Scribble Is a Negative Regulator of Interferon-I Dependent Notch1 Activation in Adult Hematopoietic Stem Cells and Progenitors

Hematopoietic stem cells (HSC) are highly quiescent cells with the ability to rapidly enter cell cycle and differentiate through changes in their polarity and the disposition of intracellular molecular fate determinants in response to microenvironment (ME) cues. Interferons type 1 (IFN-I) are ME cytokines produced during the physiological response mounted to combat a viral infection. In bone marrow hematopoiesis, IFN-I induces activation and proliferation of HSC. Clinically, patients treated with IFN-I, as well as individuals suffering from IFN-I associated chronic disease, often exhibit sustained hematological cytopenias and HSC failure. The precise molecular mechanisms that govern HSC behavior in response to IFN-I are still unclear. Our data highlights that Scribble deficient HSC are less sensitive to IFN-I mediated activation. By using hematopoietic specific deletion of Scribble in murine hematopoiesis (Vav-Cre;Scribble KO); we demonstrated that Scribble deficient LSK CD150 +/CD48 - HSC are less responsive to polyinositide-polycytidine (pI:C) induced IFN-I mediated activation and retain cellular quiescence (G0:45±5.4% vs 63±2.7% in WT and Scribble KO, respectively, p<0.05). IFN-I induced upregulation of Sca-1 expression was also significantly hampered in Scribble deficient HSC. Functionally, serial transplantation experiments demonstrated that in response to poly I:C, Scribble deficient HSC display increased competitive repopulating potential (26±1.3% vs 38±1.2% BM chimerism for WT and KO BM in secondary recipients and 38±2.5% vs 48±2.7% BM chimerism in tertiary recipients, p<0.01). The maintenance of cellular quiescence and function for Scribble deficient HSC are independent of canonical IFN-I driven STAT-1 signaling, as we report no differences in STAT-1 activation, nuclear translocation or the expression of STAT-1 canonical target genes in response to pI:C. Unsupervised transcriptomics analysis of Scribble-deficient HSC supported dysregulation of Notch signaling. Furthermore, Scribble deficiency in non-activated LSK HSC and progenitors (HSPC) was associated with constitutive activation and cleavage of Notch1 (Notch1 ICD;~3 fold) at levels comparable to IFN-I mediated activation of WT HSPC. However, Scribble deficient HSPC did not exhibit further Notch1 cleavage activation upon in vivo IFN-I induction. Pharmacological in vivo γ-secretase inhibition (YO-01027) prevented the protective effect of Scribble deficiency on IFN-I dependent loss of HSC quiescence. These data indicate that Notch1 activation, and subsequent cleavage, is indispensable for Scribble deficient HSC quiescence in response to IFN-I. Active Cdc42 is a critical regulator of HSC quiescence and fate, and previous studies have demonstrated that Scribble controls HSC asymmetric division potential and fate through the PDZ mediated scaffolding of cytosolic Yap1 with activated Cdc42 (Cdc42-GTP). Next to determine whether poly I:C mediated Notch1 cleavage linked with Cdc42 activity, we analyzed the protein interactions between cleaved Notch1 and Cdc42-GTP in relation with Scribble. Our findings revealed that Scribble associates with non-cleaved, membrane bound Notch but upon in vivo IFN-I induction Notch1 is cleaved, activated and translocates with Scribble-free, activated Cdc42 to the nucleus of HSC. Deletion of HSC Scribble associated with a reduced (~45%, p<0.001) proximity interaction between cleaved Notch1 and Cdc42-GTP. Collectively our findings identify that Scribble controls IFN-I mediated HSPC activation through induction of Notch1 cleavage and Cdc42 activity, and highlight such interaction as a new potential target to dampen inflammation driven HSC exhaustion. Disclosures Cancelas: Cerus Co: Research Funding; TerumoBCT: Research Funding; Hemanext: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytosorbents Inc: Research Funding; Fresenius-Kabi LLC: Research Funding; Westat Inc: Research Funding; Vascular Solutions Inc.: Research Funding; Hemerus LLC: Research Funding; University of South Florida/MEQU Inc: Research Funding.

Is There a Histone Code for Cellular Quiescence?

Many of the cells in our bodies are quiescent, that is, temporarily not dividing. Under certain physiological conditions such as during tissue repair and maintenance, quiescent cells receive the appropriate stimulus and are induced to enter the cell cycle. The ability of cells to successfully transition into and out of a quiescent state is crucial for many biological processes including wound healing, stem cell maintenance, and immunological responses. Across species and tissues, transcriptional, epigenetic, and chromosomal changes associated with the transition between proliferation and quiescence have been analyzed, and some consistent changes associated with quiescence have been identified. Histone modifications have been shown to play a role in chromatin packing and accessibility, nucleosome mobility, gene expression, and chromosome arrangement. In this review, we critically evaluate the role of different histone marks in these processes during quiescence entry and exit. We consider different model systems for quiescence, each of the most frequently monitored candidate histone marks, and the role of their writers, erasers and readers. We highlight data that support these marks contributing to the changes observed with quiescence. We specifically ask whether there is a quiescence histone “code,” a mechanism whereby the language encoded by specific combinations of histone marks is read and relayed downstream to modulate cell state and function. We conclude by highlighting emerging technologies that can be applied to gain greater insight into the role of a histone code for quiescence.

Exit from quiescence couples epithelial stress amplification to fluidization

Cellular quiescence is a state of reversible cell cycle arrest that is associated with tissue dormancy. Timely regulated entry into and exit from quiescence is important for processes such as tissue homeostasis, tissue repair, stem cell maintenance, developmental processes and immunity. Here we show that quiescent human keratinocyte monolayers contain an actinomyosin-based system that facilitates global viscoelastic flow upon serum-stimulated exit from quiescence. Mechanistically, serum exposure causes rapid amplification of pre-existing contractile sites leading to a burst in monolayer stress that subsequently drives monolayer fluidization. The stress magnitude after quiescence exit correlates with quiescence depth, and a critical stress level must be reached to overcome the cell sheet displacement barrier. The study shows that static quiescent cell monolayers are mechanically poised for motility and identifies global stress amplification as a mechanism for tissue fluidization.

Extracellular Fluid Flow Induces Shallow Quiescence through Physical and Biochemical Cues

ABSTRACTThe balance between cell quiescence and proliferation is fundamental to tissue physiology and homeostasis. Recent studies have shown that quiescence is not a passive and homogeneous state but actively maintained and heterogeneous. These cellular characteristics associated with quiescence were observed primarily in cultured cells under a static medium. However, cells in vivo face different microenvironmental conditions, particularly, under interstitial fluid flows distributed through extracellular matrices. Interstitial fluid flow exerts shear stress on cells and matrix strain, and results in continuous replacement of extracellular factors. In this study, by analyzing individual cells under varying fluid flow rates in microfluidic devices, we found that extracellular fluid flow alters cellular quiescence depth through flow-induced physical and biochemical cues. Specifically, increasing the flow rate drives cells to shallower quiescence and become more likely to reenter the cell cycle upon growth stimulation. Furthermore, we found that increasing shear stress or extracellular factor replacement individually, without altering other parameters, also results in shallow quiescence. We integrated the experimental results into a mathematical model to gain insight and predict the effects of varying extracellular fluid flow conditions on cellular quiescence depth. Our findings uncover a previously unappreciated mechanism that likely underlies the heterogeneous responses of quiescent cells for tissue repair and regeneration in physiological tissue microenvironments.

Adipocytes disrupt the translational programme of acute lymphoblastic leukaemia to favour tumour survival and persistence

The specific niche adaptations that facilitate primary disease and Acute Lymphoblastic Leukaemia (ALL) survival after induction chemotherapy remain unclear. Here, we show that Bone Marrow (BM) adipocytes dynamically evolve during ALL pathogenesis and therapy, transitioning from cellular depletion in the primary leukaemia niche to a fully reconstituted state upon remission induction. Functionally, adipocyte niches elicit a fate switch in ALL cells towards slow-proliferation and cellular quiescence, highlighting the critical contribution of the adipocyte dynamic to disease establishment and chemotherapy resistance. Mechanistically, adipocyte niche interaction targets posttranscriptional networks and suppresses protein biosynthesis in ALL cells. Treatment with general control nonderepressible 2 inhibitor (GCN2ib) alleviates adipocyte-mediated translational repression and rescues ALL cell quiescence thereby significantly reducing the cytoprotective effect of adipocytes against chemotherapy and other extrinsic stressors. These data establish how adipocyte driven restrictions of the ALL proteome benefit ALL tumours, preventing their elimination, and suggest ways to manipulate adipocyte-mediated ALL resistance.

Circadian Proteins Cry and Rev-erb Deepen Cellular Quiescence by Down-regulating Cyclin D and Cdk4,6

The proper balance and transition between cellular quiescence and proliferation are critical to tissue homeostasis, and their deregulations are commonly found in many human diseases, including cancer and aging. Recent studies showed that the reentry of quiescent cells to the cell cycle is subjected to circadian regulation. However, the underlying mechanisms are largely unknown. Here, we report that two circadian proteins, Cryptochrome (Cry) and Rev-erb, deepen cellular quiescence in rat embryonic fibroblasts, resulting in stronger serum stimulation required for cells to exit quiescence and reenter the cell cycle. This finding was opposite from what we expected from the literature. By modeling a library of possible regulatory topologies linking Cry and Rev-erb to a bistable Rb-E2f gene network switch that controls the quiescence-to-proliferation transition and by experimentally testing model predictions, we found Cry and Rev-erb converge to downregulate Cyclin D/Cdk4,6 activity, leading to an ultrasensitive increase of the serum threshold to activate the Rb-E2f bistable switch. Our findings suggest a mechanistic role of circadian proteins in modulating the depth of cellular quiescence, which may have implications in the varying potentials of tissue repair and regeneration at different times of the day.

Evolutionary Protection of Krüppel-Like Factors 2 and 4 in the Development of the Mature Hemovascular System

A properly functioning hemovascular system, consisting of circulating innate immune cells and endothelial cells (ECs), is essential in the distribution of nutrients to distant tissues while ensuring protection from invading pathogens. Professional phagocytes (e.g., macrophages) and ECs have co-evolved in vertebrates to adapt to increased physiological demands. Intercellular interactions between components of the hemovascular system facilitate numerous functions in physiology and disease in part through the utilization of shared signaling pathways and factors. Krüppel-like factors (KLFs) 2 and 4 are two such transcription factors with critical roles in both cellular compartments. Decreased expression of either factor in myeloid or endothelial cells increases susceptibility to a multitude of inflammatory diseases, underscoring the essential role for their expression in maintaining cellular quiescence. Given the close evolutionary relationship between macrophages and ECs, along with their shared utilization of KLF2 and 4, we hypothesize that KLF genes evolved in such a way that protected their expression in myeloid and endothelial cells. Within this Perspective, we review the roles of KLF2 and 4 in the hemovascular system and explore evolutionary trends in their nucleotide composition that suggest a coordinated protection that corresponds with the development of mature myeloid and endothelial systems.