Scribble Controls HSC Self-Renewal through Polarity-Dependent Activation of the Hippo Signaling Pathway

Search of molecular targets to expand functional hematopoietic stem cells (HSC) for transplantation and hematological cancer therapy remains paramount, particularly when manipulation of cellular quiescence is required. HSC are highly quiescent cells with the ability to rapidly enter the cell cycle in response to microenvironment cues and divide through changes in their polarity. Despite cellular polarity being one of the most basic properties of all living cells, the role that polarity regulators have on polarization and function of HSC remains controversial. Scribble, of the Scribble Polarity Complex, controls the spatial organization of intracellular proteins and negatively regulates the cell cycle. By using a combination of constitutive and inducible hematopoietic-specific Scribble-deficient animal models, hematopoietic reconstitution assays, structure-function mutants of Scribble and intracellular protein trafficking analysis, we identified the functional relevance of Scribble in HSC activity. We observed that Scribble is polarized in HSC and deletion of Scribble leads to a ~ 50% reduction in the bone marrow (BM) HSC population (p<0.001). Scribble-deficient HSC are less quiescent (G₀: 93 vs 88%, p<0.05; S/G₂/M 4 vs 10%, for WT and KO, respectively, p<0.01) and contain ~70% reduction of dormant, long-term HSC (p<0.01). This data indicates that Scribble is required to maintain HSC quiescence. Functionally, stroma-dependent long-term BM cultures revealed that Scribble-deficient BM contains a ~ 60% reduction in the number of early cobblestone area-forming cells (CAFC) while their content of late (28-35 day) CAFC (primitive progenitors/HSC) is 3-5 fold increased. Serial transplantation experiments demonstrated that Scribble-deficient BM HSC have increased competitive repopulating potential (46% vs 90% BM chimerism for WT and KO BM from tertiary recipient mice, respectively, p<0.001). Moreover, deletion of hematopoietic Scribble protects mice from 5-fluorouracil (5-FU)-mediated hematopoietic exhaustion and death by BM aplasia (>30 days median survival vs 16 days for KO mice, p<0.001). This set of functional data indicates that Scribble deficient HSC show enhanced long-term self-renewal capacity in stroma-dependent cultures and in vivo following transplantation or chemotherapeutic stress without the development of stem cell exhaustion or leukemia. Comparative transcriptional analysis of Scribble-deficient and WT HSC identified transcriptional regulation of the Hippo-dependent signaling pathway. We observed that Yap1 is polarized in a Scribble-dependent manner within WT HSC and co-localizes in the cytoplasm with the active form of the upstream inhibitory kinase, Lats1. Notably, deletion of Scribble in HSC leads to a disruption of the phosphorylated Lats1/Yap1 complex resulting in Yap1 translocation to the nucleus. Subsequently, cytoplasmic polarization of Yap1 can be restored when Scribble-deficient HSC are rescued with full length or PDZ domain containing mutants of Scribble. Expression of the N-terminus leucine-rich repeat (LRR) domain of Scribble successfully restored activation of Lats1 as well as phosphorylated Lats1 co-localization with Scribble, however, was not sufficient to prevent Yap1 translocation. Combined deletion of Yap1 and Taz using an inducible hematopoietic-specific model generates hematopoietic stem and progenitor cells (HSC/P) with enhanced cellular proliferation (HSC G₀: 88 vs. 58%, p<0.0001; G₁: 6 vs. 25%, p<0.0001; S/G₂/M 6 vs. 18%, p<0.0001 for WT controls and Yap1/Taz double deficient BM, respectively) leading to an increase in colony forming units and early CAFC potential (2 and 3 fold respectively; p<0.0001). Yap1/Taz double KO HSC are prone to exhaustion evidenced by their inability to regenerate murine hematopoiesis after serial administration of 5-FU (12 days median survival vs 20 days for WT control mice, p<0.001). Triple deficiency of Scribble, Yap1 and Taz restores the effect of both Scribble and Yap1/Taz deficiencies to a normal hematopoietic response after myeloablation. Altogether, this data identifies Scribble as a negative regulator of cell cycle entry and self-renewal of HSC, and provides compelling evidence of key polarity and signaling programs used to maintain HSC quiescence that can be utilized as novel targets to improve HSC based transplantations and hematological cancer therapies.