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

Abstract

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. DisclosuresCancelas: 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.