Maintenance Of Stem Cell Niche Research Articles

Inhibition of ribosome biogenesis by actinomycin D affects Arabidopsis root development

Actinomycin D has been reported to selectively inhibit rRNA synthesis and ribosome biogenesis, induce G2 checkpoint of cell cycle arrest in HeLa cells. In Arabidopsis, actinomycin D was also used as agent to preferentially inhibit the ribosome biosynthesis and ribosomal function. However, the function of actinomycin D on Arabidopsis root development remains to be elucidated. In this study, we exposed Arabidopsis seedlings to actinomycin D with the aim of evaluating the effects of ribosome biogenesis on root development. The results demonstrated that actinomycin D inhibited Arabidopsis root growth by reduced meristematic activity in a dose dependent manner. Exposure to actinomycin D decreased the expression of WOX5 and key stem cell niche-defining transcription factors SHR and PLT1, thus the loss function of QC identity and stem cell niche maintenance. In addition, dead cells were observed after actinomycin D treatment in root stele initials and DNA damage response was constitutively activated. Collectively, we propose that ribosome biogenesis plays key role in primary root growth through maintenance of root stem cell niche and DNA damage response in Arabidopsis.

Evolution of RGF/GLV/CLEL Peptide Hormones and Their Roles in Land Plant Growth and Regulation.

Rooting is a key innovation during plant terrestrialization. RGFs/GLVs/CLELs are a family of secreted peptides, playing key roles in root stem cell niche maintenance and pattern formation. The origin of this peptide family is not well characterized. RGFs and their receptor genes, RGIs, were investigated comprehensively using phylogenetic and genetic analyses. We identified 203 RGF genes from 24 plant species, representing a variety of land plant lineages. We found that the RGF genes originate from land plants and expand via multiple duplication events. The lineage-specific RGF duplicates are retained due to their regulatory divergence, while a majority of RGFs experienced strong purifying selection in most land plants. Functional analysis indicated that RGFs and their receptor genes, RGIs, isolated from liverwort, tomato, and maize possess similar biological functions with their counterparts from Arabidopsis in root development. RGFs and RGIs are likely coevolved in land plants. Our studies shed light on the origin and functional conservation of this important peptide family in plant root development.

Compromised Host Stem Cell Competitiveness Affords Fanconi Stem Cell Engraftment in C-Kit Mutant Humanized Mice

Humanized mouse model is a powerful tool for the study of human hematopoiesis and hematologic diseases in vivo. However, existing models continue to be limited by absent engraftment of stem cells with inherent growth defects, especially in bone marrow failure (BMF) disorders such as low-grade myelodysplastic syndromes (MDS) and Fanconi Anemia (FA). Previously, we presented a highly efficient MDS/AML PDX model in cytokine-humanized MISTRG mice, that express human M-CSF, IL-3, GM-CSF and THPO in the background of the SIRPα - humanized rag -/- IL2Rg -/- mice. MISTRG mice support efficient, faithful and long-term MDS hematopoietic stem and progenitor cell (HSPC) engraftment with multi-lineage representation. Similar to MDS, FA stem cells are defective and sensitive to toxic stress conferred by inflammation or irradiation. Irradiation, required in conventional mice as pre-conditioning, increases the risk of secondary malignancies, induces activation of inflammatory pathways and may damage the bone marrow microenvironment.In this study, we present MISTRG6kit W41 mice that in addition to the above cytokines express human IL-6, which improves lymphoid development, and that were CRISPR modified to introduce the white spotting 41 mutation in c-Kit (c-kit W41), which results in partial loss of c-kit function in the murine host stem and progenitor cells. To characterize this mouse model, we compare the engraftment capacity between MISTRG6 and MISTRG6kit W41 mice. We find that the HSPCs from both healthy donor and low grade MDS patient can be more efficiently propagated in MISTRG6Kit W41 mice and that they can be serially transplanted. Human cells are detectable at higher levels in PB of MISTRG6Kit W41 mice as early as 8 weeks post transplantation. As previously described, c-kit mutation enhances human erythroid engraftment while multi-lineage engraftment is maintained in engrafted MISTRG6Kit W41 mice. Therefore, MISTRG6Kit W41 mice offer a better host environment that supports human hematopoietic expansion without irradiation.FA is an inherited genetic disease carrying FA gene mutations associated with DNA damage and cancer predisposition and marked by BMF, MDS and AML. FA HSPCs cannot be expanded or grown for sufficient periods in vitro, and existing mouse models have failed to demonstrate engraftment of primary cells from FA patient. We transplanted a MDS sample from a FANCC mutant patient into MISTRG6Kit W41 mice giving rise to a myeloid predominant graft that could be successfully serially transplanted. Moreover, non-transformed FA HSPCs from 2 FANCA and 2 FANCG mutant patients were successfully engrafted in MISTRG6Kit W41 mice.In summary, MISTRG mice with human IL-6 expression and c-Kit W41 mutation provide a more efficient and convenient way for pre-clinical studies of BMFs such as FA and low-grade MDS with maintenance of an intact humanized stem cell niche, and this is the first time we saw primary cells from FA patient propagate in mouse model. We believe that our model would be highly beneficial to the study of additional diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita or Schwachman-Diamond syndrome. DisclosuresNo relevant conflicts of interest to declare.

MSC-Derived Extracellular Vesicles in Tumors and Therapy.

Simple SummaryTherapeutic functions of mesenchymal stroma-/stem-like cells (MSCs) are mediated predominantly through paracrine effects by the release of various different components. Upon recruitment of MSCs to damaged tissue sites or tumors, several bioactive molecules and organelles that are secreted by MSCs among others are cytokines, chemokines, metabolites, and extracellular vesicles including exosomes. The MSC-mediated cargo of released exosomes contains specific proteins and nucleic acids with varying regulatory microRNAs according to the tissue origin and the MSC microenvironment. These MSC-released exosomes are taken up by different target cells in damaged tissues to promote a regulatory network of tissue repair, including immune modulation and induction of angiogenesis. Conversely, in tumors, MSC-derived exosomes can confer predominant signals to suppress neovascularization and to relay further tumor-inhibitory effects. However, MSCs that adapted to the tumor tissue by mutual interaction with cancer cells progressively alter to an aberrant phenotype with the release of exosomes carrying tumor-supportive material.Exosomes derived from mesenchymal stroma-/stem-like cells (MSCs) as part of extracellular vesicles are considered cell-free biocompatible nanovesicles that promote repair activities of damaged tissues or organs by exhibiting low immunogenic and cytotoxic effects. Contributions to regenerative activities include wound healing, maintenance of stem cell niches, beneficial regenerative effects in various diseases, and reduction of senescence. However, the mode of action in MSC-derived exosomes strongly depends on the biological content like different regulatory microRNAs that are determined by the tissue origin of MSCs. In tumors, MSCs use indirect and direct pathways in a communication network to interact with cancer cells. This leads to mutual functional changes with the acquisition of an aberrant tumor-associated MSC phenotype accompanied by altered cargo in the exosomes. Consequently, MSC-derived exosomes either from normal tissue-originating MSCs or from aberrant tumor-associated MSCs can confer different actions on tumor development. These processes exhibiting tumor-inhibitory and tumor-supportive effects with a focus on exosome microRNA content will be discriminated and discussed within this review.

Cadmium affects cell niches maintenance in Arabidopsis thaliana post-embryonic shoot and root apical meristem by altering the expression of WUS/WOX homolog genes and cytokinin accumulation

Cadmium (Cd) is one of the most widespread polluting heavy metals in both terrestrial and aquatic environments and represents an extremely significant pollutant causing severe environmental and social problems due to its high toxicity and large solubility in water. In plants, the root is the first organ that get in contact with Cd. It is absorbed by the root system and translocated to the shoot and leaves through xylem loading, causing a variety of genetic, biochemical, and physiological damages. Cd inhibits both the root and shoot growth, but the mechanisms underlying this inhibition remain elusive. In this context in the present work we focused the attention on the effects of Cd on meristem size and organization of both shoot and root. To this aim morpho-histological and molecular analyses were carried out on 5 days old seedlings exposed or not to Cd (100 μM and 150 μM for 24) of wild type and transgenic lines expressing molecular markers with an important role in shoot and root pattern organization. More precisely, we monitored the expression pattern of WUS/CLV3 and WOX5 transcription factors involved in the establishment and maintenance of stem cell niche and the control of meristem size and of TCSn::GFP cytokinin-sensitive sensor as relevant components of hormone circuit controlling shoot and root growth. The results highlighted that the treatments with Cd impacts shoot and root size and shape by altering the paralogous WOX genes expression via cytokinin accumulation.

N,N-dimethyl-hexadecylamine modulates Arabidopsis root growth through modifying the balance between stem cell niche and jasmonic acid-dependent gene expression

N,N-dimethyl-hexadecylamine (DMHDA) is released as part of volatile blends emitted by plant probiotic bacteria and affects root architecture, defense and nutrition of plants. Here, we investigated the changes in gene expression of transcription factors responsible of maintenance of the root stem cell niche and jasmonic acid signaling in Arabidopsis seedlings in response to this volatile. Concentrations of DMHDA that repress primary root growth were found to alter cell size and division augmenting cell tissue layers in the meristem and causing root widening. DMHDA triggered the division of quiescent center cells, which correlated with repression of SHORT ROOT (SHR), SCARECROW (SCR), and PLETHORA 1 (PLT1) proteins and induction of WUSCHEL-RELATED HOMEOBOX 5 (WOX5) transcription factor. Interestingly, an activation of the expression of the jasmonic acid-related reporter genes JAZ1/TIFY10A-GFP and JAZ10pro::JAZ10-GFP suggests that the halted growth of the primary root inversely correlated with expression patterns underlying the defense reaction, which may be of adaptive importance to protect roots against biotic stress. Our data help to unravel the gene expression signatures upon sensing of a highly active bacterial volatile in Arabidopsis seedlings.

Mechanisms of stress response in the root stem cell niche.

As plants are sessile organisms unable to escape from environmental hazards, they need to adapt for survival. The stem cell niche in the root apical meristem is particularly sensitive to DNA damage induced by environmental stresses such as chilling, flooding, wounding, UV, and irradiation. DNA damage has been proven to cause stem cell death, with stele stem cells being the most vulnerable. Stress also induces the division of quiescent center cells. Both reactions disturb the structure and activity of the root stem cell niche temporarily; however, this preserves root meristem integrity and function in the long term. Plants have evolved many mechanisms that ensure stem cell niche maintenance, recovery, and acclimation, allowing them to survive in a changing environment. Here, we provide an overview of the cellular and molecular aspects of stress responses in the root stem cell niche.

Single-cell analysis of cell identity in the Arabidopsis root apical meristem: insights and opportunities

A fundamental question in developmental biology is how the progeny of stem cells become differentiated tissues. The Arabidopsis root is a tractable model to address this question due to its simple organization and defined cell lineages. In particular, the zone of dividing cells at the root tip-the root apical meristem-presents an opportunity to map the gene regulatory networks underlying stem cell niche maintenance, tissue patterning, and cell identity acquisition. To identify molecular regulators of these processes, studies over the last 20 years employed global profiling of gene expression patterns. However, these technologies are prone to information loss due to averaging gene expression signatures over multiple cell types and/or developmental stages. Recently developed high-throughput methods to profile gene expression at single-cell resolution have been successfully applied to plants. Here, we review insights from the first published single-cell mRNA sequencing and chromatin accessibility datasets generated from Arabidopsis roots. These studies successfully reconstruct developmental trajectories, phenotype cell identity mutants at unprecedented resolution, and reveal cell type-specific responses to environmental stimuli. The experimental insight gained from Arabidopsis paves the way to profile roots from additional species.

Current perspectives of limbal-derived stem cells and its application in ocular surface regeneration and limbal stem cell transplantation.

Limbal stem cells are involved in replenishing and maintaining the epithelium of the cornea. Damage to the limbus due to chemical/physical injury, infections, or genetic disorders leads to limbal stem cell deficiency (LSCD) with partial or total vision loss. Presently, LSCD is treated by transplanting limbal stem cells from the healthy eye of the recipient, living‐related, or cadaveric donors. This review discusses limbal‐derived stem cells, the importance of extracellular matrix in stem cell niche maintenance, the historical perspective of treating LSCD, including related advantages and limitations, and our experience of limbal stem cell transplantation over the decades.

Diversity and robustness of bone morphogenetic protein pattern formation.

ABSTRACTPattern formation by bone morphogenetic proteins (BMPs) demonstrates remarkable plasticity and utility in several contexts, such as early embryonic development, tissue patterning and the maintenance of stem cell niches. BMPs pattern tissues over many temporal and spatial scales: BMP gradients as short as 1-2 cell diameters maintain the stem cell niche of the Drosophila germarium over a 24-h cycle, and BMP gradients of several hundred microns establish dorsal-ventral tissue specification in Drosophila, zebrafish and Xenopus embryos in timescales between 30 min and several hours. The mechanisms that shape BMP signaling gradients are also incredibly diverse. Although ligand diffusion plays a dominant role in forming the gradient, a cast of diffusible and non-diffusible regulators modulate gradient formation and confer robustness, including scale invariance and adaptability to perturbations in gene expression and growth. In this Review, we document the diverse ways that BMP gradients are formed and refined, and we identify the core principles that they share to achieve reliable performance.

RAB GTPASE HOMOLOG8D is required for the maintenance of both the root stem cell niche and the meristem.

Previous studies have suggested that the plastid translation elongation factor, elongation factor thermo unstable (EF-Tu), encoded by RAB GTPASE HOMOLOG8D (RAB8D) is essential for plant growth. Here, through analyzing the root phenotypes of two knock-down alleles of RAB8D (rab8d-1 and rab8d-2), we further revealed a vital role for RAB8D in primary root development through the maintenance of both the stem cell niche (SCN) and the meristem. Our results showed that RAB8D deficiency affects the root auxin response and SCN maintenance signaling. RAB8D interacts with GENOMES UNCOUPLED1 (GUN1) invivo. Further analysis revealed that GUN1 is over-accumulated and is required for both stem cell death and maintenance of root architecture in rab8d Arabidopsis mutants. The ATAXIA-TELANGIECTASIA-MUTATED (ATM)-SUPPRESSOR OF GAMMA RESPONSE1 pathway is involved in the regulation of root meristem size through upregulating SIAMESE-RELATED5 expression in the rab8d-2 allele. Moreover, ETHYLENE RESPONSE FACTOR115 is highly expressed in rab8d-2, which plays a role in further quiescent center division. Our observations not only characterized the role of RAB8D in root development, but also uncovered functions of GUN1 and ATM in response to plastid EF-Tu deficiency.

A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes.

A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes.

Glucagon-like Peptide-2 (GLP-2) Treatment Reduces Gvhd-Related Mortality in a Pre-Clinical Transplant Model Via Expansion of Macrophages with Tolerogenic Potential

The gastrointestinal (GI) tract is a major target in GVHD. Conditioning-induced damage and mucosal barrier disruption are important factors in GVHD, however therapies targeting these processes have not been identified. Glucagon-like-peptide 2 (GLP-2) is an enterocyte-specific growth factor produced by L cells that has regenerative potential in models of GI damage. Its impact on the mucosal immune system has not been elucidated. We sought to examine the therapeutic and immunologic effect of GLP-2 in murine GVHD. We employed a major MHC-mismatched GVHD model (C57BL/6J → BALB/cJ). Mice were treated with 800nmol/kg/day of Elsiglutide (a GLP-2 analogue, provided by Helsinn) or vehicle beginning on D+1 for 30 days. Treatment with GLP-2 significantly improved survival and GVHD scores (Fig. 1A), while increasing small intestine mass and villi length (Fig 1B). GLP-2 also reduced T-cell infiltration into the jejunum (Fig. 1C). Analysis of intestinal immune cells by 28-color flow cytometry revealed dramatic differences between treatment groups in both myeloid- and T-cells. On D+14, GLP-2 led to an increased proportion of donor CSF-1R+ macrophages in the lamina propria (LP) (Fig. 2A) - cells that support the maintenance of the intestinal stem cell niche (Sehgal, Nat Commun, 2018). On D+21 the LP donor myeloid compartment was further altered, especially in MHC IIlow F4/80+ CD64+ macrophages (Fig. 2B, C). Here GLP-2 treatment expanded macrophages with lower expression of the co-stimulatory molecules CD80 and CD86 as well as the phagocytic marker CD206, whilst increasing the inhibitory molecule SIRPα, consistent with a tolerogenic phenotype. GLP-2 treatment also increased CX3CR1 expression on MHC IIlow macrophages with reduced Ly6C - a phenotype associated with physiologic macrophage maturation and linked to the resolution of colitis (Zigmond, Immunity, 2012). Vehicle-treated mice, conversely, had predominance of Ly6Chigh MHC IIlow LP macrophages reminiscent of an early infiltrating phenotype and near absence of mature macrophages, suggesting an impaired monocyte-macrophage transition that was restored by GLP-2. In addition, GLP-2 treatment led to significant changes in donor intraepithelial lymphocytes on D+21 (Fig. 2D), where CD8 T cells exhibited decreased CD27, CD103 and CXCR3 expression but higher PD-1, suggesting less activation. To assess potential mechanisms for the differences in macrophage and T-cell phenotype, we examined the impact of GLP-2 on the intestinal microbiota. A syngeneic BALB/cJ model was used to explore the effects of GLP-2 independent of GVHD. Stool samples from D+0, D+14, and D+28 were subjected to 16S rRNA sequencing. Vehicle-treated mice had distinct β-diversity clusters at all time-points, showing a transplant effect on the microbiota (Fig. 3A). GLP-2-treated mice had near-complete cluster overlap between D+0 and D+14, suggesting attenuation of the impact of conditioning. GLP-2 treated mice were significantly enriched for Akkermansia muciniphila and Bacteroidales S24-7 family at D+14 and D+28 (Fig. 3B). These taxa have been associated with anti-inflammatory properties and A. muciniphila abundance is linked to epithelial mucin production, which is increased by GLP-2. We then assessed the role of microbial communities in the protective effect of GLP-2 by conducting an allogeneic transplant with 3 caging conditions; 1) vehicle and GLP-2 treated mice caged together, 2) caged separately, or 3) caged separately plus oral antibiotics. We observed a clear cage effect where co-housing the treatment groups improved the survival of vehicle treated mice (Fig. 3C), suggesting transferal of the therapeutic effect via the microbiome. Antibiotic administration also dampened the beneficial effect of GLP-2. Finally, we conducted a GvL experiment by co-transplanting Luc-A20 and monitoring tumor progression via bioluminescence imaging. Both GLP-2 and vehicle-treated mice eliminated the tumor, whereas mice receiving T-cell depleted bone marrow showed tumor progression (Fig. 3D). In summary, our results demonstrate high therapeutic potential of GLP-2 in GVHD. GLP-2 administration led to reduced mortality, modified the microbiome and altered the intestinal immune response to a more tolerogenic state. This novel mechanism sheds light on the role of the enteroendocrine system in maintaining gut homeostasis and sets the stage for therapeutic clinical trials. Figure 1 Disclosures Uhlemann: Allergan: Research Funding; GSK: Research Funding; Merck: Research Funding. Reshef:Gilead: Consultancy; Magenta: Consultancy; Novartis: Consultancy; Monsato: Consultancy; Atara: Consultancy; BMS: Consultancy.

Isolation and enrichment of melanocytes from human corneal limbus using CD117 (c-Kit) as selection marker

Limbal melanocytes (LM) are located in the basal epithelial layer of the corneoscleral limbus and interact with adjacent limbal epithelial progenitor cells. The exploration of their biological role in the maintenance of the limbal stem cell niche has been limited by the difficulty of LM isolation and cultivation. Here, we report on a facile protocol for the efficient isolation and enrichment of pure populations of human LMs by fluorescence-activated cell sorting (FACS) using antibodies raised against the cell surface marker CD117 (c-Kit). The enriched LMs retain self-renewal capacity and sustained melanin production, and are suitable to study the potential of LMs in stem cell-based corneal tissue engineering.

RGF1-RGI1, a Peptide-Receptor Complex, Regulates Arabidopsis Root Meristem Development via a MAPK Signaling Cascade

Root growth is maintained by the continuous division of cells in the apical meristem. ROOT MERISTEM GROWTH FACTOR 1 (RGF1) is a critical peptide hormone regulating root stem cell niche maintenance. Previous studies discovered that five closely related leucine-rich repeat receptor-like protein kinases (LRR-RLKs), named RGF1 INSENSITIVES (RGIs) or RGF1 RECEPTORS (RGFRs), are able to perceive the RGF1 signal and redundantly control root stem cell niche maintenance. RGF1 regulates root meristem activity mainly via two downstream transcription factors, PLETHORA 1 (PLT1) and PLT2. Regulatory proteins connecting cell surface RGF1-RGI1 and nuclear PLTs, however, were not identified. Here, we report that the mitogen-activated protein (MAP) kinase kinase 4 (MKK4) and MAP kinase 3 (MPK3) were co-immunoprecipitated with RGI1-FLAG after Arabidopsis seedlings were treated with RGF1. Genetic and biochemical assays confirmed that MKK4 and MKK5, and their downstream targets MPK3 and MPK6, are essential RGI-dependent regulators of root meristem development. In addition, we found that the MKK4/MKK5-MPK3/MPK6 module functions downstream of YDA, a MAPKKK. Our results demonstrate that RGF1-RGI1 regulate the expression of PLT1/PLT2 via a YDA-MKK4/MKK5-MPK3/MPK6 signaling cascade.

Abstract 17: Ex-vivo assessment of the effect of Simvastatin in intestinal epithelium of Lynch Syndrome patients

Abstract Background: Lynch Syndrome (LS) is a hereditary condition secondary to germline mutations in DNA mismatch repair (MMR) genes, which predisposes patients to develop different tumor types but mainly colorectal and endometrial cancers. Due to the genetic predisposition, chemopreventive strategies become critical in these individuals. One such chemopreventive compound, statin, a potent pharmacologic inhibitor of cholesterol biosynthesis, is known to exert other pleotropic effects such as modulation of cell proliferation, apoptosis, and inflammation, mainly due to inhibition of the mevalonate pathway. Although, chemoprevention studies in the past were performed in cancer stem cell models the effects have not been previously assessed in the physiologic and MMR-deficient stem cell niche. Here, we have successfully established ex-vivo cultures of crypt-villus structures (termed ‘mini-guts' or organoids) in order to interrogate the chemopreventive effects of statins in Lynch Syndrome patients. Methods: Fresh endoscopic biopsy samples from a total of six LS patients followed at The University of Texas MD Anderson Cancer Center were obtained and ex-vivo organoid cultures were established. Each organoid line (passage < P6) was treated with two different doses of Simvastatin (5 and 10 µM) or DMSO (control) for 48 hours. Total RNA was isolated from organoids treated with control and Simvastatin 10 µM and RNASeq analysis was performed using Illumina HiSeq 4000. Validation of several critical genes was performed by qRT-PCR assay. Results: RNAseq analysis accounted for a total of 4,386 genes dysregulated in organoids treated with Simvastatin as compared to control. Stem cell markers, LGR5 and ASCL2, and differentiation markers, KRT20 (enterocytes) and MUC2 (goblet), were found to be induced by simvastatin. Gene Set Enrichment Analysis (GSEA) using Hallmark pathway gene sets showed significant enrichment for E2F target genes apart from canonical cholesterol homeostasis pathway. Ingenuity pathways analysis (IPA) revealed a list of top 10 genes which are upregulated/downregulated and implicated in cancer pathway. Validation of selected E2F Hallmark pathway genes (E2F1, TOP2A, Survivin, and KI-67) and IPA cancer pathway genes (KLF2, KLF6, EZH2, p57, and p21) showed a significant and dose dependent modulation upon treatment with Simvastatin. Conclusions: Treatment with Simvastatin exhibited a profound effect on key genes involved in maintenance of stem cell niche in patient-derived organoids from LS. Simvastatin had a significant effect on E2F target genes implicated in regulation of cell cycle and proliferation via modulation of Ki-67, TOP2A, and Survivin. Along with increase in cyclin-dependent kinase inhibitor p21, there was significant induction of p57, a candidate tumor suppressor gene. Hence, Simvastatin, while it maintains the stem niche, exerts its anti-proliferative effects by specifically modulating genes involved in cell cycle and apoptosis. Citation Format: Prashant Bommi, Wenhui Wu, Laura Reyes, Kyera Evans, Selvi Thirumurthi, Patrick Lynch, Krishna Sinha, Eduardo Vilar. Ex-vivo assessment of the effect of Simvastatin in intestinal epithelium of Lynch Syndrome patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 17.

Abstract 6084: Wave3 tyrosine phosphorylation activates the invasion-metastasis cascade of triple negative breast cancer tumors through the maintenance of the cancer stem cell niche

Abstract The acquisition of metastatic phenotypes is responsible for the death of ~90% of breast cancer (BC) patients. In fact, metastatic BC is the 2nd leading cause of cancer-related deaths in women in the United States, annually accounting for more than 40,100 deaths and 266,000 new cases of invasive BC. Typically, metastases are incurable and result in a median survival of only 1.5 to 3 years for BC patients. Clinically, ~30% of BC patients diagnosed with early-stage, noninvasive disease will ultimately progress to late-stage, metastatic disease, an event that severely limits treatment options and associates with dismal clinical outcomes. This problem is exacerbated by the fact the BCs are heterogeneous and comprised of at least 5 genetically distinct subtypes. Amongst individual BC subtypes, those classified as TNBCs are especially lethal due to their highly metastatic behavior and propensity to recur rapidly. As a group, TNBCs lack expression of hormone receptors (ER-α and PR) and ErbB2/HER2, which has prevented the development of FDA-approved targeted drug therapies effective against this BC subtype. Likewise, recurrent TNBCs frequently acquire resistance to standard-of-care chemotherapeutic agents (e.g., doxorubicin, cyclophosphamides, and taxanes) through mechanisms that remain incompletely understood. We established Wiskott-Aldrich Syndrome Protein-3 (WAVE3) as a novel promoter of TNBC development and metastatic progression. Our previously published studies have shown that phosphorylation of WAVE3 at specific tyrosine residues by c-Abl non-receptor tyrosine kinase is required for cancer cell migration and invasion. In this study, we show that WAVE3 phosphorylation is required for the activation of the invasion-metastasis cascade of TNBCs, both in vitro and in in vivo mouse models. Genetic inactivation of WAVE3 via CRISPR/Cas9 gene editing suppressed migration, invasion and the formation of 3D tumorspheres of TNBC cells, in vitro, as well as tumor growth and metastasis in vivo. While re-expression of phospho-active WAVE3 in the WAVE3-deficient TNBC cells restored the oncogenic activity of WAVE3, re-expression of phospho-mutant WAVE3 or the pharmacologic inhibition of WAVE3 phosphorylation did not. Further studies revealed that phosphorylation of WAVE3 was required for the activation of several hallmarks of cancer through the upregulation of survival signaling pathways. Blocking WAVE3 expression or phosphorylation inhibited epithelial-mesenchymal transition (EMT) as well as disrupted the breast cancer stem cell (BCSC) niche by inhibiting expression of BCSC-specific markers. Finally, loss of WAVE3 phosphorylation was sufficient to resensitize TNBC cells to standard of care chemotherapy-induced apoptosis and cell death through the targeting of the BCSCs. Together, our data support the targeting of WAVE3 as a novel therapeutic strategy to treat metastasis in patients with TNBC tumors by specifically targeting BCSCs. Citation Format: Wei Wang, Urna Kansakar, Vesna Markovic, Khalid Sossey-Alaoui. Wave3 tyrosine phosphorylation activates the invasion-metastasis cascade of triple negative breast cancer tumors through the maintenance of the cancer stem cell niche [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6084.

R-Spondin 1 (RSPO1) Increases Mouse Intestinal Organoid Unit Size and Survival in vitro and Improves Tissue-Engineered Small Intestine Formation in vivo.

Introduction: Cell therapy and tissue engineering has recently emerged as a new option for short bowel syndrome (SBS) treatment, generating tissue engineered small intestine (TESI) from organoid units (OU) and biodegradable scaffolds. The recombinant human R-Spondin 1 (rhRSPO1) protein may be a key player in this process due to its mitogenic activity in intestinal stem cells.Objective: Aiming at optimizing the TESI formation process and advancing this technology closer to the clinic, we evaluated the effects of rhRSPO1 protein on OU culture and TESI formation.Methods: Intestinal OU were isolated from C57BL/6 mice and cultured in Matrigel in the presence or absence of recombinant human rhRSPO1. Throughout the culture, OU growth and survival rates were evaluated, and cells were harvested on day 3. OU were seeded onto biodegradable scaffolds, in the presence or absence of 5 μg of rhRSPO1 and implanted into the omentum of NOD/SCID mice in order to generate TESI. The explants were harvested after 30 days, weighed, fixed in formalin and embedded in paraffin for histological analysis and immunofluorescence for different cell markers.Results: After 3 days, rhRSPO1-treated OU attained a larger size, when compared to the control group, becoming 5.7 times larger on day 6. Increased survival was observed from the second day in culture, with a 2-fold increase in OU survival between days 3 and 6. A 4.8-fold increase of non-phosphorylated β-catenin and increased relative expression of Lgr5 mRNA in the rhRSPO1-treated group confirms activation of the canonical Wnt pathway and suggests maintenance of the OU stem cell niche and associated stemness. After 30 days of in vivo maturation, rhRSPO1-treated TESI presented a larger mass than constructs treated with saline, developing a more mature intestinal epithelium with well-formed villi and crypts. In addition, the efficiency of OU-loaded rhRSPO1-treated scaffolds significantly increased, forming TESI in 100% of the samples (N = 8), of which 40% presented maximum degree of development, as compared to 66.6% in the control group (N = 9).Conclusion: rhRSPO1 treatment improves the culture of mouse intestinal OU, increasing its size and survival in vitro, and TESI formation in vivo, increasing its mass, degree of development and engraftment.

Gasdermin A3–Mediated Cell Death Causes Niche Collapse and Precocious Activation of Hair Follicle Stem Cells

Hair follicles undergo recurrent growth, regression, and resting phases throughout postnatal life, which is supported by hair follicle stem cells. The niche components of hair follicle stem cells are important to maintain their quiescence and stemness. Gsdma3 gain-of-function mutations in mice cause chronic skin inflammation, aberrant hair cycle, and progressive hair loss, reminiscent of scarring alopecia in humans. However, the mechanism underlying these defects remains elusive. Here, we used a combined Cre/loxP and rtTA/TRE system to study the spatiotemporal effect of Gsdma3 overexpression on distinct hair cycle stages. We found that Gsdma3-mediated cell death affects anagen initiation, anagen progression, and catagen-telogen transition. Induced Gsdma3 expression causes bulge inner layer collapse and precocious hair follicle stem cell activation, leading to subsequent hair follicle degeneration. Although macrophages and dendritic cells are recruited to the bulge region, invivo depletion of these cells using a neutralizing antibody does not alleviate cell death in the bulge or hair germ, indicating that macrophages are less likely to cause immediate hair follicle deletion. Our data suggest that dysregulated Gsdma3 causes bulge inner layer necrosis to induce club hair shedding and immediate anagen reentry without going through telogen morphology, which implicates a role for Gsdma3 in hair follicle stem cell niche maintenance.

Intersected functional zone of transcriptional regulators patterns stemness within stem cell niche of root apical meristem.

Root stem cell niche (SCN) consists of a quiescent center (QC) and surrounding stem cells. Disrupted symplastic communication leads to loss of stemness in the whole SCN. Several SCN regulators were reported to move between cells for SCN maintenance. However, single mutant of these regulators is insufficient to abolish QC stemness despite the high differentiation rate in surrounding stem cells. To dissect the mechanism behind such distinct stemness in SCN, we combined the mis-expression strategy with pWOX5:icals3m system in which QC is symplastically isolated. We found the starch accumulation in QC could be synergistically repressed by WUSCHEL-RELATED HOMEOBOX 5 (WOX5), SHORT-ROOT (SHR), SCARCROW (SCR), and PLETHORA (PLT). Like PLTs, other core regulators also exhibited dimorphic functions by inhibiting differentiation at a higher dose while promoting cell division at a low protein level. Being located in the center of the intersected expression zones, QC cells receive the highest level of core regulators, forming the most robust stemness within SCN. WUSCHEL-RELATED HOMEOBOX 5 was sufficient to activate PLT1/2 expression, contributing to the QC-enriched PLTs. Our results provide experimental evidence supporting the long-standing hypothesis that the combination of spatial expression, synergistic function and dosage effect of core regulators result in spatially distinct stemness in SCN.