ABSTRACT
Following tissue damage, epithelial stem cells (SCs) are mobilized to enter the wound, where they confront harsh inflammatory environments that can impede their ability to repair the injury. Here, we investigated the mechanisms that protect skin SCs within this inflammatory environment. Characterization of gene expression profiles of hair follicle SCs (HFSCs) that migrated into the wound site revealed activation of an immune-modulatory program, including expression of CD80, major histocompatibility complex class II (MHCII), and CXC motif chemokine ligand 5 (CXCL5). Deletion of CD80 in HFSCs impaired re-epithelialization, reduced accumulation of peripherally generated Treg (pTreg) cells, and increased infiltration of neutrophils in wounded skin. Importantly, similar wound healing defects were also observed in mice lacking pTreg cells. Our findings suggest that upon skin injury, HFSCs establish a temporary protective network by promoting local expansion of Treg cells, thereby enabling re-epithelialization while still kindling inflammation outside this niche until the barrier is restored.
Subject(s)
B7-1 Antigen , Hair Follicle , Inflammation , Skin , Stem Cells , T-Lymphocytes, Regulatory , Wound Healing , Animals , T-Lymphocytes, Regulatory/immunology , Mice , Wound Healing/immunology , Skin/immunology , Skin/injuries , Skin/pathology , Stem Cells/immunology , Stem Cells/metabolism , Inflammation/immunology , Hair Follicle/immunology , B7-1 Antigen/metabolism , Mice, Inbred C57BL , Mice, Knockout , Re-Epithelialization/immunology , Cell Movement/immunology , Cell ProliferationABSTRACT
During development, progenitors simultaneously activate one lineage while silencing another, a feature highly regulated in adult stem cells but derailed in cancers. Equipped to bind cognate motifs in closed chromatin, pioneer factors operate at these crossroads, but how they perform fate switching remains elusive. Here we tackle this question with SOX9, a master regulator that diverts embryonic epidermal stem cells (EpdSCs) into becoming hair follicle stem cells. By engineering mice to re-activate SOX9 in adult EpdSCs, we trigger fate switching. Combining epigenetic, proteomic and functional analyses, we interrogate the ensuing chromatin and transcriptional dynamics, slowed temporally by the mature EpdSC niche microenvironment. We show that as SOX9 binds and opens key hair follicle enhancers de novo in EpdSCs, it simultaneously recruits co-factors away from epidermal enhancers, which are silenced. Unhinged from its normal regulation, sustained SOX9 subsequently activates oncogenic transcriptional regulators that chart the path to cancers typified by constitutive SOX9 expression.
Subject(s)
Adult Stem Cells , Proteomics , Animals , Mice , Adult Stem Cells/metabolism , Cell Differentiation , Chromatin/genetics , Epigenesis, Genetic , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolismABSTRACT
Macrophages and dendritic cells have long been appreciated for their ability to migrate to and engulf dying cells and debris, including some of the billions of cells that are naturally eliminated from our body daily. However, a substantial number of these dying cells are cleared by 'non-professional phagocytes', local epithelial cells that are critical to organismal fitness. How non-professional phagocytes sense and digest nearby apoptotic corpses while still performing their normal tissue functions is unclear. Here, we explore the molecular mechanisms underlying their multifunctionality. Exploiting the cyclical bouts of tissue regeneration and degeneration during the hair cycle, we show that stem cells can transiently become non-professional phagocytes when confronted with dying cells. Adoption of this phagocytic state requires both local lipids produced by apoptotic corpses to activate RXRα, and tissue-specific retinoids for RARγ activation. This dual factor dependency enables tight regulation of the genes requisite to activate phagocytic apoptotic clearance. The tunable phagocytic program we describe here offers an effective mechanism to offset phagocytic duties against the primary stem cell function of replenishing differentiated cells to preserve tissue integrity during homeostasis. Our findings have broad implications for other non-motile stem or progenitor cells which experience cell death in an immune-privileged niche.
ABSTRACT
Immune and tissue stem cells retain an epigenetic memory of inflammation that intensifies sensitivity to future encounters. We investigated whether and to what consequence stem cells possess and accumulate memories of diverse experiences. Monitoring a choreographed response to wounds, we found that as hair follicle stem cells leave their niche, migrate to repair damaged epidermis, and take up long-term foreign residence there, they accumulate long-lasting epigenetic memories of each experience, culminating in post-repair epigenetic adaptations that sustain the epidermal transcriptional program and surface barrier. Each memory is distinct, separable, and has its own physiological impact, collectively endowing these stem cells with heightened regenerative ability to heal wounds and broadening their tissue-regenerating tasks relative to their naïve counterparts.
Subject(s)
Epidermal Cells/cytology , Epigenesis, Genetic , Hair Follicle/cytology , Stem Cells/physiology , Adaptation, Physiological , Animals , Cell Movement , Chromatin/metabolism , Epidermal Cells/physiology , Homeostasis , Inflammation , Mice , Regeneration , Stem Cell Niche , Transcriptome , Wound HealingABSTRACT
Through recurrent bouts synchronous with the hair cycle, quiescent melanocyte stem cells (McSCs) become activated to generate proliferative progeny that differentiate into pigment-producing melanocytes. The signaling factors orchestrating these events remain incompletely understood. Here, we use single-cell RNA sequencing with comparative gene expression analysis to elucidate the transcriptional dynamics of McSCs through quiescence, activation, and melanocyte maturation. Unearthing converging signs of increased WNT and BMP signaling along this progression, we endeavored to understand how these pathways are integrated. Employing conditional lineage-specific genetic ablation studies in mice, we found that loss of BMP signaling in the lineage leads to hair graying due to a block in melanocyte maturation. We show that interestingly, BMP signaling functions downstream from activated McSCs and maintains WNT effector, transcription factor LEF1. Employing pseudotime analysis, genetics, and chromatin landscaping, we show that following WNT-mediated activation of McSCs, BMP and WNT pathways collaborate to trigger the commitment of proliferative progeny by fueling LEF1- and MITF-dependent differentiation. Our findings shed light upon the signaling interplay and timing of cues that orchestrate melanocyte lineage progression in the hair follicle and underscore a key role for BMP signaling in driving complete differentiation.
Subject(s)
Bone Morphogenetic Proteins/physiology , Cell Differentiation/genetics , Melanocytes/cytology , Signal Transduction/genetics , Stem Cells/cytology , Animals , Cell Lineage/genetics , Gene Expression Profiling , Lymphoid Enhancer-Binding Factor 1/metabolism , Mice , Microphthalmia-Associated Transcription Factor/metabolism , Single-Cell AnalysisABSTRACT
Tissue homeostasis and regeneration rely on resident stem cells (SCs), whose behaviour is regulated through niche-dependent crosstalk. The mechanisms underlying SC identity are still unfolding. Here, using spatiotemporal gene ablation in murine hair follicles, we uncover a critical role for the transcription factors (TFs) nuclear factor IB (NFIB) and IX (NFIX) in maintaining SC identity. Without NFI TFs, SCs lose their hair-regenerating capability, and produce skin bearing striking resemblance to irreversible human alopecia, which also displays reduced NFIs. Through single-cell transcriptomics, ATAC-Seq and ChIP-Seq profiling, we expose a key role for NFIB and NFIX in governing super-enhancer maintenance of the key hair follicle SC-specific TF genes. When NFIB and NFIX are genetically removed, the stemness epigenetic landscape is lost. Super-enhancers driving SC identity are decommissioned, while unwanted lineages are de-repressed ectopically. Together, our findings expose NFIB and NFIX as crucial rheostats of tissue homeostasis, functioning to safeguard the SC epigenome from a breach in lineage confinement that otherwise triggers irreversible tissue degeneration.
Subject(s)
Alopecia/pathology , Cell Differentiation , Chromatin/metabolism , Hair Follicle/cytology , NFI Transcription Factors/physiology , Stem Cells/cytology , Alopecia/genetics , Alopecia/metabolism , Animals , Cells, Cultured , Chromatin/genetics , Female , Hair Follicle/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Regeneration , Stem Cells/metabolismABSTRACT
Sharma investigates vesicular trafficking to lysosomes and how pathogens hijack the endolysosomal system during infection.
Subject(s)
Cell Biology/history , Endosomes/metabolism , Lysosomes/metabolism , Biological Transport, Active , History, 21st Century , Humans , Portraits as TopicABSTRACT
Wang studies lysosomal degradation pathways using C. elegans as a model system.
Subject(s)
Autophagy , Caenorhabditis elegans Proteins , Caenorhabditis elegans , Cell Biology/history , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Female , History, 21st Century , Humans , Portraits as TopicABSTRACT
Mierzwa studies mechanisms of cell division in different cell types and tissue contexts.
Subject(s)
Art/history , Cell Biology/history , History, 20th Century , History, 21st Century , Portraits as TopicABSTRACT
McLaughlin studies how neurons respond to acute and chronic stress.
Subject(s)
Neuroprotection , Neurosciences/history , Stress, Physiological , Women, Working/history , Female , History, 20th Century , History, 21st Century , Humans , Neurons , Portraits as TopicABSTRACT
Davis uses microscopy and imaging approaches to study immune cell interactions.
Subject(s)
Allergy and Immunology , Killer Cells, Natural , History, 21st Century , HumansABSTRACT
Chipuk studies the interaction between mitochondrial dynamics and cell death in cancer.
Subject(s)
Mitochondria , Humans , New YorkABSTRACT
Auwerx studies the signaling networks that underlie mitochondrial function and metabolism.
Subject(s)
Translational Research, Biomedical , Animals , History, 20th Century , History, 21st Century , HumansABSTRACT
Purpose: Anaplastic lymphoma kinase (ALK) is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an in vitro screen for synergistic drug combinations that target neuroblastomas with mutations in ALK to determine whether drug combinations could enhance antitumor efficacy.Experimental Design: We screened combinations of eight molecularly targeted agents against 17 comprehensively characterized human neuroblastoma-derived cell lines. We investigated the combination of ceritinib and ribociclib on in vitro proliferation, cell cycle, viability, caspase activation, and the cyclin D/CDK4/CDK6/RB and pALK signaling networks in cell lines with representative ALK status. We performed in vivo trials in CB17 SCID mice bearing conventional and patient-derived xenograft models comparing ceritinib alone, ribociclib alone, and the combination, with plasma pharmacokinetics to evaluate for drug-drug interactions.Results: The combination of ribociclib, a dual inhibitor of cyclin-dependent kinase (CDK) 4 and 6, and the ALK inhibitor ceritinib demonstrated higher cytotoxicity (P = 0.008) and synergy scores (P = 0.006) in cell lines with ALK mutations as compared with cell lines lacking mutations or alterations in ALK Compared with either drug alone, combination therapy enhanced growth inhibition, cell-cycle arrest, and caspase-independent cell death. Combination therapy achieved complete regressions in neuroblastoma xenografts with ALK-F1174L and F1245C de novo resistance mutations and prevented the emergence of resistance. Murine ribociclib and ceritinib plasma concentrations were unaltered by combination therapy.Conclusions: This preclinical combination drug screen with in vivo validation has provided the rationale for a first-in-children trial of combination ceritinib and ribociclib in a molecularly selected pediatric population. Clin Cancer Res; 23(11); 2856-68. ©2016 AACR.
Subject(s)
Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Neuroblastoma/drug therapy , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Aminopyridines/administration & dosage , Anaplastic Lymphoma Kinase , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D/genetics , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 6/genetics , Drug Synergism , Humans , Mice , Mutation , Neuroblastoma/genetics , Neuroblastoma/pathology , Purines/administration & dosage , Pyrimidines/administration & dosage , Receptor Protein-Tyrosine Kinases/genetics , Retinoblastoma Protein/genetics , Signal Transduction/drug effects , Small Molecule Libraries/administration & dosage , Sulfones/administration & dosage , Xenograft Model Antitumor AssaysABSTRACT
UNLABELLED: Neuroblastomas harboring activating point mutations in anaplastic lymphoma kinase (ALK) are differentially sensitive to the ALK inhibitor crizotinib, with certain mutations conferring intrinsic crizotinib resistance. To overcome this clinical obstacle, our goal was to identify inhibitors with improved potency that can target intractable ALK variants such as F1174L. We find that PF-06463922 has high potency across ALK variants and inhibits ALK more effectively than crizotinib in vitro. Most importantly, PF-06463922 induces complete tumor regression in both crizotinib-resistant and crizotinib-sensitive xenograft mouse models of neuroblastoma, as well as in patient-derived xenografts harboring the crizotinib-resistant F1174L or F1245C mutations. These studies demonstrate that PF-06463922 has the potential to overcome crizotinib resistance and exerts unprecedented activity as a single targeted agent against F1174L and F1245C ALK-mutated xenograft tumors, while also inducing responses in an R1275Q xenograft model. Taken together, these results provide the rationale to move PF-06463922 into clinical trials for treatment of patients with ALK-mutated neuroblastoma. SIGNIFICANCE: The next-generation ALK/ROS1 inhibitor PF-06463922 exerts unparalleled activity in ALK-driven neuroblastoma models with primary crizotinib resistance. Our biochemical and in vivo data provide the preclinical rationale for fast-tracking the development of this agent in children with relapsed/refractory ALK-mutant neuroblastoma.
Subject(s)
Drug Resistance, Neoplasm/drug effects , Lactams, Macrocyclic/administration & dosage , Neuroblastoma/drug therapy , Protein Kinase Inhibitors/administration & dosage , Receptor Protein-Tyrosine Kinases/genetics , Aminopyridines , Anaplastic Lymphoma Kinase , Animals , Cell Line, Tumor , Crizotinib , Humans , Lactams , Lactams, Macrocyclic/pharmacology , Mice , Mutation , Neuroblastoma/genetics , Neuroblastoma/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Pyrazoles/administration & dosage , Pyrazoles/pharmacology , Pyridines/administration & dosage , Pyridines/pharmacology , Receptor Protein-Tyrosine Kinases/metabolism , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: The presence of an ALK aberration correlates with inferior survival for patients with high-risk neuroblastoma. The emergence of ALK inhibitors such as crizotinib has provided novel treatment opportunities. However, certain ALK mutations result in de novo crizotinib resistance, and a phase I trial of crizotinib showed a lack of response in patients harboring those ALK mutations. Thus, understanding mechanisms of resistance and defining circumvention strategies for the clinic is critical. EXPERIMENTAL DESIGN: The sensitivity of human neuroblastoma-derived cell lines, cell line-derived, and patient-derived xenograft (PDX) models with varying ALK statuses to crizotinib combined with topotecan and cyclophosphamide (topo/cyclo) was examined. Cultured cells and xenografts were evaluated for effects of these drugs on proliferation, signaling, and cell death, and assessment of synergy. RESULTS: In neuroblastoma murine xenografts harboring the most common ALK mutations, including those mutations associated with resistance to crizotinib (but not in those with wild-type ALK), crizotinib combined with topo/cyclo enhanced tumor responses and mouse event-free survival. Crizotinib + topo/cyclo showed synergistic cytotoxicity and higher caspase-dependent apoptosis than crizotinib or topo/cyclo alone in neuroblastoma cell lines with ALK aberrations (mutation or amplification). CONCLUSIONS: Combining crizotinib with chemotherapeutic agents commonly used in treating newly diagnosed patients with high-risk neuroblastoma restores sensitivity in preclinical models harboring both sensitive ALK aberrations and de novo-resistant ALK mutations. These data support clinical testing of crizotinib and conventional chemotherapy with the goal of integrating ALK inhibition into multiagent therapy for ALK-aberrant neuroblastoma patients.