic sites, supporting a model of polyclonal seeding followed by selective expansion of pre-existing highly metastatic subpopulations. Single-cell transcriptomic profiling identified two major metastatic subpopulations of distinct transcriptional programs, characterized by neural crest stem cell-like and lipid metabolism signatures. Both programs were enriched for invasion-associated genes and maintained across organs through distinct regulatory networks. Spatial mapping by barcode RNA-FISH linked these transcriptional states to their tissue context and showed that OLFML3 expression partially co-localized with a dominant subpopulation at the tumor-liver interface, marking the invasive fronts of metastatic growth. Together, these findings establish a framework in which clonal identity, transcriptional state, and spatial organization jointly shape metastatic melanoma progression.

Supplementary Figure 5: Granzyme B and Ki-67 expression in CD8+ T cells in the MPDOs.

Tumor volume measurements (as assessed by calipers) and number of treatment days at each experimental time point.

Abstract The Toll-like receptor (TLR) 7/8 agonist resiquimod shows promise for treating cutaneous T-cell lymphoma and actinic keratosis, yet its mechanism of action remains unclear. We demonstrated that topical resiquimod significantly inhibited melanoma growth across various genetic and syngeneic mouse models, prolonged survival, and reduced lymph node metastasis in vivo. Resiquimod suppressed B16 melanoma growth, with an effect superior to that of imiquimod. The therapeutic effect was CD8+ T cell–dependent, as evidenced by the loss of efficacy upon CD8+ T-cell depletion or in Rag2−/− mice. Resiquimod increased intratumoral CD45+ inflammatory cells, particularly antigen-experienced PD-1+CD62L−CD8+ effector T cells, and enhanced their Ki-67 and granzyme B expression. Resiquimod significantly expanded Pmel- and Trp2-specific CD8+ T cells in the presence of dendritic cells (DC). Topical treatment of melanoma-bearing mice induced systemic protection in rechallenge experiments. In addition, combining topical resiquimod with anti–PD-1 antibodies led to superior inhibition of tumor growth and metastasis across multiple melanoma models. Proteomic analysis revealed increased granzyme B and CD26 and decreased phosphorylated FOXO3a after treatment. In patient-derived organoids and melanoma slice cultures, resiquimod induced significant tumor killing and CD8+ T-cell activation, further augmented by PD-1 antibodies. Our findings support the conclusion that resiquimod promotes CD8+ T-cell priming via DCs and enhances the therapeutic efficacy of anti–PD-1 checkpoint blockade in melanoma.