Altered BM hematopoiesis and immune suppression are hallmarks of myelodysplastic syndrome (MDS). While the BM microenvironment influences malignant hematopoiesis, the mechanism leading to MDS-associated immune suppression is unknown. We tested whether mesenchymal stromal cells (MSCs) contribute to this process. Here, we developed a model to study cultured MSCs from patients with MDS (MDS-MSCs) compared with those from aged-matched normal controls for regulation of immune function. MDS-MSCs and healthy donor MSCs (HD-MSCs) exhibited a similar in vitro phenotype, and neither had a direct effect on NK cell function. However, when MDS- and HD-MSCs were cultured with monocytes, only the MDS-MSCs acquired phenotypic and metabolic properties of myeloid-derived suppressor cells (MDSCs), with resulting suppression of NK cell function, along with T cell proliferation. A MSC transcriptome was observed in MDS-MSCs compared with HD-MSCs, including increased expression of the ROS regulator, ENC1. High ENC1 expression in MDS-MSCs induced suppressive monocytes with increased INHBA, a gene that encodes for a member of the TGF-β superfamily of proteins. These monocytes also had reduced expression of the TGF-β transcriptional repressor MAB21L2, further adding to their immune-suppressive function. Silencing ENC1 or inhibiting ROS production in MDS-MSCs abrogated the suppressive function of MDS-MSC–conditioned monocytes. In addition, silencing MAB21L2 in healthy MSC-conditioned monocytes mimicked the MDS-MSC–suppressive transformation of monocytes. Our data demonstrate that MDS-MSCs are responsible for inducing an immune-suppressive microenvironment in MDS through an indirect mechanism involving monocytes.

Steroid-refractory intestinal acute graft-versus-host disease (aGVHD) is a frequently fatal condition, with little known about mechanisms driving failed steroid responses in gut mucosa. To uncover molecular insights in steroid-refractory aGVHD, we compared gene expression profiles of rectosigmoid biopsies from patients at diagnosis of clinical stage 3–4 lower intestinal aGVHD with repeat biopsies when the patients became steroid refractory and normal controls. We also performed single-gene analyses of factors associated with tolerance (programmed cell death ligand 1 [ PDL1 ], indoleamine 2,3-dioxygenase [IDO1], and T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibition motif domains [ TIGIT ]) and found that significantly higher expression levels of these aGVHD inhibitory genes ( PDL1 , IDO1 , TIGIT ) at aGVHD onset became decreased in the steroid-refractory state. We examined genes triggered by microbial ligands to stimulate gut repair, amphiregulin ( AREG ) and the aryl hydrocarbon receptor ( AhR ), and found that both AREG and AhR gene expression levels were increased at aGVHD onset and remained elevated in steroid-refractory aGVHD. We also identified higher expression levels of metallothioneins, metal-binding enzymes induced in stress responses, and M2 macrophage genes in steroid-refractory aGVHD. We observed no differences in T cell subsets between onset and steroid-refractory aGVHD. Patients with a rapidly fatal course showed greater DNA damage and a distinct microbial signature at aGVHD onset, whereas patients with more prolonged survival exhibited a gene expression profile consistent with activation of Smoothened . Our results extend the paradigm beyond T cell–centric therapies for steroid-refractory gastrointestinal aGVHD and highlight potentially new mechanisms for therapeutic exploration.

Purpose: In patients with metastatic prostate cancer (mPC), ATM and BRCA2 mutations dictate differences in PARPi inhibitor response and other therapies. We interrogated the molecular features of ATM- and BRCA2-mutated mPC to explain the divergent clinical outcomes and inform future treatment decisions. Experimental Design: We examined a novel set of 1,187 mPCs after excluding microsatellite-instable (MSI) tumors. We stratified these based on ATM (n = 88) or BRCA2 (n = 98) mutations. As control groups, mPCs with mutations in 12 other homologous recombination repair (HRR) genes were considered non-BRCA2/ATM HRR-deficient (HRDother, n = 193), whereas lack of any HRR mutations were considered HRR-proficient (HRP; n = 808). Gene expression analyses were performed using Limma. Real-world overall survival was determined from insurance claims data. Results: In noncastrate mPCs, only BRCA2-mutated mPCs exhibited worse clinical outcomes to AR-targeted therapies. In castrate mPCs, both ATM and BRCA2 mutations exhibited worse clinical outcomes to AR-targeted therapies. ATM-mutated mPCs had reduced TP53 mutations and harbored coamplification of 11q13 genes, including CCND1 and genes in the FGF family. BRCA2-mutated tumors showed elevated genomic loss-of-heterozygosity scores and were often tumor mutational burden high. BRCA2-mutated mPCs had upregulation of cell-cycle genes and were enriched in cell-cycle signaling programs. This was distinct from ATM-mutated tumors. Conclusions: Tumoral ATM and BRCA2 mutations are associated with differential clinical outcomes when patients are stratified by treatments, including hormonal or taxane therapies. ATM- and BRCA2-mutated tumors exhibited differences in co-occurring molecular features. These unique molecular features may inform therapeutic decisions and development of novel therapies.

Recent single-cell transcriptomic studies revealed new insights into cell-type heterogeneities in cellular microenvironments unavailable from bulk studies. A significant drawback of currently available algorithms is the need to use empirical parameters or rely on indirect quality measures to estimate the degree of complexity, i.e., the number of subgroups present in the sample. We fill this gap with a single-cell data analysis procedure allowing for unambiguous assessments of the depth of heterogeneity in subclonal compositions supported by data. Our approach combines nonnegative matrix factorization, which takes advantage of the sparse and nonnegative nature of single-cell RNA count data, with Bayesian model comparison enabling de novo prediction of the depth of heterogeneity. We show that the method predicts the correct number of subgroups using simulated data, primary blood mononuclear cell, and pancreatic cell data. We applied our approach to a collection of single-cell tumor samples and found two qualitatively distinct classes of cell-type heterogeneity in cancer microenvironments.

The senescence-associated secretory phenotype (SASP) is a hallmark of senescent cells and plays a critical role in the development and progression of various age-related diseases, including cancer, cardiovascular disorders, and neurodegenerative diseases. In this study, we characterize SASP heterogeneity using single-cell RNA sequencing (scRNA-seq) data, focusing on the transcriptional signatures associated with elevated expression of individual SASP genes in mature senescent cells, as well as time-dependent variation in SASP expression across the early and mature senescent states in the WI-38 human lung fibroblast cell line. We generated multiple gene sets, each representing the transcriptional landscape linked to high expression of a specific SASP gene, and integrated them into an ensemble that reflects the temporal dynamics of SASP gene expression. Applying SASP scores derived from this ensemble of gene sets (SASP scores/EGS) to publicly available scRNA-seq datasets from human lung, skin, and eye tissues enabled the identification of senescent fibroblasts and revealed IGFBP7 as a consistently upregulated marker in p21⁺ or p16⁺ fibroblasts across diverse human tissues. Our framework supports improved detection of both early and mature fibroblast replicative senescent cells, offering valuable insights into aging and age-related disease research.

Composition and rates of all detectable genomic features detectable through the Caris diagnostic platform.