Cell Polarity in Hematopoietic Stem Cell Quiescence, Signaling and Fate Determination A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements to the degree of Doctor of Philosophy (Ph.D.) in the Department of Cancer and Cell Biology of the College of Medicine March 27, 2020 By Mark Jordan Althoff B.S. Murray State University, 2013 Dissertation Committee: Jose A. Cancelas, MD, PhD (Chair) Yi Zheng, PhD Marie-Dominique Filippi, PhD Hartmut Geiger, PhD Daniel T. Starczynowski, PhD Richard Q. Lu, PhD ii Abstract Hematopoietic stem cells (HSC) self-renew and differentiate through changes in polarity. Polarity has been described as a major driver of asymmetric cell division, and in particular, Cdc42 allocation accurately predicts HSC asymmetric division potential. Few proteins responsible for establishing or maintaining cellular polarity (outside of Cdc42) have been investigated among HSC, and of those that have, many have been deemed functionally dispensable. Scribble is a multi-modular cytoplasmic scaffolding protein that coordinates the spatial organization of cell fate determinants and acts as a molecular hub for a variety of signaling proteins. The contributions of Scribble on cellular polarity establishment and maintenance in neuronal stem cells and epithelial cells is well characterized, however such mechanisms are yet to be defined in HSC. We discovered that Scribble controls HSC fate and function by acting as a molecular hub for signaling proteins like the Hippo pathway kinase, Lats1, and the effectors, Yap1 and Taz. The Hippo pathway controls proliferation and growth of multiple mammalian tissues, yet its role in HSC remains controversial. We found that Yap1 is predominantly polarized in the cytosol of HSC through a Scribble PDZ domain-mediated interaction. Deletion of Yap1 and Taz induces a loss of HSC quiescence, self-renewal and reconstitution following serial myeloablative 5-fluorouracil treatments, indicating a functional dependency for these effectors. We provide the first functional evidence that Scribble and Yap1 coordinate to control cytoplasmic Cdc42 activity, regulating both HSC quiescence and fate determination in vivo. Deletion of Scribble disrupted Yap1 co- polarization with Cdc42 and decreased Cdc42 activity, resulting in apoptosis of non-self-renewing daughter cells. This data suggests that Scribble/Yap1 co-polarization is indispensable for Cdc42- dependent activity on HSC asymmetric division and fate. The combined genetic loss of Scribble, Yap1 and Taz in HSC further decreases Cdc42 expression and activity, and is associated with transcriptional upregulation of Rac-specific guanine nucleotide exchange factors, and subsequent Rac activation and restoration of HSC fitness. Our data indicate that Scribble coordinates the cytosolic functions of Yap1 and Taz with Cdc42 activity and is required for HSC fate determination. iii We also identify a potential novel mechanism by which Scribble coordinates HSC activity in response to stress. Scribble deficient HSC retained cellular quiescence after interferons type I (IFN-I) stimulation. IFN-I are microenvironment cytokines produced during the physiological response mounted to combat a viral infection. In bone marrow hematopoiesis, IFN-I induce 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 the deficiency of Scribble in HSC rendered them insensitive to IFN-I mediated activation. As a result, Scribble deficient HSC treated with IFN-I are functionally more fit, displaying increased competitive reconstitution abilities during serial transplantations. No discernible differences in Stat-1 (the major effector of IFN-I signaling) activity were observed when measuring phosphorylation status, nuclear translocation and transcriptional response within wild- type (Wt) and Scribble deficient HSC following IFN-I exposure. Ly6a transcript levels are appropriately upregulated following IFN-I stimulation, however the encoded stem cell antigen-1 (Sca-1) protein localization was significantly decreased on the membrane surface. These data provide compelling evidence for a role of Scribble in coordinating HSC endosomal membrane trafficking to drive IFN-I mediated HSC activation. iv v Preface To address the role of Scribble in HSC polarity and function, I undertook two major research projects. The first project seeks to understand the molecular mechanisms associated with establishing HSC polarity to coordinate stem cell divisions and fate, employing both constitutive and inducible hematopoietic-specific Scribble-deficient animal models, reconstitution assays, and intracellular protein trafficking analysis using structure-function mutants of Scribble. The second project was aimed towards integrating the clinical implications of Scribble-mediated cellular polarity and HSC stress-response to Interferon signaling. As a result, the work presented in this dissertation has been previously published in, or is in preparation for, the following peer-reviewed journals: 1. *Singh A, *Althoff MJ, Cancelas JA. Signaling Pathways Regulating Hematopoietic Stem Cell and Progenitor Aging. 2018. Current Stem Cell Reports. 4(2):166-81. 2. Nayak RC, Hegde, S, Althoff MJ, Wellendorf AM, Mohmoud F, Perentesis J, Reina- Campos M, Reynaud D, Zheng Y, Diaz-Meco MT, Moscat J, Cancelas JA. The signaling axis atypical protein kinase C λ/ι-Satb2 mediates leukemic transformation of B-cell progenitors. 2019. Nat Commun. 10(1): Article Number 46. 3. Althoff MJ, Nayak R, Hegde S, Wellendorf AM, Bohan B, Filippi MD, Xin M, Lu QR, Geiger H, Zheng Y, Diaz-Meco MT, Moscat J, Cancelas JA. Yap1/Scribble polarization is required for hematopoietic stem cell division and fate. Under Review, Blood. 4. Althoff MJ, Wellendorf A, Diaz-Meco MT, Moscat J and Cancelas JA. Scribble mediates IFN-I induced activation of HSC through its regulation of Sca-1 and Akt activity independent of Stat1 effector response. Manuscript in Preparation. vi Acknowledgements First, I would like to take a moment to thank the University of Cincinnati and the Cancer and Cell biology (CCB) graduate program for their continuous guidance and support through my PhD studies. A large proportion of my gratitude has to go to my PhD mentor, Dr. Cancelas. Without a doubt, Dr. Cancelas has personalized my training and intellectual development to highlight my best attributes, framing me into a productive research scientist. He has an unrivaled devotion for his pupils and a tremendous dedication to health sciences, both attributes I plan to maintain throughout my academic career. I have to extend my appreciation to the remaining members of my Thesis Committee (Dr. Zheng, Dr. Filippi, Dr. Geiger, Dr. Starczynowski and Dr. Lu) for their expert direction and continual support. Lastly, I have to thank my growing family, Sara and Isla, for their unwavering support and the confidence they instill in me to follow my dream of becoming an independent research scientist. vii Table of Contents Abstract iii Preface vi Acknowledgements vii Table of Contents viii List of Figures and Tables x Chapter 1: Hematopoietic Stem Cell Polarity 1 Part 1: Hematopoiesis 1 Hematopoietic Stem Cells 1 HSC self-renewal and multi-lineage differentiation 6 Aberrant, dysregulated and malignant hematopoiesis 11 Transplantation as curative form of treatment 15 Part 2: Polarity and hematopoietic stem cell fate 18 Polarity and Asymmetric Division 18 Asymmetric inheritance of fate determinants and asymmetric cell division in HSC 20 The Scribble Polarity Complex 23 Chapter 2: Hypothesis and Goals 27 Chapter 3: Yap1-Scribble polarization is required for HSC division and fate 28 Abstract 29 Introduction 30 Results 31 The combined activity of the paralogues Yap1 and Taz is necessary for HSC function. 31 HSC Scribble scaffolds cytosolic Yap1 with upstream inhibitory components of the Hippo Signaling pathway. 33 The PDZ domain of Scribble is necessary for Yap1 cytoplasmic polarization while the LRR domain of Scribble is required for active Lats recruitment. 34 Scribble Scaffolds Polarized Yap1 and Activated Cdc42. 35 HSC Scribble deficiency results in enhanced long term self-renewal capacity. 37 Scribble deficiency decreases survival of nascent non self-renewing HSC clones. 38 viii Additional deficiency of Scribble restores HSC fitness of Yap1/TazΔ/Δ HSC and associates with Rac activation. 40 Discussion 41 Material and Methods 44 Acknowledgements 52 Author contributions 52 Disclosure of conflicts of interest 52 Figures and legends 53 Chapter 4: Scribble mediates IFN-I induced activation of HSC through its regulation of Sca-1 and Akt activity independent of Stat1 effector response 77 Abstract 78 Introduction 79 Results 81 Scribble deficient HSC are less responsive to IFN-I mediated HSC-activation and exhibit increased competitive repopulation following IFN-I stimulation. 81 Scribble deficient HSC mount an appropriate Stat1 signaling response following IFN-I stimulation. 82 Scribble deficient HSC display lower Akt activity and Sca-1 membrane expression. 83 Discussion 83 Material and Methods 86 Acknowledgements 91 Author contributions 91 Disclosure of conflicts of interest 91 Figures and Legends 92 Chapter 5: Discussion, Implications and Future Directions 95 Chapter 6: Conclusions