Ördög, Tamás

The intestinal epithelium is exposed to diverse combinations of microbiota-derived compounds; however, the mechanisms by which the host integrates these signals remain poorly defined. Studying two highly abundant microbial metabolites, we identified the purine metabolite hypoxanthine as an effector metabolite that directly drives signaling and the short-chain fatty acid butyrate as a regulatory metabolite that conditions host responsiveness. Specifically, hypoxanthine activates the adenosine A1 receptor–TRPC4 axis in enterochromaffin (EC) cells, triggering calcium influx and serotonin release resulting in accelerated gastrointestinal transit locally and increased platelet activation systemically. In contrast, butyrate epigenetically upregulates specific G protein–coupled receptors and ion channels to enhance response to hypoxanthine and the neurotransmitters norepinephrine and dopamine. These findings define a cooperative signaling framework and highlight the role of EC cells as a distinct epithelial signaling hub that senses and integrates microbial metabolite signals to drive physiological responses. Our findings provide a mechanistic foundation for therapeutic strategies that leverage combinatorial microbial signaling.

Abstract Human genomic studies link reduced CUB domain-containing protein 1 (CDCP1) expression with myocardial recovery in heart failure. While CDCP1 regulates cardiac fibroblast proliferation in vitro, it’s in vivo role in cardiac fibrosis remains unclear. Using a Cdcp1 -knockout (KO) angiotensin II/phenylephrine mouse model, we show that Cdcp1 deletion reduces echocardiographic left ventricular mass, histologic cardiac fibrosis, and pro-fibrotic gene expression, along with decreased fibroblast activation and inflammatory markers. Spatial transcriptomics identified a pressure overload–expanded fibroblast subpopulation enriched for growth factor and TGF-β signaling (FB5), which was markedly attenuated in Cdcp1 -KO hearts, alongside reduction of a pro-inflammatory cardiomyocyte subtype (CM4). Complementary studies in human ventricular fibroblasts demonstrate that CDCP1 knockdown reduced extracellular matrix gene expression and collagen I deposition. These findings establish CDCP1 as a regulator of cardiac fibrotic remodeling in vivo and open avenues for its further investigation as a potential therapeutic target.

Abstract Repetitive injury is hypothesized to lead to progressive tissue fibrosis and end-stage organ failure. Whether tissue-resident mesenchymal cell populations retain epigenetic memory of prior injuries that contribute to this pathological process is unknown. Here we used a genetic lineage labeling approach to mark the lung mesenchyme prior to injury, then performed multimodal analyses on isolated lung mesenchyme during the initiation, progression, and resolution of the fibrotic response. Our results demonstrate the remarkable epigenetic and transcriptional plasticity of the lung mesenchyme during fibrotic activation and de-activation. Despite this plasticity, we also find that the lung mesenchyme exhibits an enhanced fibrotic program upon reinjury. We identify RUNX1 as a critical driver of both fibrotic activation and fibrotic memory. Comparison of fresh isolated and cultured lung mesenchyme demonstrates that RUNX1 is spontaneously activated in standard culture conditions, previously masking these roles of RUNX1. Targeted knockdown of RUNX1 dampens fibrotic mesenchymal cell activation immediately after cell isolation, but with reduced efficacy after only days of culture, confirming its functional importance to both early activation and long-term memory. Collectively, our findings implicate RUNX1 in the initiation and memory of fibrotic mesenchymal cell activation that together prime enhanced mesenchymal cell responses upon repeated injury.

Abstract The importance of bone marrow (BM) microenvironment and its immune interactions within and their role in chronic myelomonocytic leukemia (CMML) disease progression have not been well studied. Our previous work in CMML identified type 1 helper T cell (Th1) and CD4 terminal effector cell suppression in peripheral blood from untreated patients, in comparison to age-matched normal controls (Mangaonkar A et al. Blood Adv 2020). Furthermore, clonal dendritic cell (DC) aggregates in CMML are present in approximately 20-30% patients at diagnosis, display significant cell heterogeneity (plasmacytoid DCs + myeloid DCs and other myeloid/immune cells), express indoleamine 2,3-dioxygenase-1 (IDO), associate with T regulatory cell (Treg) expansion, and transformation to AML [blast transformation or BT, Lucas N et al. Leukemia 2019, Mangaonkar A et al. Blood Adv 2020, and Mangaonkar A et al. Blood 2023 (abstract)]. The purpose of this study was to define the myeloid-immune cell interactions within the BM microenvironment critical for CMML progression to AML. To phenotypically identify cell types and their interactions within the CMML BM, we used single-cell type spatial omics through two distinct modalities on archival formalin-fixed paraffin-embedded (FFPE) BM biopsy specimens. Ten serial CMML samples at chronic phase [CP, n=5, median BM blast% 5 (2-15)] and at BT [n=5, median BM blast% 60 (25-74)] were used for both techniques. Spatial transcriptomics was conducted through GeoMx Digital Spatial Profiler (DSP) platform that uses UV-photocleavable, spatial-barcoded oligo probes to profile the whole transcriptome in defined regions of interest (ROI). CD123 (marker for DC-myeloid aggregates) immunofluorescence antibody was used to identify and segment ROI. 16-color flow cytometry was done to validate cell-specific expression. Spatial cellular phenotyping of immune cells on the same samples (serial cuts) was done through multiplex imaging by co-detection by indexing (CODEX) using 27 markers: CD31, Ki67, CD11b, CD20, CD44, Ecad, CD123, CD107a, CD8, CD11c, CD45RO, FoxP3, CD14, PDL1, CD56, CD68, CD163, PD-1, CD38, HLA-DR, CD4, IDO, CD45, PanCK, Na/K/ATPase, CD3e, and DAPI. CD8T+ cell anergy was demonstrated through CFSE cell proliferation assay. Benjamini-Hochberg correction was used to adjust for multiple hypotheses. After filtering poor quality ROIs [ROIs with total read count < 1000 were removed, ROIs with trim, stitch, alignment < 80% or saturation < 50% were removed], 3rd-quartile normalization [BiocGenerics (v. 0.48.1)] and batch correction [limma (v. 3.58.1)], we compared transcriptomics signatures between CD123+ and CD123- ROIs in all samples. Results showed that LIPA, SLC4A1, HBA2, TIMD4 and HMOX1 werethe top five genes upregulated in CD123+ areas. Among all gene transcripts, TIMD4 expression increased the most (Log2FC=0.6, P-value=8.79E-10, FC=1.52, FDR=1.68E-07) when CD123+ ROIs were compared between CMML-CP and CMML-BT serial samples, suggesting a biological relevance in disease progression. Previous work in metastatic peritoneal cancer has established that TIM-4 expressing macrophages suppress Th1/Th2 ratios and impair CD8+ T cell function (Chow A et al. Cancer Cell 2021). To confirm expression at the protein level, we used a multicolor flow cytometry panel and confirmed that TIM4 is expressed on macrophages (Lineage-negative, HLA-DR+, CD68+ cells) in independent CMML BM aspirate samples (n=3). Next, we conducted spatial phenotyping using CODEX on serial cuts from the same serial samples used for spatial transcriptomics. Anatomical co-localization of macrophages with leukemic cells, monocytes and T cells was visually inspected and confirmed. Cell-interaction analysis (after Benjamini-Hochberg correction) indicated that macrophages-monocyte, macrophage-leukemic cells, and macrophage-memory CD4+T cells were significantly (P<0.05) increased in CMML-BT versus CMML-CP. CD8T+ cell anergy was confirmed through a CFSE cell proliferation assay in untreated CMML PBMC samples. In conclusion, our data suggests that TIM4-expressing macrophages and their interactions with monocytes/monocyte precursors and immune cells within a DC aggregate create a protective leukemic stem cell niche and T cell tolerance, promoting blast transformation in CMML.

Obesity is an epidemic with myriad health effects, but little is understood regarding individual obese phenotypes and how they may respond to therapy. Epigenetic changes associated with obesity have been detected in blood, liver, pancreas, and adipose tissues. Previous work using human organoids found that dietary glucose hyperabsorption is a steadfast trait in cultures derived from some obese subjects, but detailed transcriptional or epigenomic features of the intestinal epithelia associated with this persistent phenotype are unknown. This study evaluated differentially expressed genes and relative chromatin accessibility in intestinal organoids established from donors classified as non-obese, obese, or obese hyperabsorptive by body mass index and glucose transport assays. Transcriptomic analysis indicated that obese hyperabsorptive subject organoids have significantly upregulated dietary nutrient absorption transcripts and downregulated type I interferon targets. Chromatin accessibility and transcription factor footprinting predicted that enhanced HNF4G binding may promote the obese hyperabsorption phenotype. Quantitative RT-PCR assessment in organoids representing a larger subject cohort suggested that intestinal epithelial expression of CUBN, GIP, SLC5A11, and SLC2A5 were highly correlated with hyperabsorption. Thus, the obese hyperabsorption phenotype was characterized by transcriptional changes that support increased nutrient uptake by intestinal epithelia, potentially driven by differentially accessible chromatin. Recognizing unique intestinal phenotypes in obesity provides a new perspective in considering therapeutic targets and options with which to manage the disease.

Abstract Purpose: Digital proteomic profiling was performed to identify spatial context in relationship to patient response and survival after anti–PD-1 therapy in metastatic colorectal cancer. Experimental Design: Primary colorectal cancers with deficient mismatch repair from patients treated with anti–PD-1 antibodies were analyzed (N = 30) using digital spatial profiling (GeoMx nCounter). At the invasive margin, 71 proteins were profiled in 10 regions of interest/slide that were segmented into 3 compartments labeled with pan-cytokeratin (epithelia), CD45 (stromal cells), and SYTO13 (nuclei). In an independent cohort (n = 13), digital spatial profiling data and single-cell transcriptomic data were analyzed. Differential protein abundance, after Benjamini–Hochberg correction, was examined by clinical response and progression-free survival (PFS) using multivariable Cox regression. Results: Protein abundance varied significantly between epithelial and stromal compartments. Nonresponders to anti–PD-1 showed higher fibronectin and smooth muscle actin abundance in the epithelial compartment that was associated with significantly shorter PFS (adjusted HR: 6.49 and 4.52, respectively; P < 0.05). In CD45+ stroma, increased expression of proteins related to T cells (CD3 and CD4), NK cells (CD56), antigen presentation (CD40), immune activation (CD27, ICOS), and apoptosis (GZMA) were found in responders (vs nonresponders) to anti–PD-1; each marker was significantly associated with longer patient PFS (0.02 < adjusted HR < 0.17; P < 0.05). In a separate cohort, consistent results by compartment were found for fibronectin and CD56. Gene expression data revealed that fibronectin and smooth muscle actin were primarily derived from cancer-associated fibroblasts. Conclusions: Spatially resolved protein profiles within microenvironments of deficient mismatch repair colorectal cancers can influence patient response and survival after anti–PD-1, highlighting their potential clinical significance.

ABSTRACTBackgroundThe carbon‐13 spirulina gastric emptying breath test (GEBT) is approved to identify delayed, but not accelerated, gastric emptying (GE). We compared the utility of the GEBT to scintigraphy for diagnosing abnormal GE in patients with diabetes mellitus.MethodsTwenty‐eight patients with diabetes ate a 230‐kcal test meal labeled with technetium 99 m and 13C‐spirulina, after which 10 scintigraphic images and breath samples (baseline, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min) were collected on 2 occasions 1 week apart. We assessed the accuracy of 13C‐spirulina GEBT excretion rate (percent dose multiplied by 1000 [kPCD] min−1) values to predict scintigraphic half‐life and distinguish between normal, delayed, and accelerated GE and the intraindividual reproducibility of the GEBT.Key ResultsScintigraphy revealed normal, delayed, and accelerated GE, respectively, in 17 (30%), 29 (52%), and 10 (18%) test results. GE T½ values measured with scintigraphy and GEBT were highly concordant within individuals; the intraindividual reproducibility was 34% (scintigraphy) and 15% (GEBT). Compared to current criteria, the kPCD150 (150 min) and kPCD180 values provided equally sensitive (90%) and more specific (81% vs. 67%) approach for distinguishing between delayed versus normal/accelerated GE. A new metric (kPCD60–kPCD15 min) was 90% sensitive and 83% specific for distinguishing between accelerated versus normal/delayed GE. These findings were used to create nomograms and an algorithm for interpreting GEBT results.Conclusions and InferencesAmong patients with poorly controlled diabetes, the 13C‐spirulina GEBT can accurately and precisely assess GE and effectively distinguish between normal, delayed, and accelerated GE.

Abstract Background Malignant peripheral nerve sheath tumors (MPNSTs) can arise from atypical neurofibromas (ANF). Loss of the polycomb repressor complex 2 (PRC2) is a common event. Previous studies on PRC2-regulated genes in MPNST used genetic add-back experiments in highly aneuploid MPNST cell lines which may miss PRC2-regulated genes in NF1-mutant ANF-like precursor cells. A set of PRC2-regulated genes in human Schwann cells (SCs) has not been defined. We hypothesized that PRC2 loss has direct and indirect effects on gene expression resulting in MPNST, so we sought to identify PRC2-regulated genes in immortalized human Schwann cells (iHSCs). Methods We engineered NF1-deficient iHSCs with loss of function SUZ12 or EED mutations. RNA sequencing revealed 1327 differentially expressed genes to define PRC2-regulated genes. To investigate MPNST pathogenesis, we compared genes in iHSCs to consistent gene expression differences between ANF and MPNSTs. Chromatin immunoprecipitation sequencing was used to further define targets. Methylome and proteomic analyses were performed to further identify enriched pathways. Results We identified potential PRC2-regulated drivers of MPNST progression. Pathway analysis indicates many upregulated cancer-related pathways. We found transcriptional evidence for activated Notch and Sonic Hedgehog (SHH) signaling in PRC2-deficient iHSCs. Functional studies confirm that Notch signaling is active in MPNST cell lines, patient-derived xenografts, and transient cell models of PRC2 deficiency. A combination of MEK and γ-secretase inhibition shows synergy in MPNST cell lines. Conclusions We identified PRC2-regulated genes and potential drivers of MPNSTs. Our findings support the Notch pathway as a druggable target in MPNSTs. Our identification of PRC2-regulated genes and pathways could result in more novel therapeutic approaches.

3520 Background: In patients with metastatic colorectal cancer (CRC) and deficient DNA mismatch repair (dMMR), we previously found transcriptomic signatures reflecting stromal compartment composition and proliferative capacity that can predict the efficacy of immune checkpoint blockade (ICB). To identify proteomic markers of response and/or resistance to ICB, we performed spatially resolved proteomic analysis of the tumor microenvironment. Methods: Patients with surgically resected, primary dMMR metastatic CRCs received pembrolizumab (Mayo Clinic, N=30). In FFPE tissues, Digital Spatial Profiling of 71 proteins was performed using panels covering immune cells, immune activation, cell death, PI3/AKT signaling, MAPK signaling, and pan-tumor markers (GeoMx nCounter). In ten regions of interest/slide (~600 µm2) at the tumor invasive margin, protein expression was determined in four cellular compartments: epithelium (panCK+), immune cells (CD45+; leucocyte common antigen), stroma (panCK-), and nonimmune stroma (panCK-/CD45-). After normalization, protein abundance per compartment was analyzed by response (RECIST 1.1) [OmicVerse Python]. Differentially expressed proteins (binary) were examined by progression-free survival (PFS) using multivariable Cox regression with covariate adjustment (Lifelines Python). Results: Pre-treatment panels including 71 proteins were analyzed by treatment response and PFS after PD-1 inhibition. Proteins found to be significantly associated with response (p< 0.01) and PFS are described. Across all compartments, responders showed upregulation of NK cells and T cell subsets (CD3+, CD4+, CD8+), and ICOS; strongest association was found for CD4+: HRadj: 0.16; 95%CI: 0.08-0.31; p< 0.001. Nonresponders showed higher phospho-JNK (HRadj: 2.09; 95%CI: 1.28-3.42; p=0.003) and SMA. In the epithelial compartment, upregulation of the Treg marker CD25 and reduced caspase-9 were observed in responders. Among responders, the stromal compartment was highly enriched in HLA-DR, dendritic cells (CD11c), PD-L1, immune activation markers (CD40, CD44, CD80, CD27), CD95/Fas, and GZMB. In the stromal compartment, downregulation of BCL6 and phospho-GSK3B was observed in responders. The immune compartment of responders showed increased beta2-microglobulin and Ki-67, while the non-immune stromal compartment showed increase in the macrophage marker CD68. Importantly, proteins found to be upregulated in responders were significantly and independently associated with longer PFS whereas the opposite was true for nonresponders. Conclusions: In metastatic dMMR CRCs, spatially defined protein markers were significantly associated with anti-PD-1 response, and independently associated with patient PFS (strongest predictive utility for CD4 in immune stroma and phospho-JNK in epithelium).

Abstract Neurofibromatosis type 1 (NF1) syndrome is an autosomal dominant cancer predisposition syndrome defined by germline deletion of one NF1 allele. Somatic loss of heterozygosity in the remaining NF1 allele in Schwann lineage cells gives rise to benign tumors known as plexiform neurofibromas. These tumors have a high risk of transforming into malignant peripheral nerve sheath tumors (MPNST) through the sequential deletion of CDKN2A/B followed by recurrent mutations in SUZ12 or EED, constituting the inactivation of polycomb repressor complex 2 (PRC2). PRC2 is a transcriptional repressor complex involved in the silencing of several development and differentiation genes. Loss of this complex leads to a depletion in trimethylation marks on Histone H3 Lysine 27 (H3K27me3) and the subsequent gain of acetylation marks on the same lysine residue (H3K27Ac). These molecular hallmarks are shared with diffuse intrinsic pontine gliomas (DIPG) harboring the H3K27M mutation which prevents effective deposition of H3K27me3, thus having similar molecular phenotypes to MPNST. However, the full range of epigenetic consequences of these events are currently not well characterized. Additionally, given that MPNSTs originate from cells of neural crest origin and DIPGs from cells of neural origin, it is not know to what degree epigenetic changes stemming from H3K27me3-loss are general across tumors or are context-dependent based on cell-of-origin. In order to contribute to this effort, the Largaespada lab has engineered Schwann cell lines to harbor deletions in NF1 and either SUZ12 or EED and assayed the H3K27ac landscape of these cells in addition to patient-derived MPNST cell lines. Utilizing the ranked ordering of super enhancers (ROSE) algorithm, we have identified common super enhancers within each MPNST cell line. These super enhancer elements are located nearby a variety of putative oncogenes responsible for the maintenance of cell identity. These elements were then compared to publicly available data from DIPG primary and patient-derived samples to understand common regulatory events driven by loss of H3K27me3 as well as molecular signatures present only in MPNST or in DIPG that may be a result of PRC2 or H3K27M alone. We further characterized these super enhancers by assessing the expression levels of their associated genes to understand their role in transcriptional activation. These efforts provide a roadmap of the super enhancer landscape of both MPNSTs and DIPGs to investigate novel mechanisms of H3K27me3-dependent tumorigenesis while also contributing to knowledge of potential effects of H3K27me3-loss that dependent on the cell-of-origin for each respective tumor. Citation Format: Christopher M. Stehn, Minu Bhunia, Kyle Williams, Alex T. Larsson, Ariel Caride, Jeong-Heon Lee, Tamas Ordog, David Largaespada. Characterizing the super enhancer landscape of H3K27me3 loss in MPNST and DIPG [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B009.