Displaced Niche Location and Decreased Quiescence Maintenance of Hematopoietic Stem Cells Due to Dysregulation of Notch Adhesive Interaction with Stromal Environment in Mice with Notch O-Fucosylation Deficiency

Abstract 29

O-fucose modification and Fringe mediated O-fucose extension of Notch EGF-like repeats is essential for Notch binding with Notch ligand and Notch signaling transactivation. Previously we have shown that mice with conditional deficiency of Notch O-fucose modification develop a myeloproliferative disorder (MPD) with some mice displaying features of MPD-like leukemia. We disclosed that this MPD is mainly contributed by the cell-autonomous loss of response of myeloid progenitors bearing non-fucosylated Notch to Notch ligand induced suppression of granulo-monocytic differentiation. More recently, several Notch loss-of-function mutations have been identified in human chronic myelomonocytic leukemia (CMML). To gain a better insight of the role of Notch loss-of-function in stem cell dysfunction and MPD progression, here we studied the significance of O-fucose deficiency of Notch on progenitor proliferation and survival, and on HSC quiescence maintenance and niche location.

We used a mouse model of pan-Notch signaling loss-of-function by Mx-Cre1 induced deficiency of Pofut1, an enzyme that modifies EGF O-fucosylation of all 4 Notch receptors. Pofut1-null hematopoietic stem and progenitor cells (HSPCs) had enhanced myeloid specification and proliferation in vitro, and displayed an increased activation of ERK and Stat5 in response to IL3 and GM-CSF when compared to the control HSPCs. The enhanced myeloid specification of Pofut1-null HSCs could be rescued by either activated Notch1 or Notch2. In addition, the HSPCs from Pofut1-null marrow and spleen displayed a 30% reduction of apoptosis. However, the increased proliferation and survival of Pofut1-null HSPCs were only partially reversed by the blocking of G-CSF, a cytokine that was up-regulated in the serum of Pofut1-null mice, supporting a role of cell-autonomous mechanism in its contribution to the increased proliferation and survival of Pofut1-null HSPCs. In line with this notion, we found that Pofut1-null mice had ∼ 50% increase in frequencies of the multi-potential progenitors (MPP) and the short-term self-renewable HSC (ST-HSC) but a 70% reduction of the more primitive long-term self-renewable HSC (LT-HSC). This change of HSC frequency was accompanied by an increased HSC cell cycling and a loss of adhesion to Notch ligand-expressing stromal cells despite that the Pofut1-null HSCs had a normal chemotactic response to SDF-1 and normal expression of CXCR4 as well as integrin adhesion molecules. Consistent with these findings, frequencies of circulating and splenic-residing HSCs were increased in Pofut1-null mice. To explore the mechanism by which loss of O-fucose of Notch regulates the stem cell activity in the bone marrow niches, we performed two-photon intravital microscopy to visualize the niche location of transplanted HSCs. We found that the Pofut1-null HSCs were positioned further from the endosteal niche and the niche supporting osteoblasts, when compared to control HSCs. In addition, Pofut1-null HSCs were not responsive to the inhibition of HSC expansion imparted by the osteoblasts in an in vitro co-culture assay.

In summary, loss of O-fucosylation of Notch not only results in skewed myeloid specification and differentiation, but also promotes HSC proliferation and suppresses HSC quiescence. We conclude that the HSC phenotypes observed in mice with Pofut1 deficiency result as a consequence of the displacement of HSCs expressing non-fucosylated Notch from the suppressive endosteal niche that is otherwise enhanced by the adhesion between HSCs with the niche supporting cells through Notch and Notch ligand interaction.