Aberrant Differentiation Research Articles

Loss of function of GATA3 induces basal-like mammary tumors.

Purpose: GATA3 is a transcription factor essential for mammary luminal epithelial cell differentiation. Expression of GATA3 is absent or significantly reduced in basal-like breast cancers. Gata3 loss-of-function impairs cell proliferation, making it difficult to investigate the role of GATA3 deficiency in vivo. We previously demonstrated that CDK inhibitor p18INK4c (p18) is a downstream target of GATA3 and restrains mammary epithelial cell proliferation and tumorigenesis. Whether and how loss-of-function of GATA3 results in basal-like breast cancers remains elusive.Methods: We generated mutant mouse strains with heterozygous germline deletion of Gata3 in p18 deficient backgrounds and developed a Gata3 depleted mammary tumor model system to determine the role of Gata3 loss in controlling cell proliferation and aberrant differentiation in mammary tumor development and progression.Results: Haploid loss of Gata3 reduced mammary epithelial cell proliferation with induction of p18, impaired luminal differentiation, and promoted basal differentiation in mammary glands. p18 deficiency induced luminal type mammary tumors and rescued the proliferative defect caused by haploid loss of Gata3. Haploid loss of Gata3 accelerated p18 deficient mammary tumor development and changed the properties of these tumors, resulting in their malignant and luminal-to-basal transformation. Expression of Gata3 negatively correlated with basal differentiation markers in MMTV-PyMT mammary tumor cells. Depletion of Gata3 in luminal tumor cells also reduced cell proliferation with induction of p18 and promoted basal differentiation. We confirmed that expression of GATA3 and basal markers are inversely correlated in human basal-like breast cancers.Conclusions: This study provides the first genetic evidence demonstrating that loss-of-function of GATA3 directly induces basal-like breast cancer. Our finding suggests that basal-like breast cancer may also originate from luminal type cancer.

Paeoniflorin Prevents Aberrant Proliferation and Differentiation of Intestinal Stem Cells by Controlling C1q Release from Macrophages in Chronic Colitis

Paeoniflorin Prevents Aberrant Proliferation and Differentiation of Intestinal Stem Cells by Controlling C1q Release from Macrophages in Chronic Colitis

Abstract P088: Combined inhibition of histone deacetylases and EZH2 for the treatment of Wilms tumors

Abstract Wilms tumor (WT) is the most common childhood kidney cancer. It is believed to arise from immature kidney cells–nephron progenitor cells (NPCs)—which have failed to differentiate properly. Altered epigenetics is central to oncogenesis in many pediatric cancers. The critical contribution of epigenetic dysregulation to pediatric tumors provides a compelling rationale for the therapeutic potential of epigenetic drugs. Histone deacetylases (HDACs, especially HDAC1/2) and Enhancer of Zeste Homolog 2 (EZH2), a histone H3K27 methyltransferase, have been demonstrated to play a critical role in self-renewal and differentiation of mouse nephron progenitor cells (NPCs). In addition, altered expression and mutations of HDACs and EZH2 have been linked to many human cancers, including WT. As such, they are among the most promising therapeutic targets for cancer treatment. For sinstance, the treatment of Panobinostat, an FDA-approved pan-HDAC inhibitor, leaded to apoptosis and growth suppression of WT cells. EZH2 is highly expressed in WT and has been demonstrated to be associated with WT progression. We reasoned that WT results from unrestrained proliferation of progenitor cells due to overactive HDAC1, HDAC2, and EZH2. To prove this, we collected 5 human WT specimens. Immunostaining demonstrated dramatically higher level of HDAC1, HDAC2, SIX1, and SIX2 in the tumor tissues compared with adjacent normal tissues, strongly suggesting a critical role for the overactive HDAC1/2 in the SIX1/2 activation to promote cell proliferation in WT. Gain-of-function of SIX1 or SIX2 has been demonstrated to be closely correlated with high proliferation and aberrant differentiation of WT. Importantly, SIX2 marks the cancer stem cell population in WT. In addition, knockdown of HDAC1/2 by siRNAs or CRISPR gene editing resulted in downregulation of SIX1/2 in human embryonic kidney 293 cells and G401 cells derived from a Wilms tumor. Published studies also showed that treatment of Trichostain (a potent pan HDAC inhibitor) significantly reduced the Six1 expression in vertebrate neural crest and Hodgkin lymphoma cells. All these support the regulation of SIX1/2 expression by HDAC1/2. Concomitant inhibition of HDACs and EZH2 has proven to be highly synergistic and very potent for the treatment of many types of human cancers. Promisingly, combined treatment benzamide-based HDAC1/2-selective inhibitor Merck60 and Tazemetostat (an FDA-approved EZH2 inhibitor) synergistically suppressed cell growth in a wide range of dose combinations in 293T and G401 cells. In summary, our studies demonstrated elevated expression and activity of HDAC1/2 in WT, positive regulation of SIX1/SIX2 by HDAc1//2 during tumorigenesis, and synergic effect of HDAC1/2 and EZH2 inhibition in suppressing tumor cell proliferation. Thus, targeting HDAC1/2 and EZH2 may provide a promising therapeutic approach to treat WT and other pediatric kidney cancer with low toxicity and low side effects. Furthermore, availability of epigenetic drugs will facilitate the translation of this research into effective therapies. Citation Format: Hongbing Liu, Nhi Ngo, Chao-Hui Chen, Samir S. El-Dahr. Combined inhibition of histone deacetylases and EZH2 for the treatment of Wilms tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P088.

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation

Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized β-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII–PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.

Phosphatidylcholine\xa0restores neuronal plasticity\xa0of neural stem cells under inflammatory stress

The balances between NSCs growth and differentiation, and between glial and neuronal differentiation play a key role in brain regeneration after any pathological conditions. It is well known that the nervous tissue shows a poor recovery after injury due to the factors present in the wounded microenvironment, particularly inflammatory factors, that prevent neuronal differentiation. Thus, it is essential to generate a favourable condition for NSCs and conduct them to differentiate towards functional neurons. Here, we show that neuroinflammation has no effect on NSCs proliferation but induces an aberrant neuronal differentiation that gives rise to dystrophic, non-functional neurons. This is perhaps the initial step of brain failure associated to many neurological disorders. Interestingly, we demonstrate that phosphatidylcholine (PtdCho)-enriched media enhances neuronal differentiation even under inflammatory stress by modifying the commitment of post-mitotic cells. The pro-neurogenic effect of PtdCho increases the population of healthy normal neurons. In addition, we provide evidences that this phospholipid ameliorates the damage of neurons and, in consequence, modulates neuronal plasticity. These results contribute to our understanding of NSCs behaviour under inflammatory conditions, opening up new venues to improve neurogenic capacity in the brain.

Vascular repair and regeneration in cardiometabolic diseases.

Chronic cardiometabolic assaults during type 2 diabetes (T2D) and obesity induce a progenitor cell imbalance in the circulation characterized by overproduction and release of pro-inflammatory monocytes and granulocytes from the bone marrow alongside aberrant differentiation and mobilization of pro-vascular progenitor cells that generate downstream progeny for the coordination of blood vessel repair. This imbalance can be detected in the peripheral blood of individuals with established T2D and severe obesity using multiparametric flow cytometry analyses to discern pro-inflammatory vs. pro-angiogenic progenitor cell subsets identified by high aldehyde dehydrogenase activity, a conserved progenitor cell protective function, combined with lineage-restricted cell surface marker analyses. Recent evidence suggests that progenitor cell imbalance can be reversed by treatment with pharmacological agents or surgical interventions that reduce hyperglycaemia or excess adiposity. In this state-of-the-art review, we present current strategies to assess the progression of pro-vascular regenerative cell depletion in peripheral blood samples of individuals with T2D and obesity and we summarize novel clinical data that intervention using sodium-glucose co-transporter 2 inhibition or gastric bypass surgery can efficiently restore cell-mediated vascular repair mechanisms associated with profound cardiovascular benefits in recent outcome trials. Collectively, this thesis generates a compelling argument for early intervention using current pharmacological agents to prevent or restore imbalanced circulating progenitor content and maintain vascular regenerative cell trafficking to sites of ischaemic damage. This conceptual advancement may lead to the design of novel therapeutic approaches to prevent or reverse the devastating cardiovascular comorbidities currently associated with T2D and obesity.

INTS7-ABCD3 Interaction Stimulates the Proliferation and Osteoblastic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells by Suppressing Oxidative Stress.

Increased adipocyte and decreased osteoblast differentiation, combined with the ectopic proliferation of bone marrow mesenchymal stem cells (BM-MSCs), represent the primary causes of osteoporosis. The dysregulation of numerous intracellular bioactive factors is responsible for the aberrant differentiation and growth of BM-MSCs. In this study, we focused on a new stimulative factor, integrator complex subunit 7 (INTS7), and its cooperative protein ATP-binding cassette subfamily D member 3 (ABCD3)/high-density lipoprotein-binding protein (HDLBP) in mouse BM-MSCs. We aimed to uncover the effects of the INTS7–ABCD3/HDLBP interaction on BM-MSC biological behaviors and the potential mechanism underlying these effects. Functional in vitro experiments showed that the suppression of the INTS7–ABCD3 interaction rather than HDLBP could impair BM-MSC proliferation and induce cell apoptosis. Moreover, Alizarin Red S and Oil Red O staining, respectively, revealed that INTS7 and ABCD3 knockdown but not HDLBP knockdown could decrease osteoblastic differentiation and accelerate the adipogenic differentiation of BM-MSCs. Mechanistically, reactive oxygen species (ROS) and histone γ-H2AX quantities significantly increased, whereas the levels of antioxidants declined due to INTS7 and ABCD3 inhibition in BM-MSCs. These findings indicated that the suppression of oxidative stress could be involved in the INTS7/ABCD3 co-regulatory mechanisms for BM-MSC proliferation and differentiation, identifying new potential candidates for osteoporosis therapy.

Management practice, quality of life and associated factors in psoriasis patients attending a dermatological center in Ethiopia.

Psoriasis is a chronic inflammatory disease characterized by keratinocyte hyperproliferation and aberrant differentiation with great negative impact on patients' quality of life (QoL). This study aimed at assessing factors influencing management practice, and QoL and its associated factors among ambulatory psoriatic patients visiting All Africa Leprosy, Tuberculosis and Rehabilitation Training (ALERT) Center in Addis Ababa, Ethiopia. A cross sectional study was conducted in 207 patients with psoriasis attending the dermatology clinic of ALERT Center in Addis Ababa, Ethiopia. Data were collected using structured questionnaire and patients' chart review. Dermatology Life Quality Index (DLQI) was used to measure patients' QoL. Patients' characteristics were summarized using descriptive statistics and predictors of QoL were identified by binary logistic regression. Among 207 study participants, 122 (58.9%) were females. The mean age of the study population was 37.92 (SD = 14.86) years (ranging from 16 to 68 years). The mean age at which diagnosis of psoriasis made was 32 (SD = 13.7) years ranging from 10 to 62 years. The duration of the disease in 112 (54.1%) patients were more than or equal to 5 years. Majority of study participants 145 (70.0%) had plaque psoriasis followed by sebopsoriasis, 24 (11.6%). The majority of plaque psoriasis (80%) cases were managed by topical corticosteroids with or without salicylic acid or coal tar and only 21 (14.5%) treated by methotrexate alone. The mean DLQI was 6.25 corresponding to a moderate effect. Symptoms and feelings were the most affected domains of QoL. Factors associated with poor QoL were female [AOR = 0.17 (95%CI: 0.06, 0.48)], low, above average and high family income ([AOR = 0.12 (95% CI: 0.02, 0.56)], [AOR = 0.06 (95% CI:0.01, 0.32)], and [AOR = 0.03 (95% CI: 0.01, 0.22)]), respectively, and primary education level [AOR = 0.14 (95% CI: 0.03, 0.64)] while being on systemic therapy [AOR = 4.26 (CI: 1.18, 15.35)] was predictor of better QoL. Poor QoL was predominant in females [AOR = 0.17 (95%CI: 0.06, 0.48)], low income [AOR = 0.12 (95% CI: 0.02, 0.56] patients, and patients with primary education level [AOR = 0.14 (95% CI: 0.03, 0.64)]. Patients on systemic therapy [AOR = 4.26 (CI: 1.18, 15.35)] had good QoL. Our study identified that topical corticosteroids were the mainstay of psoriasis treatment in the dermatology clinic of ALERT Center in Addis Ababa, Ethiopia. Moderate effect QoL was achieved by study participants based on DLQL score.

The Transcriptional and Epigenetic Reprogramming of Aged Hematopoietic Stem Cells Drives Myeloid Rewiring in Clonal Hematopoiesis-Associated Cytopenias

Patients (pts) with myelodysplastic syndromes (MDS) have few therapy options. Interventions to improve outcomes should consider strategies that arrest MDS in its early phases, when symptoms are minimal and prolonged survival is expected.To develop prevention strategies that arrest MDS before the disease outcomes become irreversibly dismal, we dissected the molecular and biological mechanisms that maintain MDS in one of its premalignant phases, clonal cytopenia of undetermined significance (CCUS). Recognizing that CCUS is an aging-related disease, we first sought to determine, at the single-cell level, how CCUS affects the transcriptional and epigenetic profile of the aging hematopoietic stem and progenitor cell (HSPC) compartment and overcomes aging-induced degenerative phenotypes. We performed single-cell RNA sequencing (scRNA-seq) analysis of Lin -CD34 + HSPCs isolated from the bone marrow (BM) of 3 young healthy donors (yHDs), 4 elderly HDs (eHDs), and 3 elderly pts with CCUS carrying mutations in common MDS driver genes.We found that the frequencies of hematopoietic stem cells (HSCs) and megakaryocytic (Mk)/erythroid (Er) progenitors were increased at the expense of myeloid (My) progenitors in eHDs as compared with yHDs (Fig. a). In contrast, CCUS pts had a predominant My-biased HSPC distribution (Fig. a). However, immunophenotypic quantification in large cohorts of eHDs and CCUS pts revealed that CCUS pts' BM had significantly fewer CD34 +CD38 - HSC populations and CD34 +CD38 + My progenitors, suggesting that My bias in CCUS results from the aberrant My differentiation of HSCs rather than My cell expansion. Further differential expression analysis among the scRNA-seq clusters showed that, compared with yHD HSCs, eHD HSCs were characterized by a significant upregulation of genes involved in the TNFα-induced activation of NF-κB (e.g., MCL1; Fig. b), which is consistent with previous findings that aged HSCs undergo transcriptional reprogramming to maintain their survival in response to changes in the systemic environment (He et al. Blood 2020). In contrast, CCUS HSCs, compared with eHD HSCs, overexpressed regulators of translation, respiratory electron transport, and mitochondrial translation initiation (Fig. c), which underscores these cells' state of proliferation and metabolic activation and their ability to overcome aging-induced phenotypic alterations.To evaluate whether the aberrant lineage differentiation in eHD and CCUS HSPCs arose from the altered fate determination of HSCs, we performed single-cell assays for transposase-accessible chromatin sequencing to profile chromatin accessibility in sorted HSCs or Lin -CD34 + HSPCs from yHDs, eHDs, and CCUS pts. Consistent with our transcriptomic data, compared with yHD HSCs, eHD HSCs were mostly poised for Mk differentiation, whereas CCUS HSPCs were poised for lymphoid/My differentiation. Indeed, eHD HSCs had an increased activity of transcriptional factors belonging to the NF-κB family and open peaks at the distal elements of genes involved in hemostasis (Fig. d). In contrast, CCUS HSCs were poised to downregulate the expression of genes involved in NF-κB signaling and Mk/Er differentiation (Fig. e).These results suggested that CCUS HSCs are highly metabolically active to maintain My differentiation. Indeed, metabolomic analyses confirmed that intermediates of oxidative phosphorylation were significantly upregulated in CCUS CD34 + cells as compared with eHD CD34 + cells (Fig. f). Further, scRNA-seq analysis of mononuclear cells isolated from the BM of 3 CCUS and 3 eHD samples revealed the widespread upregulation of genes involved in protein processing and mitochondrial metabolism. This analysis also revealed impaired terminal My differentiation despite the HSPC My bias, with decreased frequencies of monocytic cells, and an intriguing expansion of cytotoxic cell subsets in the BM of CCUS pts.In conclusion, our results demonstrate that CCUS HSCs carrying MDS driver mutations evade aging-induced phenotypic degeneration, become metabolically active, and have aberrant My skewing. Our study clarifies the molecular mechanisms underlying MDS initiation and offers an opportunity for early therapeutic intervention. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PLAGL2 Independently Drives Aberrant Erythropoiesis and Initiation of Preleukemic State

BackgroundThe identification and understanding of early drivers in malignancy is crucial to prevent or revert preleukemic events. Del(20q) is one of the most common primary cytogenetic abnormalities found in preleukemic malignancies from myeloproliferative neoplasms to myelodysplastic syndrome (MDS). Previous studies have identified a “common retained region” within 20q11.21 that is often amplified in a subset of MDS patients. PLAGL2 is one of the 4 genes identified to be within the minimally conserved amplified region. (MacKinnon et al. 2010) Indeed, in previously published datasets of MDS hematopoietic stem and progenitor cells (HSPCs) transcriptome, PLAGL2 is significantly elevated in del(20q) patients compared to healthy controls. However, we have found that its level is also higher in HSPCs of cytogenetically normal MDS patients with low blasts. Given these findings, we sought to define the role of PLAGL2 as a potential early driver of myeloid malignancies.ResultsIn healthy cord blood (CB) HSPCs, PLAGL2 overexpression enhanced proliferation ex vivo, better maintained stemness and decreased apoptosis. Colony formation assays also identified increased output of the erythroid lineage. Xenotransplanted CB CD34+ HSPCs overexpressing PLAGL2 exhibited increased engraftment competitiveness and led to splenomegaly with signs of hypercellularity after 20 weeks, features consistent with clinical observations of hematological malignancy. Grafts derived from PLAGL2 overexpressing cells reproducibly maintained a significantly larger CD34+ HSPC compartment. Intriguingly we also identified that ~50% of PLAGL2-overexpressing grafts exhibited a significant erythroid (CD71+/CD235a+) component where none was observed in the control group. This unique finding of aberrant erythropoiesis is reminiscent of clinical observations in patients with 20q11.21 amplification, where a high proportion of erythroblasts in the marrow and in some cases progression to erythroleukemia was noted. To evaluate the progression of PLAGL2-overexpressing grafts, further secondary transplantations were carried out and showed the persistence of only immature erythroid progenitors (CD71+/CD235a-) coupled with a near complete absence of lymphopoiesis in the same grafts. Together, our data strongly suggests ectopic levels of PLAGL2 can independently drive the expansion of human HSPCs and enforce features of myeloid malignancy.To uncover the molecular mechanism underlying PLAGL2 function, we performed RNA-seq and Cut&Run in human CB CD34+ HSPCs overexpressing PLAGL2. Geneset enrichment analysis of the transcriptome and over-representation analysis of bound genes both identified signatures consistent with LSCs. We compared these findings with identically-derived omics profiles of HSPCs overexpressing PLAG1, a closely related family member that our lab has identified as a potent expander of HSCs ex vivo but not capable of promoting malignant features. We found a strong common feature in the downregulation of ribosomal components and translation machinery, then functionally validated reduced protein synthesis in PLAGL2 overexpressing HSPCs through OP-Puro assays. We have shown dampened mRNA translation to be one of the mechanisms by which PLAG1 enhances stemness and survival of HSCs, one that potentially extends to PLAGL2 as well. However, we also identified discordant signatures, notably PLAGL2's unique capacity to reduce mitochondrial translation, a pathway associated with ineffective erythropoiesis and MDS and one that we are currently exploring as a means by which PLAGL2 can enforce malignant phenotypes.Finally, to investigate the potential of PLAGL2 as a therapeutic target in MDS, we performed shRNA knockdown in MDSL, a human MDS cell line. In vitro competitive assays with mixed wildtype cells showed steady dropout of PLAGL2 depleted cells. Currently, we are continuing to purse the dependence of primary MDS cells on PLAGL2 through in vivo xenograft models.ConclusionWe have identified PLAGL2's potential as an early independent driver of myeloid malignancy and aberrant erythroid differentiation. An understanding of PLAGL2 and its downstream mechanisms will not only further our understanding on the development of early myeloid malignancies but also potentially provide another avenue to treat or prevent leukemia before it manifests. DisclosuresDick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Integrative Analysis of Primary SF3B1 mt Ring Sideroblasts Provides Fundamental Insights into MDS-RS Pathogenesis and Dyserythropoiesis

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders characterized by aberrant HSC differentiation, cytopenia, and an increased risk of progression to leukemia. The low-risk subtype MDS with ring sideroblasts (MDS-RS) is specifically characterized by expanded and ineffective erythropoiesis, with more than 80% of patients displaying mutations in the core spliceosome component SF3B1 (SF3B1 mt).A hallmark of the MDS-RS bone marrow (BM) is the progressive accumulation of ring sideroblasts (RS), erythroblasts displaying perinuclear mitochondria loaded with aberrant ferritin-iron complexes. Whilst several in vitro and in vivo model systems exist for studying the impact of SF3B1 mt on erythropoiesis and RS development, primary SF3B1 mt erythroid biology remains largely unexplored due to the inability to purify live SF3B1 mt cells or fully replicate BM conditions in vitro.To address this issue, we developed an innovative two-step method to isolate live ring sideroblasts from SF3B1 mt MDS-RS BM aspiration material with extremely high purity (as determined through droplet digital PCR-based genotyping [Fig. 1A] and morphology-based detection through Prussian blue staining [Fig. 1B,C]). Unexpectedly, evaluation of matching peripheral blood samples showed that circulating ring sideroblasts are strikingly common in MDS-RS (Fig. 1D), with their abundance being significantly positively associated with clinically-determined BM RS frequencies and serum erythropoietin levels, as well as negatively associated with hemoglobin levels.Through high-throughput Chromium 3'-based single-cell RNA sequencing (scRNAseq) analysis of purified RS, we then showed that these cells comprise a heterogeneous population encompassing all stages of the erythroid differentiation continuum, from early progenitors to orthochromatic erythroblasts (Fig. 1E). The RS transcriptome was shown to be dynamically regulated towards the maintenance of cell survival during late terminal erythroid differentiation (exemplified through parkin 1 [PINK1] expression), with SF3B1 K700E erythroblasts employing multiple strategies to preserve homeostasis despite undergoing extreme oxidative stress. These observations were confirmed through a parallel whole-transcript RNAseq investigation comprising CD34 + and GPA +-enriched samples obtained from normal bone marrow (NBM) donors and SF3B1 K700E MDS-RS patients, as well as purified RS samples. This bulk RNAseq experiment validated the RS transcriptomic signature observed in scRNAseq (Fig. 1F) and allowed for a detailed investigation of RNA splicing. SF3B1 K700E-associated alternative splicing in CD34 + and RS was consistent with previous literature, but also highly context-dependent and with substantial changes in scope and magnitude throughout erythroid differentiation (Fig. 1G-I).Finally, we substantiated these RNAseq results through Tandem Mass Tag-based semi-quantitative proteomic analysis of purified RS and GPA-enriched cells from NBM donors and MDS-RS patients. We confirmed that ring sideroblast survival is heavily dependent on redox balance modulation and suppression of ER stress via an increased dependence on glutamine, mirroring the molecular mechanisms observed in malignancy. Additionally, our data strongly indicate that the RS population is a major modulator of the MDS-RS BM microenvironment due to expression of stress factors (with particular emphasis on GDF15, erythroferrone and IL-18). In conclusion, our integrative analysis of primary RS constitutes a unique platform for the study of MDS-RS, with special interest for the investigation of potential drivers of disease severity or treatment avenues. [Display omitted] DisclosuresKretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment.

Integrated Multidimensional Flow Cytometry (MFC) and Next-Generation Sequencing (NGS) to Reconstruct Evolutionary Paterns from Dysplasia to Acute Myeloid Leukemia (AML)

Integrated Multidimensional Flow Cytometry (MFC) and Next-Generation Sequencing (NGS) to Reconstruct Evolutionary Paterns from Dysplasia to Acute Myeloid Leukemia (AML)

Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Mechanical Checkpoint Regulates Monocyte Differentiation in Fibrotic Matrix

Myelofibrosis (MF) is a progressive, myeloid malignancy characterized by deposition of collagen and reticulin fibers in the bone marrow (BM). Previous studies have shown that monocytosis is associated with poor prognosis in MF, highlighting a potential pathogenic role for monocytes in MF. Although many studies have addressed the role of cell-intrinsic and soluble extracellular factors in MF development, it is currently unknown if mechanical properties of fibrotic BM contribute to aberrant differentiation of myeloid cells and of monocytes in particular.We first defined the stiffness and viscoelastic properties of healthy and fibrotic BM. Stiffness is defined as the resistance of a matrix to deformation, while viscoelasticity is the rate of dissipation of an applied stress over time. Independent of stiffness, an applied stress relaxes rapidly in a more viscous, liquid-like matrix, whereas in a more elastic, solid-like material, stress relaxes slowly.We next generated a cohort of fibrotic and non-fibrotic mice by transplanting retrovirally transduced JAK2V617F or empty vector (EV) control hematopoietic stem and progenitor cells (HSPCs) into lethally irradiated recipients. Femurs from these mice were harvested seven months post-transplant, as well as from age- and sex-matched healthy primary mice. Nanoindentation was performed to measure BM stiffness and viscoelasticity. Fibrotic BM showed higher stiffness, as well as trending higher elastic, solid-like properties, compared to BM of control mice.We then aimed to study the effect of matrix stiffness and viscoelasticity on monocytes. Human BM-derived monocytes were encapsulated in stiff, viscous or stiff, elastic hydrogels and cultured in the presence of GM-CSF, IL-4, and PGE2 for 3 days, followed by nanoString and flow cytometry analyses. Cells in elastic gels upregulated gene sets associated with co-stimulatory molecules and cytokine receptor signaling, MHC class II antigen presentation, and regulation of extracellular matrix (ECM), compared to cells in viscous gels of the same stiffness. The fraction of dendritic cells (DCs) was significantly upregulated, as indicated by double-positive CD11c+CD1c+ (40.9% viscous vs 69.5% elastic of CD11b+HLA-DR+ cells) and CD80+ cells (20.9% viscous vs 62.7% elastic of CD11b+HLA-DR+ cells), and surface expression of HLA-DR (gMFI 2587 viscous vs 6334 elastic). Consistent with these findings, the fraction of pro-fibrotic SLAMF7+ cells (4.2% viscous vs 17.3% elastic) were also significantly higher in elastic gels. Together, these data suggest that stiff, elastic ECM drives pro-inflammatory polarization and differentiation of monocytes into antigen-presenting cells.Next, we examined the role of the cytoskeleton on human monocyte differentiation. Cortical F-actin was significantly upregulated in cells in stiff, elastic gels compared to viscous gels. Cells were exposed to a highly selective small molecular inhibitor of the γ-isoform of PI3K. Treatment with the PI3Ky inhibitor significantly reduced F-actin staining of cells in elastic gels, upregulated immature monocyte markers, reduced surface expression of HLA-DR, and downregulated the cytokines IL6, IL8, CCL4, which have previously been associated with disease progression in myelofibrosis.In line with the above human ex vivo data, BM isolated from fibrotic mice (described above) showed skewing towards Ly6G-Ly6C+ monocytes (a population enriched for inflammatory monocytes) within the CD11b myeloid compartment compared to control transplanted mice or to non-fibrotic mice that were transplanted with endogenously expressing Jak2V617F cells. Additionally, the percentage of conventional DCs (cDCs) was increased in fibrotic Jak2V617F mice compared to control mice. Importantly, 16 day in vivo treatment with the PI3Ky inhibitor significantly reduced the fraction of Ly6G-Ly6C+ monocytes within the CD11b compartment as well as the fraction of cDCs, compared to vehicle-treated Jak2V617F mice.In summary, fibrotic BM is stiffer and more elastic than normal BM. Our studies show that a stiff, elastic BM environment drives monocytes towards a more pro-inflammatory state which can in part be suppressed by PI3K-γ inhibition. Our results have relevance for human MF by demonstrating that a fibrotic BM niche is not just a consequence of chronic inflammation but is also inflammation-promoting.KHV and AEM contributed equally to this work. DisclosuresPozdnyakova: Scopio Labs: Consultancy. Mullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy. Wucherpfennig: Novartis: Research Funding; Nextechinvest: Membership on an entity's Board of Directors or advisory committees; Immunitas Therapeutics: Current holder of individual stocks in a privately-held company; TScan Therapeutics: Membership on an entity's Board of Directors or advisory committees; TCR2 Therapeutics: Membership on an entity's Board of Directors or advisory committees; SQZ Biotech: Membership on an entity's Board of Directors or advisory committees. Mooney: Novartis: Patents & Royalties: Licensed IP, Research Funding; Sirenex: Patents & Royalties: Licensed IP; Samyang Corp: Membership on an entity's Board of Directors or advisory committees; IVIVA: Membership on an entity's Board of Directors or advisory committees; Attivare: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Revela: Membership on an entity's Board of Directors or advisory committees; Amend Surgical: Patents & Royalties: Licensed IP; Lyell: Current equity holder in publicly-traded company, Patents & Royalties.

Single-cell profiling of T lymphocytes in deficiency of adenosine deaminase 2.

Deficiency of adenosine deaminase 2 (DADA2) is a monogenic vasculitis syndrome caused by autosomal-recessive loss-of-function mutations in the ADA2 gene (previously known as CECR1). Vasculitis, vasculopathy, and inflammation are dominant clinical features of this disease; the spectrum of manifestations includes immunodeficiency and lymphoproliferation as well as hematologic manifestations. ADA2 is primarily secreted by stimulated monocytes and macrophages. Aberrant monocyte differentiation to macrophages and neutrophils are important in the pathogenesis of DADA2, but little is known about T lymphocytes in this disease. We performed combined single-cell RNA sequencing and single-cell TCR sequencing in order to profile T cell repertoires in 10 patients with DADA2. Although there were no significant alterations of T cell subsets, we observed activation of both CD8+ and CD4+ T cells. There was no clonal expansion of T cells: most TCRs were expressed at basal levels in patients and healthy donors. TCR usage was private to individual patients and not disease specific, indicating as unlikely a common pathogenic background or predisposition to a common pathogen. We recognized activation of IFN pathways as a signature of T cells and STAT1 as a hub gene in the gene network of T cell activation and cytotoxicity. Overall, T cells in DADA2 patients showed distinct cell-cell interactions with monocytes, as compared with healthy donors, and many of these ligand-receptor interactions likely drove up-regulation of STAT1 in both T cells and other immune cells in patients. Our analysis reveals previously undercharacterized cell characteristics in DADA2.

LncRNA DDGC participates in premature ovarian insufficiency through regulating RAD51 and WT1

The list of long non-coding RNAs (lncRNAs) that participate in the function of ovarian granulosa cells (GCs) is rapidly expanding, but the mechanisms through which lncRNAs regulate GC function are not yet fully understood. Here, we recognized a minimally expressed lncRNA RP4-545C24.1 (which we named DDGC) in GCs from patients with biochemical premature ovarian insufficiency (bPOI). We further explored the role of lncRNA DDGC in GC function and its contribution to the development of bPOI. Mechanistically, silencing DDGC downregulated RAD51 by competitively binding with miR-589-5p, and this resulted in significant inhibition of DNA damage repair capacity. In addition, decreased expression of DDGC promoted ubiquitin-mediated degradation of Wilms tumor 1 (WT1) protein through interactions with heat shock protein 90 (HSP90), which led to aberrant differentiation of GCs. Moreover, DDGC was able to ameliorate the etoposide-induced DNA damage and apoptosis in vivo. Taken together, these findings provide new insights into the contribution of lncRNAs in POI pathogenesis.

Skin Barrier Dysregulation in Psoriasis.

The skin barrier is broadly composed of two elements—a physical barrier mostly localised in the epidermis, and an immune barrier localised in both the dermis and epidermis. These two systems interact cooperatively to maintain skin homeostasis and overall human health. However, if dysregulated, several skin diseases may arise. Psoriasis is one of the most prevalent skin diseases associated with disrupted barrier function. It is characterised by the formation of psoriatic lesions, the aberrant differentiation and proliferation of keratinocytes, and excessive inflammation. In this review, we summarize recent discoveries in disease pathogenesis, including the contribution of keratinocytes, immune cells, genetic and environmental factors, and how they advance current and future treatments.

200 Genome-wide analysis of long non-coding RNA expression profiles in keratinocytes from psoriasis skin

Psoriasis is a common, chronically relapsing inflammatory skin disease, affecting the patients’ life quality substantially. Psoriasis has a strong genetic background and a large number of genetic susceptibility loci have been identified. In psoriatic plaques, keratinocytes, the major cell type in the epidermis, undergo hyperproliferation and aberrant differentiation. The regulation of these processes in keratinocytes is not fully understood. Long non-coding RNAs are >200 nucleotide long transcripts, which do not code for protein but regulate other genes. Little is known about their involvement in epidermal alterations in psoriasis. In this study, we aimed to explore the potential contribution of long non-coding RNAs to epidermal alterations in psoriasis. In order to detect changes in the non- coding epidermal transcriptome, keratinocytes were sorted from skin biopsies collected from healthy skin of volunteers as well as from non-lesional and lesional skin of patients with psoriasis. Affymetrix Human Transcriptome Array 2.0 platform was used to identify differentially expressed lncRNAs. Microarray analysis identified 43 lncRNAs that were significantly up-regulated and 37 lncRNAs that were down-regulated in keratinocytes from psoriasis lesional skin as compared to healthy skin. 65 lncRNAs were deregulated in the lesional skin when compared with non-lesional skin. Analysis of overlap between the genomic loci coding for lncRNAs and known psoriasis susceptibility regions identified several lncRNAs, which are localized in genomic regions in the vicinity of known psoriasis susceptibility loci. We identified the LINC00958 as a lncRNA to be overexpressed in keratinocytes from psoriasis lesional skin, as compared with non-lesional and healthy skin. We have confirmed its overexpression in keratinocytes of psoriasis skin by qPCR and RNAscope in situ hybridization. Our results suggest that lncRNAs may contribute to epidermal dysfunction in psoriatic skin and that lncRNA modulation may be a novel treatment option for psoriasis in the future.

Anti-TRIM21 antibody is associated with aberrant B-cell function and type I interferon production in systemic lupus erythematosus.

TRIM21 is a member of the tripartite motif family proteins and is one of the autoantigens which react with anti-SS-A antibody (Ab) present in sera of patients with systemic lupus erythematosus (SLE) and Sjögren's syndrome. Previous studies have shown that TRIM21 dysfunction promotes aberrant B-cell differentiation and Ab production in SLE, and anti-TRIM21 Ab may be related to the TRIM21 dysfunction in human SLE pathogenesis. Here, we examined the relationship between anti-TRIM21 Ab and clinical and immunological characteristics in SLE patients. Twenty-seven patients with SLE (23 women and four men) before immunosuppressive therapies, who fulfilled the revised 1997 American College of Rheumatology criteria for SLE, and four healthy controls (3 women and one man) were enrolled in the study. SLE patients were divided into two groups according to the seropositivity for anti-TRIM21 Ab. Serum anti-TRIM21 Ab levels were measured using enzyme-linked immunosorbent assays. The serum levels of cytokines and immunoglobulins were measured by cytometer beads arrays. The expression levels of TRIM21 protein in peripheral mononuclear cells (PBMCs) from SLE patients were evaluated by Western blotting. Sixteen and 9 patients showed seronegativity and seropositivity for anti-TRIM21 Ab, respectively. There were no significant differences in the background parameters, including female ratio, age, disease duration, SLE activity, and laboratory data between the two groups. The serum levels of interferon (IFN)-β were significantly higher in patients with anti-TRIM21 Ab as compared with those without anti-TRIM21 Ab (P = .043). The levels of IgG1 and IgA were significantly higher in SLE patients with anti-TRIM21 Ab as compared with those without anti-TRIM21 Ab (P = .0022 and .032, respectively). The PBMCs of patients with anti-TRIM21 Ab showed a significantly lower expression of TRIM21 protein as compared with those of patients without anti-TRIM21 Ab (P = .014). Anti-TRIM21 Ab seropositivity was related to B-cell abnormalities and type I IFN overproduction in SLE patients. These findings suggest that anti-TRIM21 Ab may have an inhibitory effect on TRIM21 functions and be a novel biomarker for the level of dependence on type I IFN overproduction and B-cell abnormalities.

Stromal androgen and hedgehog signaling regulates stem cell niches in pubertal prostate development.

Stromal androgen-receptor (AR) action is essential for prostate development, morphogenesis and regeneration. However, mechanisms underlying how stromal AR maintains the cell niche in support of pubertal prostatic epithelial growth are unknown. Here, using advanced mouse genetic tools, we demonstrate that selective deletion of stromal AR expression in prepubescent Shh-responsive Gli1-expressing cells significantly impedes pubertal prostate epithelial growth and development. Single-cell transcriptomic analyses showed that AR loss in these prepubescent Gli1-expressing cells dysregulates androgen signaling-initiated stromal-epithelial paracrine interactions, leading to growth retardation of pubertal prostate epithelia and significant development defects. Specifically, AR loss elevates Shh-signaling activation in both prostatic stromal and adjacent epithelial cells, directly inhibiting prostatic epithelial growth. Single-cell trajectory analyses further identified aberrant differentiation fates of prostatic epithelial cells directly altered by stromal AR deletion. In vivo recombination of AR-deficient stromal Gli1-lineage cells with wild-type prostatic epithelial cells failed to develop normal prostatic epithelia. These data demonstrate previously unidentified mechanisms underlying how stromal AR-signaling facilitates Shh-mediated cell niches in pubertal prostatic epithelial growth and development.