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Wellstein, A.

Publications and source records attributed to Wellstein, A..

8 recordsLinked to original sources

TEAD inhibition alters the lung immune microenvironment and attenuates metastasis.

The Hippo pathway signaling mediated through YAP/TAZ, and the transcription factor TEAD is known to be involved in primary tumor progression. Here we report that novel TEAD inhibitors (iTEAD) cause a significant reduction in the outgrowth of lung metastases from triple negative breast cancer (TNBC) models mediated predominantly through changes in stromal immune signaling. TEAD inhibition did not affect the proliferation of TNBC cancer cells in vitro or the growth of the primary tumor in vivo. In normal mice that were treated with iTEAD in the absence of tumors, the lungs showed a decrease in pro-tumor inflammatory pathways. However, the IL12 signaling pathway was enhanced and its production from isolated lung tissue resident macrophages, but not bone marrow derived macrophages, was elevated. In syngeneic TNBC mouse models, inhibition of TEAD suppressed pro-tumor inflammation and the M2-like macrophage phenotype in lung tissues, and increased the infiltration of CD8+ T cells into the lung as well as Th1 CD4+ T cells, restoring an immune responsive microenvironment. iTEAD-treated T cells showed enhanced cytotoxicity and degranulation when co-cultured with cancer cells via increased IL-2 activity. Furthermore, TEAD inhibition or knockdown, enhanced T-cell macrophage crosstalk and anti-tumor activity in 3D tumorspheres which was reversed by IL12 neutralizing antibodies. Our data supports a multifaceted model of TEAD inhibition on the innate and adaptive immune cells as they respond to tumor cell signals and reveals an important stromal phenotype by which TEAD inhibitors could reverse immune suppression and eliminate seeded metastases in the lungs.

cancer biology↗

Tumour specimen cold ischemia time impacts molecular cancer drug target discovery

Tumour tissue collections are used to uncover pathways associated with disease outcomes that can also serve as targets for cancer treatment, ideally by comparing the molecular properties of cancer tissues to matching normal tissues. The quality of such collections determines the value of the data and information generated from their analyses including expression and modifications of nucleic acids and proteins. These biomolecules are dysregulated upon ischemia and decomposed once the living cells start to decay into inanimate matter. Therefore, ischemia time before final tissue preservation is the most important determinant of the quality of a tissue collection. Here we show the impact of ischemia time on tumour and matching adjacent normal tissue samples for mRNAs in 1,664, proteins in 1,818 and phosphoproteins in 1,800 cases (tumour and matching normal samples) of four solid tumour types (CRC, HCC, LUAD and LUSC NSCLC subtypes). In CRC, ischemia times exceeding 15 minutes impacted 12.5% (mRNA), 25% (protein) and 50% (phosphosites) of differentially expressed molecules in tumour versus normal tissues. This hypoxia- and decay-induced dysregulation increased with longer ischemia times and was observed across tumour types. Interestingly, the proteomics analysis revealed that specimen ischemia time above 15 minutes is mostly associated with a dysregulation of proteins in the immune response pathway and less so with metabolic processes. We conclude that ischemia time is a crucial quality parameter for tissue collections used for target discovery and validation in prognostic cancer research.

cancer biology↗

Circulating, cell-free methylated DNA indicates cellular sources of allograft injury after liver transplant

Post-transplant complications reduce allograft and recipient survival. Current approaches for detecting allograft injury non-invasively are limited and do not differentiate between cellular mechanisms. Here, we monitor cellular damages after liver transplants from cell-free DNA (cfDNA) fragments released from dying cells into the circulation. We analyzed 130 blood samples collected from 44 patients at different time points after transplant. Sequence-based methylation of cfDNA fragments were mapped to patterns established to identify cell types in different organs. For liver cell types DNA methylation patterns and multi-omic data integration show distinct enrichment in open chromatin and regulatory regions functionally important for the respective cell types. We find that multi-tissue cellular damages post-transplant recover in patients without allograft injury during the first post-operative week. However, sustained elevation of hepatocyte and biliary epithelial cfDNA beyond the first week indicates early-onset allograft injury. Further, cfDNA composition differentiates amongst causes of allograft injury indicating the potential for non-invasive monitoring and timely intervention.

molecular biology↗

Differential effects of IL4I1 protein on lymphocytes from healthy and multiple sclerosis patients

Multiple sclerosis (MS) is a chronic inflammatory disease characterized by immune mediated demyelination of the central nervous system, resulting in extensive neurological deficit and remyelination impairment. We have previously found that interleukin-four induced one (IL4I1) protein modulates CNS inflammation and enhances remyelination in mouse models of experimental demyelination. However, it remained unclear if IL4I1 regulates lymphocyte activity in MS. To assess the therapeutic potential of IL4I1 in MS, we investigated the impact of IL4I1 treatment on human lymphocytes from peripheral blood mononuclear cells (PBMCs) obtained from healthy individuals and MS patients. We found that IL4I1 increased the relative densities of Th2 and regulatory T-cells, while reducing Th17 cell density in healthy control samples. Furthermore, IL4I1-treated lymphocytes promoted CNS remyelination when grafted into demyelinated spinal cord lesions in mice. We found that baseline endogenous IL4I1 expression was reduced in people with MS. However, unlike healthy controls, IL4I1 treatment had no significant effect on IL17 or TOB1 expression in lymphocytes derived from MS patients. These results suggest that IL4I1 skews CD4+ T-cells to a regulatory state in healthy human lymphocytes, which may be essential for promoting remyelination. However, IL4I1 appears unable to exert its influence on lymphocytes in MS, indicating that impaired IL4I1-mediated activity may underlie MS pathology.

neuroscience↗

Pleiotrophin drives a pro-metastatic immune niche within the breast tumor microenvironment

Metastatic cancer cells adapt to thrive in secondary organs. To investigate metastatic adaptation, we performed transcriptomic analysis of metastatic and non-metastatic murine breast cancer cells. We found that pleiotrophin (PTN), a neurotrophic cytokine, is a metastasis-associated factor that is expressed highly by aggressive breast cancers. Moreover, elevated PTN in plasma correlated significantly with metastasis and reduced survival of breast cancer patients. Mechanistically, we find that PTN activates NF-kB in cancer cells leading to altered cytokine production, subsequent neutrophil recruitment and an immune suppressive microenvironment. Consequently, inhibition of PTN, pharmacologically or genetically, reduces the accumulation of tumor associated neutrophils and reverts local immune suppression resulting in increased T cell activation and attenuated metastasis. Furthermore, inhibition of PTN significantly enhanced the efficacy of immune checkpoint blockade + chemotherapy in reducing metastatic burden in mice. These findings establish PTN as a previously unrecognized driver of a pro-metastatic immune niche and thus represents a promising therapeutic target for the treatment of metastatic breast cancer.

cancer biology↗

Cell-free, methylated DNA in blood samples reveals tissue-specific, cellular damage from radiation treatment

Radiation therapy is an effective cancer treatment although damage to healthy tissues is common. Here we characterize the methylomes of healthy human and mouse tissues to establish sequencing-based, cell-type specific reference DNA methylation atlases. Identified cell-type specific DNA blocks were mostly hypomethylated and located within genes intrinsic to cellular identity. Cell-free DNA fragments released from dying cells into the circulation were captured from serum samples by hybridization to CpG-rich DNA panels. The origins of the circulating DNA fragments were inferred from mapping to the established DNA methylation atlases. Thoracic radiation-induced tissue damages in a mouse model were reflected by dose-dependent increases in lung endothelial, cardiomyocyte and hepatocyte methylated DNA in serum. The analysis of serum samples from breast cancer patients undergoing radiation treatment revealed distinct tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation indicating the impact on liver tissues. Thus, changes in cell-free methylated DNA can uncover cell-type specific effects of radiation and provide a quantitative measure of the biologically effective radiation dose received by healthy tissues. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/487966v3_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@129f229org.highwire.dtl.DTLVardef@d999bcorg.highwire.dtl.DTLVardef@1fc3543org.highwire.dtl.DTLVardef@10cebc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

Full-length, single-cell RNA-sequencing of human bone marrow subpopulations reveals hidden complexity

Bone marrow progenitor cell differentiation has frequently been used as a model for studying cellular plasticity and cell-fate decisions. Recent analysis at the level of single-cells has expanded knowledge of the transcriptional landscape of human hematopoietic cell lineages. Using single-molecule real-time (SMRT) full-length RNA sequencing, we have previously shown that human bone marrow lineage-negative (Lin-neg) cell populations contain a surprisingly diverse set of mRNA isoforms. Here, we report from single cell, full-length RNA sequencing that this diversity is also reflected at the single-cell level. From fresh human bone marrow unselected and lineage-negative progenitor cells were isolated by droplet-based single-cell selection (10xGenomics). The single cell-derived mRNAs were analyzed by full-length SMRT and short-read sequencing. In both samples we detected an average of 8000 different genes using short-read sequencing. Differential expression analysis arranged the single-cells of the total bone marrow into only four clusters whereas the Lin-neg population was much more diverse with nine clusters. mRNA isoform analysis of the single-cell populations using full-length sequencing revealed that Lin-neg cells contain on average 24% more novel splice variants than the total bone marrow cells. Interestingly, among the most frequent genes expressing novel isoforms were members of the spliceosome, e.g. HNRNPs, DEAD box helicases and SRSFs. Mapping the isoforms from all genes to the cell type clusters revealed that total bone marrow cells express novel isoforms only in a small subset of clusters. On the other hand, lineage-negative progenitor cells expressing novel isoforms were present in nearly all subpopulations. In conclusion, on a single-cell level lineage-negative cells express a higher diversity of genes and more alternatively spliced novel isoforms suggesting that cells in this subpopulation are poised for different fates. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=149 HEIGHT=200 SRC="FIGDIR/small/454226v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1fa661aorg.highwire.dtl.DTLVardef@454456org.highwire.dtl.DTLVardef@1897badorg.highwire.dtl.DTLVardef@116d516_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Single molecule real time (SMRT) full length RNA-sequencing reveals novel and distinct mRNA isoforms in human bone marrow cell subpopulations

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single molecule, real time (SMRT) full length RNA sequencing. This analysis revealed a [~]5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of mRNA isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was validated by mass spectrometry and validated previously unknown protein isoforms predicted e.g. for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g. CFD, GATA2, HLA-A, B & C) also distinguished cell subpopulations but were only detectable by full length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

molecular biology↗