George F. Murphy

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) resets epigenetic age, creating a major challenge for modeling age-associated cellular states and limiting the use of iPSCs in aging research beyond the genetic component. Recent work has shown that healthy organoids can re-accumulate age-associated epigenetic states over extended culture; whether disease-associated genetic states can directionally reshape these trajectories after reprogramming remains unknown. Here, we present a reproducible, in-vitro approach to re-establish epigenetic aging trajectories using iPSC-derived organoid systems, as quantified by multiple epigenetic clock models. We first demonstrate that epigenetic age progressively increases with serial passaging in a lung organoid model (alveolospheres). We then show that disease-associated mutations in multiple models drive additional, cell-intrinsic shifts in epigenetic age, implicating disease state as an independent modulator of epigenetic clocks. Together, these findings reinforce that iPSC-derived organoids recapitulate epigenetic aging trajectories over time, while highlighting disease state as a potent and potentially tractable modulator of epigenetic age in vitro . By enabling more faithful modeling of age-associated cellular states, this approach expands the utility of iPSC-based systems for dissecting the molecular mechanisms that regulate human aging.

Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene that promotes hemoglobin polymerization, leading to chronic hemolytic anemia, vaso-occlusive episodes, and progressive organ damage. The most efficacious therapies focus on reactivating fetal hemoglobin (HbF) expression to mitigate the pathological effects of sickle hemoglobin (HbS) polymerization. However, the predominantly used HbF inducer, hydroxyurea (HU), exhibits substantial interpatient variability in efficacy, and curative approaches such as gene therapy remain inaccessible to the vast majority of patients. Although all SCD patients share the same causative HBB glu7val mutation, differences in genetic background significantly influence disease severity and therapeutic response. We describe a SCD-specific induced pluripotent stem cell (iPSC) platform as a renewable and scalable preclinical model to interrogate treatment responses across the genetically diverse SCD patient population. By generating patient-specific iPSC-derived erythroblasts (iEry) representing distinct SCD genetic backgrounds, we demonstrate that this system faithfully recapitulates the heterogeneous HbF induction observed clinically in response to HU. Moreover, this platform enables the identification and evaluation of alternative therapeutic agents for HU non-responders and provides sufficient resolution to dissect drug-specific effects on erythroid differentiation and cellular phenotypes. Together, these findings support the use of iPSC-derived erythroid models as a versatile tool to advance precision therapeutic strategies for SCD. KEY POINTS: - SCD iPSC-derived erythroid cells (iEry) reflect the diversity in HU-mediated HbF induction seen in SCD patients - SCD iEry recapitulate patient-specific treatment responses and can be used to identify therapeutic alternatives for HU non-responders - iEry provide a versatile platform to study the impact of novel HbF inducers on erythroid cell characteristics and differentiation parameters

Misfolding of aggregation-prone proteins underpins diseases known as proteinopathies. One of these proteins, alpha-synuclein, is a component of aggregates in neurodegenerative conditions such as Parkinson’s disease. The melanosomal protein PMEL, which forms physiologic amyloid scaffold structures on which melanin is organized in melanosomes, similarly ectopically accumulates in the dermis in many forms of cutaneous hyperpigmentation. Here, we demonstrate in a wide range of common clinical pigmentary disorders, as well as in primary melanocyte and mouse models examined by molecular, proteomic, and electron microscopic tools, that melanocytic alpha-synuclein is a prominent component of intracellular protein aggregates bound to similar proteins as in Parkinson’s disease, as well as melanized extracellular protein deposits. Using the Real Time Quaking-Induced Conversion Assay (RT-QuIC), we demonstrate that UV induces misfolded melanosomal proteins to self-propagate, augmenting this pathology in prion-like fashion. CUT&RUN chromatin profiling and single-cell RNA-seq demonstrate that melanocytes utilize microphthalmia-associated transcription factor (MITF)-regulated autophagy to counteract protein aggregation, identifying aggregate removal as a core function of tanning. In contrast to extracellular aggregation, impaired intracellular aggregate removal contributes to melanocyte senescence, which conversely exacerbates chronic hypopigmentation and photoaging-related discoloration. These findings identify melanosomal proteinopathy as a common contributor to melanocyte dysfunction and suggest aggregate-focused management approaches.

Melanoma patients with in-transit metastasis (ITM), a stage of disease where melanoma has metastasized to sites in between the primary lesion and draining lymph node, vary significantly in their clinical outcomes, but the biology driving differential outcomes in ITM is poorly understood. To elucidate the mechanisms of differential outcomes, we utilized multimodal molecular profiling (WES, RNA-seq, highly multiplexed immunofluorescence, spatial transcriptomics) in 1) evolutionary analysis of longitudinal tumor samples and 2) identifying prognostic tumor intrinsic and microenvironmental features in a unique cohort of patients with unresectable ITM. Among other findings, we observed a persistent dedifferentiated AXL⁺/NGFR⁺ clonal lineage pre-existing and following immune checkpoint blockade in in-transit and distant metastases. Concordantly, we found that low pigmentation and high T cell exhaustion signatures were independently associated with distant progression. Our findings highlight tumor cell state and immune dysfunction as key predictors and potential biomarkers of metastatic risk in ITM. STATEMENT OF SIGNIFICANCE: What drives distant progression in melanoma is unclear. Analyzing tumor and immune features in a rare in-transit melanoma patient cohort, we identify biological signals highlighting how immune and tumor states observable in pre-distant metastasis melanomas shape long-term outcomes, and nominate potential prognostic biomarkers.

Diseases such as cancer involve alterations in cell proportions, states and interactions, as well as complex changes in tissue morphology and architecture. Histopathological diagnosis of disease and most multiplexed spatial profiling relies on inspecting thin (4–5 µm) specimens. Here we describe a high-plex cyclic immunofluorescence method for three-dimensional tissue imaging and use it to show that few, if any, cells are intact in conventional thin tissue sections, reducing the accuracy of cell phenotyping and interaction analysis. However, three-dimensional cyclic immunofluorescence of sections eightfold to tenfold thicker enables accurate morphological assessment of diverse protein markers in intact tumor, immune and stromal cells. Moreover, the high resolution of this confocal approach generates images of cells in a preserved tissue environment at a level of detail previously limited to cell culture. Precise imaging of cell membranes also makes it possible to detect and map cell–cell contacts and juxtracrine signaling complexes in immune cell niches.

Our current understanding of blood cell development and functionality stems primarily from the investigation of adult bone marrow (BM) and the fetal liver prenatally. However, emerging evidence highlights the lung as a previously underappreciated residence for hematopoietic cells. While a diversity of cells specific to the BM are known to promote the maturation and trafficking of hematopoietic cells, how the lung niche influences the development and functionality of resident cells is not known. Spatial in situ transcriptomics enables accurate mapping of cell identities and interactions within intact tissue, providing insights not accessible by dissociated single-cell profiling. Here, we present a high-resolution spatial transcriptomic atlas of the healthy adult murine lung placing specific emphasis on the hemato-endothelial landscape of this organ. As a case study, we developed a semi-automatic workflow to explicitly identify and curate rare – often multinucleated – megakaryocytes, requiring a combination of hex-binning spatial enrichment of canonical markers, expert curation, and cell boundary merging to correct for segmentation artifacts. We then characterized the spatial neighborhoods of megakaryocytes, illustrating their topological embedding within vascular, stromal, and immune microenvironments. Finally, we demonstrated the utility of this dataset for hypothesis-driven signaling studies by examining ligand–receptor interactions across pathways including BMP, VEGF, and ECM–integrin signaling. Together, this work defines the lung-blood niche and advances our understanding of the organ-specific properties of blood cells. We also provide a high-resolution spatial reference for the murine lung and demonstrate how targeted spatial in situ transcriptomics enable focused case studies of rare hematopoietic niches. KEY POINTS: This work represents the highest resolution gene expression mapping of the spatial symbiosis between the hematopoietic and pulmonary systems. Pulmonary megakaryocytes localize within distinct vascular and stromal neighborhoods.

Background: The induction of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α; encoded by PPARGC1A) expression and activation of oxidative phosphorylation (OXPHOS) is associated with disease progression and treatment resistance in patients with cutaneous melanoma. Loss of the TET2 (ten-eleven translocation 2)/5-hydroxymethylcytosine (5-hmC) epigenetic pathway is linked to melanoma aggressiveness, although the underlying mechanisms remain unclear. Objectives: To explore a relationship between TET2-mediated DNA hydroxymethylation and the induction of PPARGC1A/PGC-1α expression and activation of OXPHOS in melanoma. Methods: RNA sequencing data from 368 melanoma metastases and 102 primary melanoma tumours were analysed, with tumours categorized as ‘TET2-low’ or ‘TET2-high’, based on TET2 gene expression. Differential gene expression and gene set enrichment analyses were done, with further validation using a tissue microarray comprised of 33 clinical specimens and a publicly available gene expression dataset from 209 primary tumours. Confirmatory in vitro and in vivo studies were performed using melanoma cell lines with altered TET2 and PGC-1α expression. 5-hmC and 5-methylcytosine levels at PPARGC1A were assessed using hydroxymethylated (hMeDIPseq) and methylated DNA immunoprecipitation sequencing. Results: PGC-1α expression and activation of OXPHOS were significantly upregulated in TET2-low metastases and primary tumours. Tissue microarray analysis and gene expression studies showed an inverse relationship between TET2/5-hmC and PPARGC1A/PGC-1α expression and OXPHOS. In vitro and in vivo, PPARGC1A/PGC-1α expression and activation of OXPHOS was higher in TET2-low cells. hMeDIPseq identified significantly lower 5-hmC levels at an upstream PPARGC1A active enhancer in melanoma compared with naevi, in TET2-low compared with TET2-high cells and in cells expressing catalytically inactive TET2 compared with cells expressing wildtype TET2. Inhibition of PGC-1α expression and activation of OXPHOS mitigated migration and invasion in vitro and metastasis in vivo; TET2 loss was associated with resistance to mitogen-activated protein kinase (MAPK) pathway inhibition and enhanced sensitivity to OXPHOS inhibition. Conclusions: Loss of TET2 promotes activation of PGC-1α/OXPHOS in melanoma, driving metabolic reprogramming that supports tumour progression and resistance to MAPK inhibition in a subset of tumours. Importantly, this phenotype renders TET2-deficient melanomas selectively vulnerable to OXPHOS inhibition, identifying an actionable therapeutic opportunity. These findings establish an epigenetic–metabolic axis as a critical determinant of melanoma aggressiveness and highlight TET2/5-hmC as a potential biomarker and a targetable pathway.