ALBERT

Description: Journal of Chemical & Engineering Data DOI: 10.1021/je300647k

Description: Introduction Acute leukemias are often associated with the suppression of hematopoiesis. This condition is generally believed to reflect the “crowding-out” effect by the rapidly proliferating leukemia cells. Emerging data, however, has indicated that leukemia cells can instructively modulate the marrow microenvironment that is essential for the maintenance of hematopoietic homeostasis. One of the underlying mechanisms likely implicates leukemia-associated disruption of the marrow stem cell niche; however, the mechanisms by which acute leukemia employs to modulate niche architectural alterations are not well understood. A better understanding of the leukemia niche elements and the implicated molecular pathways involved in the niche regulation will facilitate the development of therapies to accelerate hematopoietic recovery and reconstitution in leukemia patients. Methods We used AML human NSG xenografts and the murine T-ALL model induced by the retroviral transfection of marrow progenitors with activated form of Notch1 (ICN1). In the murine T-ALL model, the transformed progenitors were co-transplanted with the wild type (WT) marrow cells into irradiated immune-sufficient C57BL/6 mice. The activated Notch1 drives leukemia transformation characterized by the expansion of an immature CD4+/CD8+ population of T progenitors. We tracked the niche location of leukemia using multi-photon intravital microscopy. The niche cell compartment and the reconstitution of co-transplanted marrow cells were characterized by flow cytometry, RT-PCR and in vitro differentiation assays. Results In NSG mice receiving primary human AML cells, we observed that leukemia development was accompanied by a suppression of the host hematopoietic stem cell (HSC) compartment. A similar hematopoietic suppression was observed in the C57BL/6 mice receiving ICN1-expressing progenitors but not in the mice receiving control cells in a disease stage-dependent manner, characterized by a loss of the HSC population at the later disease stage and a prominent suppression of B lymphopoiesis in the companion non-transformed marrow compartment. Co-culturing with leukemia cells only modestly affected HSPC differentiation, indicating elements of leukemia microenvironment are also important to mediate the hematopoietic suppression. To understand how leukemia cells modulate niche, we longitudinally tracked the niche localization and the proliferation of pre-leukemia cells in lethally irradiated host mice. We found that ICN1-expressing individual pre-leukemia cells can be identified on day1 at the peri-vascular niche after adoptive transfer, and appeared as clusters around day8 followed by a rapid expansion phase 2-3 weeks after transplantation. We then demonstrated that the CD8+ leukemia cells, which are enriched for leukemia initiating cells, but not the CD8- cells, homed to the peri-vascular niche in the secondary leukemia transplanted hosts. Further, co-transplantation of leukemia cells with WT HSCs decreased HSC homing to the marrow and displaced them from the peri-vascular niche. In addition, leukemia mice displayed a marked loss of endosteal-lining osteoblasts, accompanied with a down-regulation of the transcription factors essential for the osteoblast differentiation in both osteoblastic progenitors and the multi-potent stromal cells (MSCs), and leukemia MSCs displayed a suppressed osteoblast formation when compared to control MSCs. In contrast, expressions of Notch signaling targets as well as CXCL12 were up-regulated in the leukemia stromal cells. The requirement of Notch in the alteration of leukemia niche function is supported by the observation that gamma-secretase inhibitor (GSI) treatment of mice receiving GSI-insensitive ICN1 cells attenuated the HSC suppression. Conclusion In summary, we found a potentially common mechanism employed by different types of acute leukemia to instructively modulate niche components essential for normal hematopoiesis. Specifically, we identified that T-ALL leukemia cells driven by ICN1 induces a hematopoietic suppression through competitive displacement of the normal HSCs from the proliferating peri-vascular niche and an ablation of the endosteal osteoblasts. Our future studies will target to elucidate the mechanistic co-regulation of normal and malignant hematopoiesis by leukemia microenvironment. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND: Notch has long been recognized as an important molecule that regulates stem cell self-renewal and differentiation. Despite that Notch is required for the embryonic hematopoietic stem cell (HSC) generation, and that Notch is expressed in the adult HSC, the role of Notch pathway in adult HSC remains unclear. Recently it has been shown that Notch activation and the ability of interacting with Notch ligand Jagged1 is a signature of human primitive HSC and supports HSC regenerative potential. Here we study the physiological significance of Notch in the adult HSC population focusing on how Notch-ligand engagement regulates HSC quiescence and niche retention. METHODS: To better understand the role of Notch in adult HSC homeostasis, we examined HSC frequency, quiescence maintenance, niche occupancy, and HSC mobilization in mice with either conditional lack of RBP-JK, which mediates the canonical Notch signaling activity, or in mice with conditional lack of Pofut1 that catalyzes O-fucosylation of Notch EGF-like repeats and the generation of O-fucose glycans important for the binding of Notch ligands. We also tested Notch ligand neutralizing antibodies and Notch1 and Notch2 inhibitory antibodies to examine the effect of blocking Notch receptor-ligand engagement or the block of Notch signaling activation on HSC homeostasis. RESULTS: We report here that Pofut1-deficient hematopoietic stem and progenitor cells (HSPCs) display enhanced cell cycling and proliferation cell autonomously. These changes are accompanied by G-CSF-independent increased HSPC egress from the marrow to the periphery and other hematopoietic organs, and their enhanced sensitivity to mobilizing stimuli of G-CSF plus the CXCR4 antagonist, AMD3100. This phenotype is caused by reduced adhesion of Pofut1-deficient HSPC to Notch ligand-expressing stromal cells and the osteoblastic lineage cells. Adhesion to these cells by wild type but not Pofut1-deficient HSPCs can be blocked by recombinant Notch ligand Dll1 or Dll4. In addition, adhesion to these cells inhibits wild type but not Pofut1-deficient HSPC cycling, independent of RBP-Jk-mediated Notch signaling. Further, Pofut1-deficient HSPCs exhibit normal expression of key adhesion molecules and normal SDF-1-mediated chemotaxis, but show scattered and distal occupancy in the endosteal or osteoblastic niche. In support of roles for Notch-ligand engagement in facilitating HSPC niche retention, we show that mice receiving 4 doses of neutralizing antibodies to the Notch ligand Dll4 or Jagged1, but not Dll1, display ~2-3-fold increased HSC and progenitor egress when compared to mice receiving isotype control antibody, and further display 60% increased HSC mobilization when compared to mice receiving control antibody and treated similarly with G-CSF and AMD3100. Dll4 blockade also increases the sinusoidal endothelial cell population and HSPC cell cycling. In comparison, only a mild HSPC proliferation and egress is found in RBP-JK-deficient mice, or in mice receiving Notch2 inhibitory antibody. However, Notch2 blockade but not Notch1 blockade induces unique features of HSC and myeloid progenitor mobilization responding to G-CSF plus AMD3100. CONCLUSIONS: Based on these findings, we conclude that HSPC quiescence and retention in the marrow niche is facilitated by the interaction between Notch-expressing HSPCs and Jagged1- or Dll4-expressing niche cells, and is likely also contributed by Notch signaling activation. In addition, Notch receptor-ligand engagement in this process is strengthened by O-fucose modification of Notch receptors. Finally, the observations from our studies may provide a therapeutic indication. Inadequate mobilization in HSPC transplantation remains a clinical problem. Our findings that targeting Notch receptor-ligand interaction and/or inhibiting Notch2 activation increase HSPC emigration suggests a novel approach for enhancing mobilization of stem and progenitor cells for those patients who respond poorly to current mobilizing regimes. Disclosures Shim: Genentech: Employment, Equity Ownership. Yan:Genentech: Employment, Equity Ownership. Lowe:Genentech: Employment, Equity Ownership. Siebel:Genentech: Employment, Equity Ownership.

Description: 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. Disclosures: No relevant conflicts of interest to declare.

Description: Notch signaling is essential for lymphocyte development and is also implicated in myelopoiesis. Notch receptors are modified by O-fucosylation catalyzed by protein O-fucosyltransferase 1 (Pofut1). Fringe enzymes add N-acetylglucosamine to O-fucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. To address physiologic functions in hematopoiesis of Notch modified by O-fucose glycans, we examined mice with inducible inactivation of Pofut1 using Mx-Cre. These mice exhibited a reduction in T lymphopoiesis and in the production of marginal-zone B cells, in addition to myeloid hyperplasia. Restoration of Notch1 signaling rescued T lymphopoiesis and the marrow myeloid hyperplasia. After marrow transfer, both cell-autonomous and environmental cues were found to contribute to lymphoid developmental defects and myeloid hyperplasia in Pofut1-deleted mice. Although Pofut1 deficiency slightly decreased cell surface expression of Notch1 and Notch2, it completely abrogated the binding of Notch receptors with Delta-like Notch ligands and suppressed downstream Notch target activation, indicating that O-fucose glycans are critical for efficient Notch-ligand binding that transduce Notch signals. The combined data support a key role for the O-fucose glycans generated by Pofut1 in Notch regulation of hematopoietic homeostasis through modulation of Notch-ligand interactions.