Quiescence Exit Research Articles

Modelling quiescence exit of neural stem cells reveals a FOXG1-FOXO6 axis.

The molecular mechanisms controlling the balance of quiescence and proliferation in adult neural stem cells (NSCs) are often deregulated in brain cancers such as glioblastoma multiforme (GBM). Previously, we reported that FOXG1, a forebrain-restricted neurodevelopmental transcription factor, is frequently upregulated in glioblastoma stem cells (GSCs) and limits the effects of cytostatic pathways, in part by repression of the tumour suppressor Foxo3. Here, we show that increased FOXG1 upregulates Foxo6, a more recently discovered FOXO family member with potential oncogenic functions. Although genetic ablation of Foxo6 in proliferating NSCs had no effect on the cell cycle or entry into quiescence, we found that Foxo6-null NSCs could no longer efficiently exit quiescence following FOXG1 elevation. Increased Foxo6 resulted in the formation of large acidic vacuoles, reminiscent of Pak1-regulated macropinocytosis. Consistently, Pak1 expression was upregulated by FOXG1 overexpression and downregulated upon FOXO6 loss in proliferative NSCs. These data suggest a pro-oncogenic role for FOXO6, downstream of GBM-associated elevated FOXG1, in controlling quiescence exit, and shed light on the potential functions of this underexplored FOXO family member.

CDKL3 is a targetable regulator of cell cycle progression in cancers.

Cell cycle regulation is largely abnormal in cancers. Molecular understanding and therapeutic targeting of the aberrant cell cycle are essential. Here, we identified that an underappreciated serine/threonine kinase, cyclin-dependent kinase-like 3 (CDKL3), crucially drives rapid cell cycle progression and cell growth in cancers. With regard to mechanism, CDKL3 localizes in the nucleus and associates with specific cyclin to directly phosphorylate retinoblastoma (Rb) for quiescence exit. In parallel, CDKL3 prevents the ubiquitin-proteasomal degradation of cyclin-dependent kinase 4 (CDK4) by direct phosphorylation on T172 to sustain G1 phase advancement. The crucial function of CDKL3 in cancers was demonstrated both in vitro and in vivo. We also designed, synthesized, and characterized a first-in-class CDKL3-specific inhibitor, HZ1. HZ1 exhibits greater potency than CDK4/6 inhibitor in pan-cancer treatment by causing cell cycle arrest and overcomes acquired resistance to CDK4/6 inhibitor. In particular, CDKL3 has significant clinical relevance in colon cancer, and the effectiveness of HZ1 was demonstrated by murine and patient-derived cancer models. Collectively, this work presents an integrated paradigm of cancer cell cycle regulation and suggests CDKL3 targeting as a feasible approach in cancer treatment.

Mecp2 fine-tunes quiescence exit by targeting nuclear receptors

Quiescence (G0) maintenance and exit are crucial for tissue homeostasis and regeneration in mammals. Here, we show that methyl-CpG binding protein 2 (Mecp2) expression is cell cycle-dependent and negatively regulates quiescence exit in cultured cells and in an injury-induced liver regeneration mouse model. Specifically, acute reduction of Mecp2 is required for efficient quiescence exit as deletion of Mecp2 accelerates, while overexpression of Mecp2 delays quiescence exit, and forced expression of Mecp2 after Mecp2 conditional knockout rescues cell cycle reentry. The E3 ligase Nedd4 mediates the ubiquitination and degradation of Mecp2, and thus facilitates quiescence exit. A genome-wide study uncovered the dual role of Mecp2 in preventing quiescence exit by transcriptionally activating metabolic genes while repressing proliferation-associated genes. Particularly disruption of two nuclear receptors, Rara or Nr1h3, accelerates quiescence exit, mimicking the Mecp2 depletion phenotype. Our studies unravel a previously unrecognized role for Mecp2 as an essential regulator of quiescence exit and tissue regeneration.

Mecp2 fine-tunes quiescence exit by targeting nuclear receptors.

Quiescence (G0) maintenance and exit are crucial for tissue homeostasis and regeneration in mammals. Here, we show that methyl-CpG binding protein 2 (Mecp2) expression is cell cycle-dependent and negatively regulates quiescence exit in cultured cells and in an injury-induced liver regeneration mouse model. Specifically, acute reduction of Mecp2 is required for efficient quiescence exit as deletion of Mecp2 accelerates, while overexpression of Mecp2 delays quiescence exit, and forced expression of Mecp2 after Mecp2 conditional knockout rescues cell cycle reentry. The E3 ligase Nedd4 mediates the ubiquitination and degradation of Mecp2, and thus facilitates quiescence exit. A genome-wide study uncovered the dual role of Mecp2 in preventing quiescence exit by transcriptionally activating metabolic genes while repressing proliferation-associated genes. Particularly disruption of two nuclear receptors, Rara or Nr1h3, accelerates quiescence exit, mimicking the Mecp2 depletion phenotype. Our studies unravel a previously unrecognized role for Mecp2 as an essential regulator of quiescence exit and tissue regeneration.

Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of biological consequences beyond the classic DNA damage, we treated 9 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 5 PDOX models (P < 0.05) accompanied by γH2AX positivity in >95 % tumor cells in tumor core and >85 % in the invasive foci as well as ∼30 % apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species accumulation, were detected together with the enrichment of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies.

262: Radiation induces Mitochondrial Biogenesis and Cellular Quiescence Exit in PDOX models of pHGG

262: Radiation induces Mitochondrial Biogenesis and Cellular Quiescence Exit in PDOX models of pHGG

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence.

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensitiesand fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.

A stable microtubule bundle formed through an orchestrated multistep process controls quiescence exit

Cells fine-tune microtubule assembly in both space and time to give rise to distinct edifices with specific cellular functions. In proliferating cells, microtubules are highly dynamics, and proliferation cessation often leads to their stabilization. One of the most stable microtubule structures identified to date is the nuclear bundle assembled in quiescent yeast. In this article, we characterize the original multistep process driving the assembly of this structure. This Aurora B-dependent mechanism follows a precise temporality that relies on the sequential actions of kinesin-14, kinesin-5, and involves both microtubule–kinetochore and kinetochore–kinetochore interactions. Upon quiescence exit, the microtubule bundle is disassembled via a cooperative process involving kinesin-8 and its full disassembly is required prior to cells re-entry into proliferation. Overall, our study provides the first description, at the molecular scale, of the entire life cycle of a stable microtubule structure in vivo and sheds light on its physiological function.

A stable microtubule bundle formed through an orchestrated multistep process controls quiescence exit.

Cells fine-tune microtubule assembly in both space and time to give rise to distinct edifices with specific cellular functions. In proliferating cells, microtubules are highly dynamics, and proliferation cessation often leads to their stabilization. One of the most stable microtubule structures identified to date is the nuclear bundle assembled in quiescent yeast. In this article, we characterize the original multistep process driving the assembly of this structure. This Aurora B-dependent mechanism follows a precise temporality that relies on the sequential actions of kinesin-14, kinesin-5, and involves both microtubule-kinetochore and kinetochore-kinetochore interactions. Upon quiescence exit, the microtubule bundle is disassembled via a cooperative process involving kinesin-8 and its full disassembly is required prior to cells re-entry into proliferation. Overall, our study provides the first description, at the molecular scale, of the entire life cycle of a stable microtubule structure in vivo and sheds light on its physiological function.

Abstract 5113: Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Abstract Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of the biological activities beyond the classic DNA damage, we treated 6 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 4/6 PDOX models (P&amp;lt;0.05) and widespread γH2AX positivity in &amp;gt;95% tumor cells in tumor core and &amp;gt;85% in the invasive foci, accompanied by ~30% apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species buildup, were detected together with the accumulation of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies. Citation Format: Zi-Lu Huang, Zhi-Gang Liu, Qi Lin, Ya-Lan Tao, Xinzhuoyun Li, Patricia Baxter, Jack MF Su, Adekunle M. Adesina, Chris Man, Murali Chintagumpala, Wan Yee Teo, Yu-Chen Du, Yun-Fei Xia, Xiao-Nan Li. Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5113.

Single-cell NAD(H) levels predict clonal lymphocyte expansion dynamics.

Adaptive immunity requires the expansion of high-affinity lymphocytes from a heterogeneous pool. Whereas current models explain this through signal transduction, we hypothesized that antigen affinity tunes discrete metabolic pathways to license clonal lymphocyte dynamics. Here, we identify nicotinamide adenine dinucleotide (NAD) biosynthesis as a biochemical hub for the T cell receptor affinity-dependent metabolome. Through this central anabolic role, we found that NAD biosynthesis governs a quiescence exit checkpoint, thereby pacing proliferation. Normalizing cellular NAD(H) likewise normalizes proliferation across affinities, and enhancing NAD biosynthesis permits the expansion of lower affinity clones. Furthermore, single-cell differences in NAD(H) could predict division potential for both T and B cells, before the first division, unmixing proliferative heterogeneity. We believe that this supports a broader paradigm in which complex signaling networks converge on metabolic pathways to control single-cell behavior.

Nutrients: Signal 4 in T cell immunity.

T cells are integral in mediating adaptive immunity to infection, autoimmunity, and cancer. Upon immune challenge, T cells exit from a quiescent state, followed by clonal expansion and effector differentiation. These processes are shaped by three established immune signals, namely antigen stimulation (Signal 1), costimulation (Signal 2), and cytokines (Signal 3). Emerging findings reveal that nutrients, including glucose, amino acids, and lipids, are crucial regulators of T cell responses and interplay with Signals 1-3, highlighting nutrients as Signal 4 to license T cell immunity. Here, we first summarize the functional importance of Signal 4 and the underlying mechanisms of nutrient transport, sensing, and signaling in orchestrating T cell activation and quiescence exit. We also discuss the roles of nutrients in programming T cell differentiation and functional fitness and how nutrients can be targeted to improve disease therapy. Understanding how T cells respond to Signal 4 nutrients in microenvironments will provide insights into context-dependent functions of adaptive immunity and therapeutic interventions.

Gli1 labels progenitors during chondrogenesis in postnatal mice

Skeletal growth promoted by endochondral ossification is tightly coordinated by self-renewal and differentiation of chondrogenic progenitors. Emerging evidence has shown that multiple skeletal stem cells (SSCs) participate in cartilage formation. However, as yet, no study has reported the existence of common long-lasting chondrogenic progenitors in various types of cartilage. Here, we identify Gli1+ chondrogenic progenitors (Gli1+ CPs), which are distinct from PTHrP+ or FoxA2+ SSCs, are responsible for the lifelong generation of chondrocytes in the growth plate, vertebrae, ribs, and other cartilage. The absence of Gli1+ CPs leads to cartilage defects and dwarfishness phenotype in mice. Furthermore, we show that the BMP signal plays an important role in self-renewal and maintenance of Gli1+ CPs. Deletion of Bmpr1α triggers Gli1+ CPs quiescence exit and causes the exhaustion of Gli1+ CPs, consequently disrupting columnar cartilage. Collectively, our data demonstrate that Gli1+ CPs are common long-term chondrogenic progenitors in multiple types of cartilage and are essential to maintain cartilage homeostasis.

EPIGENETIC MECHANISMS OF TISSUE AGING

Abstract Epigenetic alterations are a hallmark of aging, affecting DNA and histone modifications, 3D genome structure, and gene expression. Our lab studies these changes in various tissues to understand their unique and shared characteristics. In this talk, I will focus on the skeletal muscle, which makes up ~40% of the body weight, contributes to ~30% of resting energy expenditure, and is affected by aging. Aging reduces muscle mass and strength, partly due to the reduced function of resident muscle stem cells (MuSCs). We used an integrative multi-omics approach to profile the transcriptome, epigenome, and 3D genome of MuSCs isolated from young, old, and geriatric mice and comprehensively understand why their regenerative potential diminishes with age. We found an elevated immune response and quiescence exit signature in MuSCs during aging and identified regulatory elements that contribute to these changes. By targeting these networks, we can potentially rejuvenate MuSC function. Overall, we show that the non-coding genome potentiates a functional drift in MuSCs, an otherwise non-immune cell to a more immune function.

Riboproteome remodeling during quiescence exit in Saccharomyces cerevisiae

The quiescent state is the prevalent mode of cellular life in most cells. Saccharomyces cerevisiae is a useful model for studying the molecular basis of the cell cycle, quiescence, and aging. Previous studies indicate that heterogeneous ribosomes show a specialized translation function to adjust the cellular proteome upon a specific stimulus. Using nano LC-MS/MS, we identified 69 of the 79 ribosomal proteins (RPs) that constitute the eukaryotic 80S ribosome during quiescence. Our study shows that the riboproteome is composed of 444 accessory proteins comprising cellular functions such as translation, protein folding, amino acid and glucose metabolism, cellular responses to oxidative stress, and protein degradation. Furthermore, the stoichiometry of both RPs and accessory proteins on ribosome particles is different depending on growth conditions and among monosome and polysome fractions. Deficiency of different RPs resulted in defects of translational capacity, suggesting that ribosome composition can result in changes in translational activity during quiescence.

TRIB2 safeguards naive T cell homeostasis during aging

Naive CD4+ Tcells are more resistant to age-related loss than naive CD8+ Tcells, suggesting mechanisms that preferentially protect naive CD4+ Tcells during aging. Here, we show that TRIB2 is more abundant in naive CD4+ than CD8+ Tcells and counteracts quiescence exit by suppressing AKT activation. TRIB2 deficiency increases AKT activity and accelerates proliferation and differentiation in response to interleukin-7 (IL-7) in humans and during lymphopenia in mice. TRIB2 transcription is controlled by the lineage-determining transcription factors ThPOK and RUNX3. Ablation of Zbtb7b (encoding ThPOK) and Cbfb (obligatory RUNT cofactor) attenuates the difference in lymphopenia-induced proliferation between naive CD4+ and CD8+ cells. In older adults, ThPOK and TRIB2 expression wanes in naive CD4+ Tcells, causing loss of naivety. These findings assign TRIB2 a key role in regulating Tcell homeostasis and provide a model to explain the lesser resilience of CD8+ Tcells to undergo changes with age.

Cell quiescence in planarian stem cells, interplay between p53 and nutritional stimuli.

Cell quiescence appeared early in evolution as an adaptive response to adverse conditions (i.e. nutrient depletion). In metazoans, quiescence has been involved in additional processes like tissue homeostasis, which is made possible by the presence of adult stem cells (ASCs). Cell cycle control machinery is a common hub for quiescence entrance, and evidence indicates a role for p53 in establishing the quiescent state of undamaged cells. Mechanisms responsible for waking up quiescent cells remain elusive, and nutritional stimulus, as a legacy of its original role, still appears to be a player in quiescence exit. Planarians, rich in ASCs, represent a suitable system in which we characterized a quiescent population of ASCs, the dorsal midline cord (DMC) cells, exhibiting unique transcriptional features and maintained quiescent by p53 and awakened upon feeding. The function of DMC cells is puzzling and we speculate that DMC cells, despite retaining ancient properties, might represent a functional drift in which quiescence has been recruited to provide evolutionary advantages.

Lrig1 regulates the balance between proliferation and quiescence in glioblastoma stem cells.

Patients with glioblastoma (GBM) face a dismal prognosis. GBMs are driven by glioblastoma stem cells (GSCs) that display a neural stem cell (NSC)-like phenotype. These glioblastoma stem cells are often in a quiescent state that evades current therapies, namely debulking surgery and chemo/radiotherapy. Leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins have been implicated as regulators of growth factor signalling across many tissue stem cells. Lrig1 is highly expressed in gliomas and importantly, polymorphisms have been identified that are risk alleles for patients with GBM, which suggests some functional role in gliomagenesis. We previously reported that Lrig1 is a gatekeeper of quiescence exit in adult mouse neural stem cells, suppressing epidermal growth factor receptor signalling prior to cell cycle re-entry. Here, we perform gain- and loss-of-function studies to understand the function of Lrig1 in glioblastoma stem cells. Using a novel mouse glioblastoma stem cell model, we show that genetic ablation of Lrig1 in cultured GBM stem cells results in higher proliferation and loss of quiescence. In vivo, mice transplanted with glioblastoma stem cells lacking Lrig1 display lower survival compared to Lrig1 WT glioblastoma stem cells, with tumours displaying increased proportions of proliferative cells and reduced quiescent subpopulations. In contrast, Lrig1 overexpression in mouse glioblastoma stem cells results in enhanced quiescence and reduced proliferation, with impaired tumour formation upon orthotopic transplantation. Mechanistically, we find that Lrig1-null cells have a deficiency in BMP signalling responses that may underlie their lack of responsiveness to quiescence cues in vivo. These findings highlight important roles for Lrig1 in controlling responsiveness to both epidermal growth factor receptor and BMPR signalling, and hence the proportions of quiescent and proliferative subpopulations in GBMs.

SETD4 cells contribute to brain development and maintain adult stem cell reservoir for neurogenesis.

SummaryCellular quiescence facilitates maintenance of neural stem cells (NSCs) and their subsequent regenerative functions in response to brain injury and aging. However, the specification and maintenance of NSCs in quiescence from embryo to adulthood remain largely unclear. Here, using Set domain-containing protein 4 (SETD4), an epigenetic determinant of cellular quiescence, we mark a small but long-lived NSC population in deep quiescence in the subventricular zone of adult murine brain. Genetic lineage tracing shows that SETD4+ cells appear before neuroectoderm formation and contribute to brain development. In the adult, conditional knockout of Setd4 resulted in quiescence exit of NSCs, generating newborn neurons in the olfactory bulb and contributing to damage repair. However, long period deletion of SETD4 lead to exhaustion of NSC reservoir or SETD4 overexpression caused quiescence entry of NSCs, leading to suppressed neurogenesis. This study reveals the existence of long-lived deep quiescent NSCs and their neurogenetic capacities beyond activation.

Global chromatin accessibility profiling analysis reveals a chronic activation state in aged muscle stem cells

SummaryRegulation of chromatin accessibility is critical for cell fate decisions. Chromatin structure responds to extrinsic environments rapidly. The traditional adult stem cell isolation approach requires tissue dissociation, which triggers stem cell activation and leads to alterations in chromatin structure. To preserve the in vivo chromatin states, we utilized the PFA-perfusion-based isolation approach and characterized the DNA regulatory landscapes during muscle stem cell quiescence exit and aging. We showed that aged SCs display a chronically activated chromatin signature. Detailed analysis of the chromatin accessibility profiles identified key enhancer elements for SC quiescence. Constant activation of the enhancer elements promotes stemness and prevents SCs from differentiation, whereas genetic deletion causes cell-cycle arrest and leads to defects in activation. Our comprehensive characterization of the chromatin accessibility and transcriptomic landscapes in SC quiescence and aging broadens our understanding of these processes and identifies key distal regulatory elements for SC function.