In the above-cited article, the variant p.Asp1603Tyr was mistakenly given as p.Ala1603Tyr in the abstract, on p. 429 (second column, fourth row), and in the legend to Fig. 1. In addition, typographical errors in Table 1 were present for the clinical characteristics of female patients for FPL pedigrees FPL143.6, FPL340.3, and FPL 414.3. The authors apologize for the errors and confirm that correction of the errors does not affect the conclusions of the article. The online version of the article (https://doi.org/10.2337/db24-0624) has been updated with the correct text.

Objective: Females have a higher risk to sustain anterior cruciate ligament (ACL) tears and a higher prevalence and incidence of developing knee osteoarthritis (OA) later in life, compared to age-matched males. Anatomical, biomechanical, and hormonal factors are known to be behind these disparities. However, studies defining a sex-specific response at the tissue level in human cartilage after ACL injury are lacking. Furthermore, mouse models of post-traumatic OA (PTOA) do not recapitulate the same sex differences observed in humans, as male mice develop cartilage degeneration faster than female mice, limiting efforts in the advancement of women's health research. Therefore, the aim of this study was to unveil the molecular response elicited in human knee articular cartilage after ACL injury based on biological sex. Our goal is to understand changes occurring after injury but before any development of PTOA. We hypothesize that there are sex differences in the molecular pathways triggered by cartilage injury. Methods: We compared the proteomic profiles of normal cartilage with injured cartilage. Normal cartilage from post-mortem donors was obtained from AlloSource (5 males, 5 females, ages 37-39). Injured cartilage samples were cartilage biopsies taken from the lateral notch during ACL-repair surgery performed up to 6 months post injury, with IRB approval and patient consent (5♀, 5♂, 22-39 y.o.). Importantly, this tissue was not exposed to direct damaging physical impact, but rather to the injurious environment triggered by an ACL tear. Specimens were snap frozen and processed for proteomic analysis using Tandem Mass Tag (TMT) for quantification of relative abundance. Differentially expressed proteins between normal and injured cartilage per sex were considered for pathway analysis when log2 fold change was <-2 and >2 and with adjusted p value <0.05. Pathway enrichment analysis was done using gene ontology (GO) and gene set enrichment analysis (GSEA) compared against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Extracellular matrix (ECM) proteins were further analyzed using the MatrisomeDB2.0 database. A second pathway analysis was done considering only those differentially expressed proteins that were not shared between male and female cartilage (sex-specific analysis). These targets had a log2 fold change of <-1 and >1, and an adjusted p value <0.05. Results: Principal component analysis indicates that samples separate mostly by condition (injured vs normal) rather than by biological sex suggesting that male and female cartilage share similar responses to injury (Fig.1A-B). This was confirmed by clustering analysis of differentially expressed genes by sex, indicating that only a small number of responsive proteins are sex specific (Fig.1C). Indeed, from the 1,660 total proteins identified in the analysis, male and female shared 1,121 upregulated and 23 downregulated proteins. Enriched pathway analysis further revealed similarities. Nevertheless, sex differences were found in some pathways. Males were enriched in ' ECM-receptor interaction ', ' complement and coagulation cascades ', and ' Focal adhesion ' (Fig2A), while females were enriched in ' Citrate cycle (TCA cycle) ', ' Fatty acid degradation ', ' Ribosome ', and ' Fatty acid metabolism ' (Fig.2B). Further analysis using the MatrisomeDB2.0 database revealed a larger number of core matrisome proteins and associated to matrisome targets in male compared to female cartilage. Although, changes in expression of some of the major ECM components and metalloproteases were shared between sexes (Table 1). From the sex-specific group of proteins, 252 were upregulated and 19 were downregulated only in males, while 242 were upregulated and 3 were downregulated only in females. Among those, male-specific upregulated proteins included ADAM9 (metallopeptidase domain 9), FBLN1 (fibulin 1), MMP2 (matrix metalloprotease 2), NID2 (nidogen 2), and CRTAC1 (cartilage acidic protein 1), while male-specific downregulated proteins included COL9A3 (collagen IX alpha 3), CILP2 (cartilage intermediate layer protein 2), and TIMP3 (tissue inhibitor of metallopeptidase 3). Among the female-specific targets, CRTAP (cartilage associated protein), DSE (dermatan sulfate epimerase), SDC2 (syndecan 2), and SLIT3 (slit guidance ligand 3) were upregulated, while only LTBP1 (latent transforming growth factor beta binding protein 1) was downregulated. Analysis of molecular functions of the sex-specific targets shows an enrichment of ECM binding in males and ribosome-related functions in females (Fig.3). Conclusions: The most striking finding from this study is that cartilage taken from ACL surgeries, which has only been exposed to an injurious environment likely including pro-inflammatory conditions, shows signs of cartilage remodeling in both sexes even in the absence of PTOA. These changes are occurring shortly after injury, as samples were taken between 0.5- and 6-months post injury. Further investigation is needed to determine whether this acute matrix remodeling is permanent or resolves over time. In terms of sex-specific analysis, male and female cartilage share mostly similar responses to ACL injury, with similar changes at the ECM level and only subtle sex-specific signatures. Nevertheless, within this small group of targets, we observed that females were enriched in proteins and pathways related to metabolism and protein synthesis, while males were enriched in proteins and pathways related to focal adhesions and cell-ECM interaction. Additional research efforts are needed to define the impact of these subtle sex differences over time; for example, whether they amplify at a later time point and lead to differential processes for cartilage degeneration. A limitation of our study is the small sample size per sex.

Purpose:To study a non-redundant role of Tcf12 in retinal health. Methods:A loss-of-function mutation in Tcf12 was identified by applying optical coherence tomography (OCT) to a forward genetic pipeline. CRISPR/Cas9-generated Tcf12ra/ra mice, expressing a replacement allele ("ra") were used to validate the findings from the mutagenesis screen. Retinal morphology was assessed using OCT, fundus photography, histology, and immunohistochemistry. Retinal function was evaluated by electroretinography (ERG). Bulk RNA sequencing and proteomic analyses were performed, with select targets validated by RT-qPCR and immunoblotting. Results:Tcf12ra/ra mice exhibited outer nuclear layer thinning on OCT, which was confirmed by histology. Fundus imaging revealed age-dependent accumulation of retinal fundus spots. Subretinal accumulation of Iba1⁺/Tmem119⁺ cells was observed, many of which stained positively for Gal3, suggestive of activated resident microglia. ERG deficits were noticed at 12 to 15 months of age in Tcf12ra/ra mice. Transcriptomic and proteomic profiling using two independent pathway analyses identified dysregulated pathways related to protein and RNA metabolism, mitochondrial and energy metabolism, cell cycle, signal transduction, cellular response to stimuli, and inflammation. RT-qPCR results showed upregulation of Tmem233 and downregulation of Doc2b, Alpk2, Agr2, and Apobec2 in Tcf12ra/ra retinas. Western blot analysis demonstrated upregulation of Selenbp1 and downregulation of Neurod1 and Faah in Tcf12ra/ra retinas. Conclusions:Deficiency in Tcf12 induces early-onset retinal structural alterations, and late-onset subretinal microglial activation and functional decline. Widespread dysregulation in metabolic and signaling pathways was documented in transcriptomic and proteomic analyses. These findings establish Tcf12 as a key contributor to retinal development and homeostasis.

Objective:Anterior cruciate ligament (ACL) tears increase the risk for developing posttraumatic osteoarthritis (PTOA). Females have greater risk for both. However, studies defining sex-specific protein responses in human cartilage after ACL injury are lacking. We hypothesize that articular cartilage's response to an injurious environment differs depending on sex. Design:We compared the proteomic profiles of normal cartilage with injured cartilage harvested from the intercondylar area during ACL surgery. Sex-specific injury effects were estimated through contrasts between Injured Male and Normal Male and between Injured Female and Normal Female. Pathway enrichment analysis was done using gene ontology (GO) and compared against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Extracellular matrix (ECM) proteins were further analyzed using the Matrisome AnalyzeR. Results:From the 2,188 proteins identified, males and females shared 1,121 upregulated and 23 downregulated proteins in injured compared to normal cartilage. Analysis of ECM proteins and enriched pathways revealed mostly similar male and female responses to an injurious environment, with evidence of early cartilage remodeling in both sexes. Nevertheless, more than 240 proteins were affected specifically by sex, and significant sex differences were found in inflammation, ECM-related, and metabolic pathways. Males were enriched mostly in "ECM-receptor interaction", while females were enriched in "Citrate cycle (TCA cycle)", "Fatty acid degradation", and "Fatty acid metabolism" pathways. Conclusion:Articular cartilage shows signs of remodeling soon after ACL injury, even when only exposed to an injurious environment rather than being physically impacted. Sex differences were observed in inflammation, metabolic pathways, and ECM synthesis.

Abstract Genome-wide association studies (GWAS) have significantly advanced our understanding of complex traits and diseases, but their interpretive power remains limited due to challenges in identifying causal genes and pathways. Integrating GWAS with multi-omics data—such as gene expression, protein-protein interactions, and gene-pathway networks have the potential to enhance biological insights and improve gene prioritization. To fulfill this potential and need, we developed the GWAS & Multi-omics Integration Pipeline (GMIP), a flexible and scalable framework that incorporates widely used tools such as PoPS, MAGMA, and benchmarker to enrich GWAS findings. However, PoPS suffers from multicollinearity in its features, which can impact performance. To overcome this, we introduce GMIP-PLSR, an extension of GMIP that uses Partial Least Squares Regression (PLSR) to manage multicollinearity effectively. We applied GMIP-PLSR across multiple GWAS datasets, demonstrating superior performance over PoPS in most cases. In a case study on NAFLD, GMIP-PLSR, using features derived from both disease-specific scRNA-seq and general PoPS features, identified gene sets with higher heritability and stronger enrichment in known NAFLD pathways, confirming its ability to enhance GWAS findings. Built on Nextflow, GMIP is computationally efficient, adaptable to diverse research environments, and provides a robust solution for gene reprioritization in post-GWAS analyses. GMIP-PLSR is available at https://github.com/mohammedmsk/GMIP .

The Foxd1+ stromal progenitor cells give rise to the majority of the renal interstitium; yet, much remains to be understood about how this self-renewing progenitor population is regulated during development. Here, we demonstrate that disruption of the nephron progenitor cell (NPC) lineage via loss of Wt1 (i.e. Six2cre;Wt1c/c) results in an expansion of Foxd1+ progenitor cells in mice. Analyses of two additional models (i.e. Wnt4-null mutants, which fail to form nephron structures similar to Six2cre;Wt1c/c kidneys, and NPC ablation via diphtheria toxin using the Six2cre;RosaDTAc/+) phenocopy the expansion in Foxd1+ cells and further confirm that mutant kidneys with defects in nephrogenesis develop an abnormal increase in the stromal progenitor population. Furthermore, single nuclei RNA-sequencing shows transcriptional changes in the Foxd1+ progenitor cells from Six2cre;Wt1c/c kidneys and identifies a distinct subcluster of the Foxd1+ stroma, which is maintained independent of signals from the nephrogenic niche in the Six2cre;RosaDTAc/+ model. Overall, these findings provide insights into the developmental regulation of the stromal progenitor population and uncover heterogeneity within the Foxd1+ cells, which undergo both cellular and molecular changes in response to defects in nephrogenesis.

Autosomal recessive familial partial lipodystrophy type 5 (FPLD5) due to a homozygous NP_001186481.1; p.E186* CIDEC variant has previously been reported in a 19-year-old female with diabetes mellitus, hypertriglyceridemia, and hepatic steatosis. Now, we report an 18-year-old Hispanic female who presented with FPL, along with hirsutism, acanthosis nigricans, and marked insulin resistance, and was found to have an extremely rare homozygous variant in CIDEC (NM_001199623.2:c.224G>T; NP_001186552.1; p.Ser75Ile) by whole exome sequencing. She also harbored a novel homozygous variant in WRN (NM_000553.4:c.1856T>G; NP_000544; p.Leu619Arg). Both serine 75 of the CIDEC protein and leucine 619 of the WRN protein were well conserved across species. She developed an invasive papillary thyroid carcinoma at the age of 17 years. Our report confirms the previously reported association of the biallelic CIDEC variant with the FPL phenotype and also highlights the extremely rare possibility of co-occurrence of FPLD5 with thyroid cancer, a clinical feature of Werner syndrome. Thus, our patient may not only need surveillance for the metabolic complications of FPLD5, such as diabetes, hypertriglyceridemia, and hepatic steatosis, but also for WRN-associated neoplasms and features of premature aging.

Mitochondria regulate cellular processes through direct and indirect interactions with other organelles. A well-studied example has been contact with the endoplasmic reticulum at mitochondrial-associated endoplasmic reticulum membranes1, which control pathways including redox and calcium homeostasis2,3. Recent studies have also reported direct mitochondria-nuclear membrane contacts in cancer cells and yeast that promote pro-survival signalling4,5. Here we identify direct interactions between mitochondria and nuclear pores. Using two unbiased proteomic screens, GST pulldown and BioID, we found that VDAC1 was the top mitochondrial candidate that interacts with the filamentous nuclear pore protein RANBP2. In vitro RANBP2 CRISPR knockout, RANBP2 truncation or site-directed mutagenesis of RANBP2-VDAC1 interacting amino acids resulted in reduced mitochondria-nucleus proximity and decreased nuclear ATP and phosphocreatine levels. This was accompanied by a decline in the levels of the nuclear phosphoproteome and downregulation of pathways involved in histone modification, cellular differentiation and transcriptional regulation in vitro. Moreover, deletion of the RANBP2 C-terminal domain in vivo in mice resulted in embryonic lethality due to cardiac and neural crest differentiation defects. Collectively, these results describe a mechanism by which mitochondria directly interact with the nuclear pore complex, a phenomenon critical for regulation of nuclear energetics and cellular differentiation. Undoubtedly, additional roles of this interaction remain to be revealed.

Abstract Rationale Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disorder characterized by cardiac hypertrophy, fibrosis, arrhythmias, and sudden cardiac death (SCD). Although current therapies primarily target sarcomere dysfunction, the contribution of epigenetic dysregulation to HCM pathogenesis and its therapeutic potential remain poorly understood. Objective To determine whether pharmacological inhibition of histone lysine demethylases (KDMs) with JIB-04 can prevent or reverse HCM progression and to identify the underlying epigenetic mechanisms. Methods and Results We evaluated the pan-KDM inhibitor JIB-04 in Myh6 R403Q/+ mice carrying the murine equivalent of the pathogenic human MYH7 R403Q mutation. JIB-04 prevented disease progression, reduced cardiac hypertrophy and fibrosis, preserved cardiac function, and completely prevented SCD in cyclosporin A– accelerated HCM. JIB-04 also reversed established disease, produced sustained therapeutic benefits after drug withdrawal, and improved cardiac function in aged mice with spontaneous HCM. Bulk RNA sequencing and ATAC-seq demonstrated partial restoration of disease-associated transcriptional programs and chromatin accessibility. Proteomic analyses identified PHF2 (KDM7C) as a candidate target of JIB-04 in both mouse and human HCM hearts. PHF2 knockdown suppressed hypertrophic, inflammatory, and fibrotic gene expression in cardiomyocytes, macrophages, and fibroblasts, respectively. Human HCM hearts exhibited increased expression of multiple JIB-04-sensitive KDMs, including PHF2. In MYH7 R403Q induced pluripotent stem cell– derived cardiomyocytes, JIB-04 normalized disease-associated gene expression, restored connexin-43 membrane localization, and improved mitochondrial respiration. Although prolonged treatment induced reversible hepatomegaly with hepatic lipid accumulation, co-administration of the antioxidant N-acetylcysteine mitigated liver toxicity while preserving the therapeutic efficacy of JIB-04. Conclusions Pharmacological KDM inhibition prevents and reverses HCM through epigenetic remodeling of disease-associated transcriptional and chromatin programs. These findings identify KDM inhibition as a promising therapeutic strategy for HCM, establish PHF2 as a candidate mediator of disease pathogenesis, and support further development of KDM-targeted therapies.

Neurodegenerative tauopathies, including Alzheimer’s disease, appear to be driven by propagation of tau assemblies, which must access the cytoplasm to recruit monomer and self-replicate, a process termed “seeding.” The prevailing model holds that tau seeds enter cells via macropinocytosis and reach the cytosol through lysosomal rupture or micro-perforation. Our findings revise this model by revealing that endocytosis is not required for seeding. Using genome-scale CRISPR screening, we identified multiple v-ATPase components whose loss reduced tau uptake (measured by flow cytometry) yet paradoxically increased seeding (measured by FRET biosensors). Acute v-ATPase inhibition with bafilomycin A1 produced the same effect in v2L tau biosensors and iPSC-derived neurons. Among regulators of endosome maturation, dominant-negative Rab5a decreased internalization while enhancing cytoplasmic templating. Cholesterol depletion produced identical results. Strikingly, transient hypothermia eliminated virtually all detectable tau uptake and dramatically increased seeding, without affecting subsequent tau monomer or aggregate degradation. We conclude that efficient endolysosomal trafficking does not appear to be required for cytoplasmic seeding under the conditions studied here. Across diverse perturbations, reduced endolysosomal flux consistently enhanced tau seeding, consistent with prior work indicating that most internalized aggregates are routed toward degradation rather than amplification. To seed effectively, tau must cross the plasma or vesicular membranes into the cytoplasm. We have found that proper endolysosomal trafficking suppresses cytoplasmic tau seeding, as all perturbations augmented this process. These findings reframe the role of the endolysosomal system in tau seeding and identify membrane transit rather than macropinocytosis itself as a critical gateway to cytoplasmic tau amplification.

Genetic lipodystrophies are a heterogeneous group of autosomal dominant and recessive disorders characterized by generalized or partial loss of body fat. Most patients with familial partial lipodystrophy (FPLD) have dominant inheritance with heterozygous pathogenic missense variants in LMNA. Here, we report two females with rare biallelic variants in LMNA presenting with divergent lipodystrophic phenotypes. Proband 1, a 32-year-old female, has near-generalized lipodystrophy (body fat 12.7%) due to compound heterozygous c.1745G>T (p.R582L) and c.1750C>T (p.R584C) LMNA variants. She was diagnosed with diabetes at age 17, hypertriglyceridemia at age 18, and metabolic dysfunction-associated steatotic liver disease (MASLD) at age 20. She was treated with metreleptin with only partial improvement in metabolic parameters. Her parents, heterozygous carriers of these variants, did not have lipodystrophy. Proband 2, a 35-year-old female, has partial lipodystrophy (body fat 21.2%) due to a homozygous c.1750C>T (p.R584C) LMNA variant. She was diagnosed with diabetes at age 19 and had a history of hypertriglyceridemia and mild hepatic steatosis. Her parents reportedly did not have lipodystrophy. These cases highlight the expression of LMNA variants in the homozygous or compound heterozygous state, manifesting in near-generalized and partial loss of body fat with distinct phenotypic heterogeneity.

Background:Most genetic studies on celiac disease (CeD) have focused on individuals of European descent. Limited data are available for the Hispanic and black populations. Methods:We analyzed whole-genome sequencing data, electronic health records (EHR), and laboratory results from the All of Us Research Program. We identified 3,481 individuals with CeD through EHR, self-reporting, or both. Of these, 2,899 carried one of the four well-established risk haplotypes, including 262 of admixed American (89% Hispanic) and 108 of African (70% black) ancestry. Five sex-, age-, and ancestry-matched controls per case were selected for the assessment of genetic and clinical risk factors. Results:An enrichment in the DQB1*02:01 allele was observed in CeD patients across all ancestries, with the strongest association in Europeans (32.3% vs. 11.6%), followed by Americans (18.5% vs. 8.1%) and Africans (15.7% vs. 8.1%). Among individuals carrying the DQ2.5 (DQA1*05:01-DQB1*02:01 haplotype), HLA-B8 was present in 72.3% of Europeans, 42.3% of Admixed Americans, and lower in Africans. This linkage disequilibrium was higher in CeD patients than in controls across all three ancestries. A polygenic risk score distinguished seropositive CeD from controls with 86% accuracy. Incorporating clinical risk factors, including family history, hypothyroidism, diarrhea, vitamin D deficiency, and anemia, increased predictive accuracy to 92%. The model identified 93% of CeD patients with tTG-IgA levels greater than 10 IU/mL. Conclusion:Linkage between HLA-B8 and DQ2.5 differs significantly among individuals of European, admixed American, and African ancestry, contributing to ancestry-dependent genetic risk for CeD.

Chronic inflammation is a hallmark of the breast cancer tumor microenvironment and is also known to be associated with disease progression and therapeutic response. Interleukin-1 (IL-1) signaling has been widely studied in breast cancer biology; however, the long-term effect of sustained IL-1 exposure on hormone receptor-positive breast cancer cells remain poorly understood. In this study, we investigated how chronic IL-1 exposure influences inflammatory response, hormone dependency, and therapeutic sensitivity in ERα+/PR+ breast cancer models, MCF7 and T47D. Chronic IL-1 exposure attenuated response to subsequent acute IL-1 treatment, but the chronically exposed cells remained sensitive to serum deprivation, retained dependence on estrogen or progesterone receptor signaling, and responded robustly to endocrine and chemotherapeutic treatments. Extensive changes in basal gene expression and histone modification revealed that chronic IL-1 exposure alters transcriptional reprogramming and chromatin remodeling. Together, these findings demonstrate that chronic IL-1 signaling drives selective inflammatory response in hormone receptor-positive MCF7 and T47D breast cancer cells. This work underscores the continued therapeutic relevance of hormone receptor-targeted strategies in chronically inflamed tumors and provides insight into how sustained inflammatory stress shapes tumor behavior and gene regulation predicted to promote tumor progression.

Using data from the All of Us Research Program, Xin Long et al. found that HLA-DQ2.5, the major genetic risk for celiac disease, is present across diverse populations. However, its linkage with HLA-B8 varies by genetic ancestry and contributes to differences in disease prevalence.

Polycomb repressive complex 2 (PRC2) mediates developmental gene repression as two classes of holocomplexes, PRC2.1 and PRC2.2. EPOP is an accessory subunit specific to PRC2.1, which also contains PCL proteins. Unlike other accessory subunits that collectively facilitate PRC2 targeting, EPOP was implicated in an enigmatic inhibitory role, together with its interactor Elongin BC. We report an unusual molecular mechanism whereby EPOP regulates PRC2.1 by directly modulating its oligomerization state. EPOP disrupts the PRC2.1 dimer and weakens its chromatin association, likely by disabling the avidity effect conferred by the dimeric complex. Congruently, an EPOP mutant specifically defective in PRC2 binding enhances genome-wide enrichments of MTF2 in mouse epiblast-like cells. Elongin BC is largely dispensable for the EPOP-mediated inhibition of PRC2.1. EPOP defines a distinct subclass of PRC2.1, which may uniquely maintain an epigenetic program by preventing the over-repression of key gene regulators along the continuum of early differentiation. PRC2 operates as two holocomplexes, PRC2.1 and PRC2.2, with EPOP serving as a PRC2.1-specific accessory subunit. Here the authors show that EPOP inhibits PRC2.1 by disrupting its dimeric structure, thereby weakening chromatin association to prevent excessive gene repression during early differentiation.

Limited genetic studies on celiac disease (CeD) are available for the Hispanic and black populations. We identified 3,481 individuals with CeD from the All of Us Research Program. Of these, 2,899 carried one of the four well-established risk haplotypes, including 262 of admixed American (89% Hispanic) and 108 of African (70% black) ancestry. An enrichment in the DQB1*02:01 allele was observed in CeD patients across all ancestries, with the strongest association in Europeans (32.3% vs. 11.6%), followed by Americans (18.5% vs. 8.1%) and Africans (15.7% vs. 8.1%). HLA-B8 conferred an additive risk for CeD independent of HLA-DQ2.5 across all three ancestries. The B8-DQ2.5 haplotype was significantly enriched in individuals with CeD but occurred at substantially lower frequencies in individuals with admixed American (3.2%) and African ancestry (1.2%) than in those with European ancestry (7.3%), accounting for ∼34% and 38% of the lower CeD prevalence, respectively. The frequency of the B8-DQ2.5 haplotype contributes to ancestry-dependent differences in CeD prevalence.

4580 Background: Pts with mUC have poor prognosis, and while EV/pembro have improved outcomes, therapeutic targets arising after EV-pembro treatment are elusive. Proteomic profiling of circulating tumor cells (CTCs) offer a minimally invasive approach to capture tumor biology changes in real-time. We evaluated CTC burden and proteomic changes in relation to treatment response in mUC. Methods: Pts with mUC were prospectively enrolled at UT Southwestern Medical Center. Peripheral blood samples were collected at baseline, 4 weeks, and 12 weeks of treatment, as well as progression of disease. CTCs were isolated using a dendrimer-based microfluidic chip assay functionalized with antibodies against EpCAM, Trop-2, Nectin-4, and FGFR3, identifying nucleated CTCs (DAPI+/CK+, CD45-). After CTCs were selected within regions of interest, spatial proteomic profiling was performed via the Nanostring GeoMx Immuno-Oncology Proteome Atlas panel. CTC variation and surface proteomics after treatment were primary objectives. CTC changes were considered concordant if they decreased >10% in pts with complete or partial response (CR or PR), remained stable (within 10% of baseline) in pts with stable disease (SD), or increased >10% in pts with progressive disease (PD), as defined by RECIST 1.1. One pt underwent post-treatment biopsy with IHC analysis. Results: Thirty pts with mUC were enrolled, with a median age of 71.5 years, predominantly male (26/30, 87%) and Caucasian (24/30, 80%). Most received EV/pembro (26/30, 86.7%), while the rest were treated with chemotherapy (3/30, 10%) or SG (1/30, 3%). Median CTC counts (cells/ml) were assessed at baseline (30.3, IQR 18.5–62.9), at week 4 (22.3, IQR 6.0–37.0), and at week 12 (29.5, IQR 15.2–44.5). Twenty-two pts had sufficient radiographic and clinical follow up. CTC changes were concordant with treatment responses in 77% (17/22) of pts, including 12/22 pts with CR/PR, 3/22 with SD, and 7/22 with PD. Proteomic profiling of serial CTCs from 9 pts treated with EV/pembro revealed dynamic proteomic changes from baseline to week 12 and at disease progression. Together, these pts demonstrated upregulation of activated EGFR (phospho-Y1068), HER2 (phospho-Y877), STAT5, FAK, and vimentin. Pts with primary PD (n=3) showed B7-H3 upregulation on CTCs, whereas pts with secondary PD(n=2) showed higher expression of vimentin, c-MET, and MHC class I on CTCs. IHC of a post-EV/pembro progression bone metastasis confirmed increased HER2 expression (2+). Conclusions: CTCs with proteomic profiling can serve as a dynamic, noninvasive biomarker in mUC, identifying distinct therapeutic targets for pts progressing on EV/pembro. The emergence of actionable pathways, including EGFR, HER2, c-MET, and B7-H3, suggests opportunities for rational, biomarker-driven therapeutic strategies.

Abstract Introduction Thymus organogenesis depends on coordinated communication between stromal and hematopoietic cells. We found that thymic mesenchyme contains a specialized stromal subset (CD45⁻Ter119⁻PDGFRA+PDGFRB+CD90+) distinct from mesenchyme of the pharyngeal, submandibular, tongue, and lung regions, which uniquely signals to thymic epithelial cells. Methods We compared stromal competence across organs using flow cytometry, single-cell transcriptomics, and recombinant reaggregate thymic organoid culture (ReRTOC). Pharmacologic reprogramming was tested in Tbx1neo2/neo2 embryos (∼36% normal Tbx1), a model of 22q11.2 deletion syndrome, using the FDA-approved drug Minoxidil. Results ReRTOC assays showed that pharyngeal and submandibular mesenchyme retain partial ability (∼60% thymic growth) to support epithelial expansion and thymopoiesis, revealing inherent plasticity. In Tbx1neo2/neo2 mutants, mesenchyme exhibited a shift from Bmp4 to Bmp5—Sox9 signaling, driving chondrogenic differentiation (Col2a1, Col9a1, Col11a1), loss of stromal organization, and reduced vascular branching with a lower SMA/Cx40 ratio. Minoxidil restored Bmp4 activity, suppressed Sox9/Bmp5 expression, and normalized thymic size and vascular complexity. CellChat analysis of wild-type thymic stroma revealed perivascular mesenchymal subsets acting as dominant signaling hubs, communicating with endothelial cells through the LAMININ, SPP1, BSP, and WNT pathways, which maintain basement membrane stability and endothelial anchoring. Conclusion Our results demonstrate that mesenchymal—endothelial communication is essential for thymic vascular patterning and that targeted mesenchymal reprogramming–through pharmacological or growth factor cues–can restore thymic vascularization and function, even during aging. Funding Source This work was supported, in part by grants from the National Institutes of Health Grant R01 AI114523 (Nicolai S.C. van Oers) and Jeffrey Modell Foundation (Christian A. Wysocki) Topic Categories Hematopoiesis and Immune System Development (HEM)

Context Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy (MDPL) syndrome is a rare, autosomal dominant disorder due to pathogenic heterozygous variants in POLD1. Clinical features of MDPL vary between patients; however, there is no previously reported genotype-phenotype association.Objective This work reports 14 new patients with lipodystrophy due to POLD1 variants and compares phenotypic differences between those with p.Ser605del and missense variants.Methods Genetic sequencing was performed on DNA of 14 patients for POLD1 variants, including exome (n = 10), genome (n = 1), and candidate gene (n = 3) sequencing. Comparisons of demographic, clinical features, and metabolic complications between carriers of POLD1 p.Ser605del and missense variants in our cases and those reported in the literature were made using the Fisher exact test for categorical variables and the t test for continuous variables.Results A total of 9 different POLD1 variants were identified in our patients, including 3 novel variants: p.Asp25Glufs*16, p.Arg507His, and p.Trp781Cys. Compared to individuals with missense variants (n = 15), those with the p.Ser605del (n = 26) POLD1 variant had significantly increased prevalence of mandibular hypoplasia (57% vs 100%, respectively; P = .015), small mouth (36% vs 100%, respectively; P = .015), crowded teeth (44% vs 91%, respectively; P = .046), and hypogonadism in male patients (0% vs 92%, respectively; P = .046). There were no differences in the prevalence of metabolic complications, such as diabetes, hypertriglyceridemia, and hepatic steatosis, in the two groups.Conclusion Individuals with the heterozygous POLD1 p.Ser605del variant had typical MDPL with more severe phenotype compared to those with missense variants with atypical MDPL.