Biology subjects

Salame, T. M.

Publications and source records attributed to Salame, T. M..

16 recordsLinked to original sources

bioRxiv2025

Systemic administration of PD-L1 blocking antibodies leads to removal of senescent microglia

Senescent microglia develop during aging and Alzheimers disease (AD), driving chronic neuroinflammation. Here we hypothesized that the previously observed disease-modifying effects of PD-1/PD-L1 blockade occur through clearance of senescent microglia. Using CyTOF, we found that a single systemic anti-PD-L1 injection leads to rapid elimination of senescent microglia in 5xFAD and aged wild-type mice, independently of Fc effector function, while increasing homeostatic microglia. These findings suggest that immune rejuvenation via PD-L1 blockade promotes disease modification in AD through senescent-microglial elimination.

neuroscience↗

bioRxiv2025

Alzheimer's disease causes bone marrow myelopoiesis dysfunction

Accumulating evidence suggests that both innate and adaptive immunity play crucial roles in combating Alzheimers disease (AD). Specifically, enhancing the homing of monocyte-derived macrophages to the affected brain has been shown to reduce local inflammation, decrease proteinopathy, rescue neurons, and mitigate cognitive decline. However, the factors limiting their spontaneous recruitment remain unclear. Using multi-omics techniques, we identified impaired myelopoiesis and monocyte development in both mice and AD patients. While not the primary cause of the disease, this impairment is associated with disease progression. In the 5xFAD mouse model, monocyte differentiation was found to be disrupted due to a maladaptive bone marrow (BM) response, driven by type I interferon (IFN-I) signaling. A similar phenotype was found in circulating monocytes from AD patients compared to healthy controls. Blocking IFN-I with monoclonal antibodies or using chimeric AD mice with BM from mice lacking the IFN-I receptor (IFNAR1) alleviated myelopoiesis dysfunction, normalized monocyte phenotypes, and reduced cognitive impairment. These improvements in myeloid function were accompanied by an increased homing of monocyte-derived macrophages in the AD brain. Our results reveal an unexpected dysfunction in BM myelopoiesis in the context of neurodegeneration and support the emerging concept that neurodegenerative diseases are not solely brain-centric.

neuroscience↗

bioRxiv2025

Sensitizing Immune-Refractory Ovarian Tumors via p53 Mutation-Tailored Immunotherapy

High-grade serous ovarian cancer demonstrates limited responsiveness to immune checkpoint inhibitors, owing in part to immunosuppressive environments shaped by nearly universal p53 aberrations. Utilizing an immunocompetent mouse model and individual p53 mutations, we identified a dependence of the p53-R270H mutation (equivalent of human R273H) on regulatory T cells (Tregs) and the PD-1/PD-L1 axis. Analysis of patient datasets associated R273H with elevated levels of two p53 targets, PD-L1 and amphiregulin (AREG), a Tregs growth factor. In contrast to p53-R172H tumors, where there was limited activity, dual antibody therapy targeting AREG and PD-L1 selectively and effectively inhibited R270H tumors. This involved polarization toward M1 macrophages, infiltration of CD8+ T cells, diminished Ly6G+ neutrophils and downregulation of interleukin-4. In patient-derived R273C organoids, the combination treatment reduced the CD4/CD8 ratio. This study is the first to establish a mutation-tailored therapeutic approach that leverages the capacity of p53 to modulate immunosuppressive mechanisms.

cancer biology↗

bioRxiv2025

Persistent ECM Scarring Reprograms Intestinal Stem Cells to Drive Chronic Inflammation

Tissue regeneration is conventionally viewed as a return to homeostasis. Here, we uncover that the extracellular matrix (ECM) in the colon undergoes a lasting pathological reprogramming following inflammation, forming a remodeled niche--modECM--that persistently disrupts intestinal stem cell (ISC) identity. Using temporal multi-omics, biomechanical profiling, and spatial fate mapping in murine colitis models, we show that modECM, characterized by Collagen XVIII accumulation and immune-driven proteolysis, redirects ISCs toward a wound-associated, squamous-like epithelial state with pro-inflammatory transcriptional signatures. Ex vivo, modECM alone reprograms ISC fate by suppressing Wnt signaling and activating immune recruitment pathways. In vivo, modECM-rich zones sustain T cell infiltration and KRT14 epithelial cell emergence from Lgr5 progenitors. This aberrant epithelial program is mirrored in inflamed rectal biopsies from ulcerative colitis patients. Our findings redefine the ECM as a long-lived instructive compartment that encodes injury memory and promotes maladaptive regeneration, positioning it as a potential therapeutic target in chronic inflammatory disease.

immunology↗

bioRxiv2025

Avoidance of pyroptosis accounts for the relatively high metastatic potential observed in early hybrid EMT states

EMT converts epithelial (E) phenotypes to invasive mesenchymal (M) states. However, analyses of circulating tumor cells (CTCs) indicated that biphenotypic (E+M) CTCs better correlate with metastasis. Similarly, investigations of murine tumors undergoing EMT concluded that early E+M states posses the highest metastatic potential. To explore this, we selected in animals with breast cancer CTCs having progressively increasing intravasation abilities. This revealed that downregulation of arrestin Arrdc4 associates with CTC aggressiveness. In xenografts, depleting Arrdc4 accelerated tumor progression, whereas overexpression hindered progression in immunocompetent, but not in immunocompromised mice. Mechanistically, high Arrdc44 suppresses glucose uptake and enhances gasdermin E, triggering pyroptosis a type of pro-inflammatory cell death. Consistently, Arrdc4s lowest levels characterize the most metastatic biphenotypic states. In patients, both epigenetic and chromosomal aberrations downregulate ARRDC4 and predict poor prognosis. In summary, the uncovered mechanism portrays pyroptosis of biphenotypic EMT cells as a rheostat of CTCs, which may resolve the controversy on the role played by EMT in metastasis.

cancer biology↗

bioRxiv2024

Immune organization in sentinel lymph nodes of melanoma patients is prognostic of distant metastases

Sentinel lymph node (sLN) biopsy is part of melanoma staging, as involved LNs indicate a higher risk of recurrence. However, how the sLN is shaped by the tumor and reciprocally affects metastatic progression is poorly understood. Here, we mapped immune organization in involved and non-involved sLNs of 69 melanoma patients using high-resolution spatial proteomics, spatial transcriptomics and deep learning, leveraging the data for prognostic evaluation. In patients with involved LNs, a robust T cell response correlated with absence of recurrence. In non-involved LNs, protection from metastases was linked to expanded sinuses colocalized with plasmablasts whereas CCR7+ cells and Tregs correlated with future development of distant metastases. We trained a model that predicts development of distant metastases with 79% and 93% AUC for non-metastatic and metastatic LNs, respectively. Our findings reveal conserved immune patterns in sLNs that prospectively identify patients at risk for metastatic disease and may aid in therapeutic decisions.

cancer biology↗

bioRxiv2024

A spatial atlas of human gastro-intestinal acute GVHD reveals epithelial and immune dynamics underlying disease pathophysiology

Acute graft-versus-host disease (aGVHD) is a significant complication of allogeneic hematopoietic stem cell transplantation (aHSCT), driven by alloreactive donor T cells in the gut. However, the roles of additional donor and host cells in this process are not fully understood. We conducted multiplexed imaging on 59 biopsies from patients with gastrointestinal GVHD and 10 healthy controls, revealing key pathological changes, including fibrosis, crypt alterations, loss of Paneth cells, accumulation of endocrine cells, and disrupted immune organization, particularly a reduction in IgA-secreting plasma cells. Interestingly, CD8T cells were enriched only in a subset of patients, while others exhibited non-canonical enrichments of macrophages and neutrophils. Post-transplantation time significantly influenced immune composition, with host cells dominating plasma and T cell compartments long after transplantation. This spatial atlas of healthy duodenum and GVHD uncovers non-canonical immune dynamics, offering insights into disease pathophysiology and potential clinical applications in GVHD and other inflammatory bowel diseases.

immunology↗

bioRxiv2024

Mutant EZH2 alters the epigenetic network and increases epigenetic heterogeneity in B cell lymphoma

Diffuse large B cell lymphomas and follicular lymphomas show recurrent mutations in epigenetic regulators; among these are loss-of-function mutations in KMT2D and gain-of-function mutations in EZH2. To systematically explore the effects of these mutations on the wiring of the epigenetic network, we applied a single-cell approach to probe a wide array of histone modifications. We show that mutant-EZH2 elicits extensive effects on the epigenome of lymphomas, beyond alterations to H3K27 methylations, and is dominant over KMT2D mutations. Utilizing the single-cell data, we present computational methods to measure epigenetic heterogeneity. We identify an unexpected characteristic of mutant-EZH2, but not KMT2D, in increasing heterogeneity, shedding light on a novel oncogenic mechanism mediated by this mutation. Finally, we present tools to reconstruct known interactions within the epigenetic network, as well as reveal potential novel cross talk between various modifications, validated by functional perturbations. Our work highlights novel roles for mutant-EZH2 in lymphomagenesis and establishes new concepts for measuring epigenetic heterogeneity and intra-chromatin connectivity in cancer cells.

cancer biology↗

bioRxiv2023

TREM2-dependent senescent microglia conserved in aging and Alzheimer's disease

Dementia in general, and Alzheimers disease (AD) in particular, are age-related diseases1,2. AD is associated with multiple causative factors3,4, among which local brain inflammation plays a significant role5. Microglia, the brain-resident immune cells6,7, are activated along the disease course7. Yet, their contribution to the disease progression is still controversial. Here, using high-throughput mass cytometry for microglial immuno-phenotyping, we identified accumulation of senescent microglia in several pathologies associated with cognitive decline. These senescent microglia have a unique profile conserved across the multiple conditions investigated, including aging, mouse models of amyloidosis, and tauopathy. Moreover, we found that the expression of markers of senescence correlates with levels of TREM2, whose polymorphism was identified by GWAS as an AD risk factor8,9. A TREM2-null AD mouse model showed lower levels of senescent microglia, relative to TREM2-intact AD mice. Senolysis using the drug ABT-73710,11 in an AD mouse model reduced the abundance of TREM2-senescent microglia without affecting levels of TREM2-dependent activated microglia, ameliorated cognitive deficits, and reduced brain inflammation. These results reveal the unexpected contribution of TREM2 to accumulation of senescent microglia in AD pathology, an effect that must be considered when targeting TREM2 as a therapeutic approach.

neuroscience↗

bioRxiv2023

p16-dependent upregulation of PD-L1 impairs immunosurveillance of senescent cells

The accumulation of senescent cells promotes aging, but a molecular mechanism that senescent cells use to evade immune clearance and accumulate remains to be elucidated. Here, we report that p16-positive senescent cells upregulate the immune checkpoint protein programmed death-ligand 1 (PD-L1) to accumulate in aging and chronic inflammation. p16-mediated inhibition of CDK4/6 promotes PD-L1 stability in senescent cells via the downregulation of ubiquitin-dependent degradation. p16 expression in infiltrating macrophages induces an immunosuppressive environment that can contribute to an increased burden of senescent cells. Treatment with immunostimulatory anti-PD-L1 antibody enhances the cytotoxic T cell activity and leads to the elimination of p16, PD-L1-positive cells. Our study uncovers a molecular mechanism of p16-dependent regulation of PD-L1 protein stability in senescent cells and reveals the potential of PD-L1 as a target for treating senescence-mediated age-associated diseases.

cell biology↗

bioRxiv2022

RORc expressing immune cells support pro-tumorigenic functions of tertiary lymphoid structures

Tertiary lymphoid structures (TLSs) are formed in many cancer types and have been correlated with better prognosis and response to immunotherapy. In liver cancer, TLSs have been reported to be pro-tumorigenic as they harbor tumor progenitor cells and nurture their growth. The processes involved in TLS development and the acquisition of a pro- or anti-tumorigenic phenotype in cancer are largely unknown. RORc expressing immune cells have been previously implicated in TLS formation, however we find that they are not necessary for TLS neogenesis in the context of inflammation-associated liver cancer. On the contrary, RORc expressing cells negatively regulate TLS formation, since in their absence TLSs form in excess. CD4 cells are essential for liver TLS formation whereas B cells are required for TLS formation specifically in the absence of RORc expressing cells. Importantly, in chronically inflamed livers lacking RORc expressing cells, TLSs become anti-tumorigenic, resulting in reduced tumor load. Comparing liver pro- and anti-tumorigenic TLSs by transcriptional, proteomic and immunohistochemical analyses, revealed enrichment of exhausted CD8 cells that retained effector functions as well as germinal center B cells and plasma cells in anti-tumorigenic TLSs. Cell depletion experiments revealed a role mainly for B cells in limiting tumor development, possibly via tumor directed antibodies. Thus, RORc expressing cells negatively regulate B cell responses, and facilitate the pro-tumorigenic functions of hepatic TLSs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/498540v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@385067org.highwire.dtl.DTLVardef@126c45corg.highwire.dtl.DTLVardef@25ad06org.highwire.dtl.DTLVardef@1829326_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

From pseudotime to true dynamics: reconstructing a real-time axis for T cells differentiation

Numerous methods have recently emerged for ordering single cells along developmental trajectories. However, accurate depiction of developmental dynamics can only be achieved after rescaling the trajectory according to the relative time spent at each developmental point. We formulate a model which estimates local cell densities and fluxes, and incorporates cell division and apoptosis rates, to infer the real time dimension of the developmental trajectory. We validate the model using mathematical simulations, and apply it on experimental high dimensional cytometry data obtained from the mouse thymus to construct the true time-profile of the thymocyte developmental process. Our method can easily be implemented in any of the existing tools for trajectory inference.

systems biology↗

bioRxiv2021

Single-cell epigenetic analysis reveals principles of chromatin states in H3.3-K27M gliomas

Cancer cells are highly heterogeneous at the transcriptional level and in their epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted Cytometry by Time of Flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of Diffused Intrinsic Pontine Glioma (DIPG). DIPG is a lethal glioma, driven by histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIGP, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem-cell and differentiation markers. Moreover, we demonstrate the use of this high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell-cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.

cancer biology↗

bioRxiv2021

Pseudo-mutant p53 as a targetable phenotype of DNMT3A-mutated pre-leukemia

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemo-resistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPCs) and lead to chemo-resistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPCs. Intriguingly, a misfolded p53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between a wild-type (WT) and a "pseudomutant" conformations of p53. By combining single cell analyses and p53 conformation-specific monoclonal antibodies we studied preL-HSPCs from primary human DNMT3A AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPCs the pseudomutant conformation is the dominant. HSPCs from non-leukemic samples expressed both conformations to a similar extent. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of p53, specifically eliminated preL-HSPCs that had dysfunctional canonical p53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable p53 dysfunction in human preL-HSPCs carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

cancer biology↗

bioRxiv2020

A functional pre-screening platform for identifying points of vulnerability in the cell death map of human melanoma tumors

Targeted drug therapy in melanoma patients carrying the BRAF V600E mutation provides temporary remission, often followed by relapse due to acquired drug resistance. Here we propose a functional approach to circumvent drug resistance by applying a personalized prescreening platform that maps points of vulnerability in each tumor, prior to drug treatment. This platform applies siRNAs targeting 81 apoptosis, autophagy and programmed necrosis genes in patient tumor cell cultures, identifying genes whose targeting maximizes cell killing by short-term BRAF inhibition. Melanoma tumors displayed large heterogeneity in the number and identities of soft-spots, providing different tumor-specific functional death signatures. The soft-spots were targeted by replacing functional siRNAs with small compound inhibitors for long-term treatment in combination with vemurafenib. This strategy reduced the number of drug-tolerant persister cells surviving treatment, and most importantly, the number of drug-resistant foci. Thus, prescreening melanoma tumors for soft-spots within the cell death network may enhance targeted drug therapy before resistance emerges, thereby reducing the odds of developing drug-resistant mutations, and preventing tumor relapse.

cancer biology↗

bioRxiv2018

CSNAP, the smallest CSN subunit, modulates proteostasis through cullin-RING ubiquitin ligases

The cullin-RING ubiquitin E3 ligase (CRL) family consists of ~250 complexes that catalyze ubiquitylation of proteins to achieve cellular regulation. All CRLs are inhibited by the COP9 signalosome complex (CSN) through both enzymatic (deneddylation) and non-enzymatic (steric) mechanisms. The relative contribution of these two mechanisms is unclear. Here, we decouple the mechanisms using CSNAP, the recently discovered ninth subunit of the CSN. We find that CSNAP reduces the affinity of CSN toward CRL complexes. Removing CSNAP does not affect deneddylation, but leads to global effects on the CRL, causing altered reproductive capacity, suppressed DNA damage response, decreased viability, and delayed cell cycle progression. Thus, although CSNAP is only 2% of the CSN mass, it plays a critical role in the steric regulation of CRLs by the CSN.

biochemistry↗