Abstract 1444: Overexpression Of Endothelial Nitric Oxide Synthase Restores Post-natal Neovascularization In Atherosclerosis.

Alteration in post-ischemic neovascularization is a common complication of atherosclerotic disease. This results, at least in part, from abrogation of bone-marrow mononuclear cells (BM-MNC) pro-angiogenic potential. Overexpression of eNOS has been shown to promote vessel growth in the setting of ischemia. We hypothesized that eNOS overexpression could restore impaired neovascularization in atherosclerotic (ApoE KO) mice. Hind limb ischemia was induced in mice by right femoral artery ligation. After two weeks we evaluated tissue perfusion of the foot by Laser Doppler, vessel density in the hind limb by micro-angiography and histology, and atherosclerotic plaque size. In vitro BM-MNC cell culture assays were performed. Tissue perfusion and vessel density were 1.5-fold increased in transgenic mice overexpressing eNOS (eNOStg) as compared to wild type (WT) (P<0.001, n=10). Transplantation of 1x106 WT- or eNOStg BM-MNC in WT recipients caused a 1.5-fold increase in tissue perfusion and vessel density compared to injection of PBS (P<0.001, n=10). Next, we used ApoE KO mice and crossbreeds of eNOStg and ApoE KO mice (eNOStg*ApoE KO). Tissue perfusion and vessel density were 1.8-fold increased in eNOStg*ApoE KO mice as compared to ApoE KO mice (P<0.001, n=10). Transplantation of both WT- or eNOStg*ApoE KO BM-MNC in ApoE KO recipients caused a 1.6- to 2-fold increase in tissue perfusion and vessel density compared to PBS (P<0.01, n=10), while transplantation of ApoE KO BM-MNC had no positive effect on neovascularization. Moreover, transplantation of WT BM-MNC significantly increased plaque size, while eNOStg*ApoE KO BM-MNC had no effect on plaque size. eNOS overexpression did not affect BM-MNC apoptosis and secretion of growth factors but increased their ability to differentiate in vitro into EPC. Conclusion: eNOS overexpression in the endothelium improves post-ischemic neovascularization in both physiological as atherosclerotic settings. Furthermore, eNOS overexpression in the bone marrow restores the impaired pro-angiogenic potential of atherosclerotic BM-MNC without adverse effects on plaque size. Therefore, overexpression of eNOS could play a vital part in the development of therapeutic angiogenesis for atherosclerotic disease.

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Mesenchymal Stem/Stromal Cells from Discarded Neonatal Sternal Tissue: In Vitro Characterization and Angiogenic Properties

Stem Cells International ◽

10.1155/2016/5098747 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 7

Author(s):

Shuyun Wang ◽

Lakshmi Mundada ◽

Eric Colomb ◽

Richard G. Ohye ◽

Ming-Sing Si

Keyword(s):

Bone Marrow ◽

Cardiac Surgery ◽

Stromal Cells ◽

Umbilical Vein ◽

Culture Method ◽

Tissue Perfusion ◽

Explant Culture ◽

Critical Congenital Heart Disease ◽

Surface Marker Expression

Autologous and nonautologous bone marrow mesenchymal stem/stromal cells (MSCs) are being evaluated as proangiogenic agents for ischemic and vascular disease in adults but not in children. A significant number of newborns and infants with critical congenital heart disease who undergo cardiac surgery already have or are at risk of developing conditions related to inadequate tissue perfusion. During neonatal cardiac surgery, a small amount of sternal tissue is usually discarded. Here we demonstrate that MSCs can be isolated from human neonatal sternal tissue using a nonenzymatic explant culture method. Neonatal sternal bone MSCs (sbMSCs) were clonogenic, had a surface marker expression profile that was characteristic of bone marrow MSCs, were multipotent, and expressed pluripotency-related genes at low levels. Neonatal sbMSCs also demonstrated in vitro proangiogenic properties. Sternal bone MSCs cooperated with human umbilical vein endothelial cells (HUVECs) to form 3D networks and tubes in vitro. Conditioned media from sbMSCs cultured in hypoxia also promoted HUVEC survival and migration. Given the neonatal source, ease of isolation, and proangiogenic properties, sbMSCs may have relevance to therapeutic applications.

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Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Blood ◽

10.1182/blood.v81.3.661.661 ◽

1993 ◽

Vol 81 (3) ◽

pp. 661-669 ◽

Cited By ~ 92

Author(s):

EF Srour ◽

JE Brandt ◽

RA Briddell ◽

S Grigsby ◽

T Leemhuis ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Fold Increase ◽

Hematopoietic Progenitor Cells ◽

Proliferative Potential ◽

Hematopoietic Progenitor ◽

Hla Dr

Abstract Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

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The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation

Blood ◽

10.1182/blood-2007-07-099648 ◽

2008 ◽

Vol 111 (1) ◽

pp. 42-49 ◽

Cited By ~ 144

Author(s):

Antje M. Wengner ◽

Simon C. Pitchford ◽

Rebecca C. Furze ◽

Sara M. Rankin

Keyword(s):

Bone Marrow ◽

Intraperitoneal Injection ◽

Fold Increase ◽

In Situ Perfusion ◽

Acute Peritonitis ◽

Femoral Bone Marrow ◽

Prior Administration

In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1α–mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 × 106 and 5.4 × 106 neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 × 106 neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.

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Reconstitution of Early Lymphoid Proliferation and Immune Function in Jak3-Deficient Mice by Interleukin-3

Blood ◽

10.1182/blood.v94.6.1906.418k32_1906_1914 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1906-1914

Author(s):

Michael P. Brown ◽

Tetsuya Nosaka ◽

Ralph A. Tripp ◽

James Brooks ◽

Jan M.A. van Deursen ◽

...

Keyword(s):

Bone Marrow ◽

Cytotoxic T Lymphocyte ◽

Severe Combined Immunodeficiency ◽

Fold Increase ◽

Receptor Activation ◽

Beneficial Effects ◽

Interleukin 7 ◽

Histocompatibility Complex ◽

Lymphoid Progenitors

Expansion of early lymphoid progenitors requires interleukin-7 (IL-7), which functions through γc-mediated receptor activation of Jak3. Jak3 deficiency is a cause of severe combined immunodeficiency (SCID) in humans and mice. IL-3 activates many of the same signaling pathways as IL-7, such as Stat5, but achieves this effect through the activation of Jak2 rather than Jak3. We hypothesized that expansion of an IL-7–responsive precursor population through a Jak3-independent pathway using IL-3 may stimulate early lymphoid progenitors and restore lymphopoiesis in Jak3−/− mice. Newborn Jak3−/− mice that were injected with IL-3 demonstrated thymic enlargement, a 2- to 20-fold increase in thymocyte numbers, and up to a 10-fold expansion in the number of CD4+, CD8+, and B220+/IgM+ splenic lymphocytes, consistent with an effect upon an early lymphoid progenitor population. In contrast to control mice, IL-3–treated Jak3−/− mice challenged with the allogeneic major histocompatibility complex (MHC) class I-bearing tumor P815 developed a specific CD8-dependent cytotoxic T lymphocyte (CTL) response. IL-3–treated mice also mounted influenza-specific CTL responses and survival was prolonged. The beneficial effects of IL-3 are proposed to be produced by stimulation of a lymphoid precursor population of IL-7R+/IL-3R+ cells that we identified in wild-type bone marrow. In vitro, we show that an early IL-7R+ lymphoid progenitor population expresses IL-3R and proliferates in response to IL-3 and that IL-3 activates Stat5 comparably to IL-7. Clinically, IL-3 may therefore be useful treatment for X-linked and Jak3-deficient SCID patients who lack bone marrow donors.

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Reconstitution of Early Lymphoid Proliferation and Immune Function in Jak3-Deficient Mice by Interleukin-3

Blood ◽

10.1182/blood.v94.6.1906 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1906-1914 ◽

Cited By ~ 22

Author(s):

Michael P. Brown ◽

Tetsuya Nosaka ◽

Ralph A. Tripp ◽

James Brooks ◽

Jan M.A. van Deursen ◽

...

Keyword(s):

Bone Marrow ◽

Cytotoxic T Lymphocyte ◽

Severe Combined Immunodeficiency ◽

Fold Increase ◽

Receptor Activation ◽

Beneficial Effects ◽

Interleukin 7 ◽

Histocompatibility Complex ◽

Lymphoid Progenitors

Abstract Expansion of early lymphoid progenitors requires interleukin-7 (IL-7), which functions through γc-mediated receptor activation of Jak3. Jak3 deficiency is a cause of severe combined immunodeficiency (SCID) in humans and mice. IL-3 activates many of the same signaling pathways as IL-7, such as Stat5, but achieves this effect through the activation of Jak2 rather than Jak3. We hypothesized that expansion of an IL-7–responsive precursor population through a Jak3-independent pathway using IL-3 may stimulate early lymphoid progenitors and restore lymphopoiesis in Jak3−/− mice. Newborn Jak3−/− mice that were injected with IL-3 demonstrated thymic enlargement, a 2- to 20-fold increase in thymocyte numbers, and up to a 10-fold expansion in the number of CD4+, CD8+, and B220+/IgM+ splenic lymphocytes, consistent with an effect upon an early lymphoid progenitor population. In contrast to control mice, IL-3–treated Jak3−/− mice challenged with the allogeneic major histocompatibility complex (MHC) class I-bearing tumor P815 developed a specific CD8-dependent cytotoxic T lymphocyte (CTL) response. IL-3–treated mice also mounted influenza-specific CTL responses and survival was prolonged. The beneficial effects of IL-3 are proposed to be produced by stimulation of a lymphoid precursor population of IL-7R+/IL-3R+ cells that we identified in wild-type bone marrow. In vitro, we show that an early IL-7R+ lymphoid progenitor population expresses IL-3R and proliferates in response to IL-3 and that IL-3 activates Stat5 comparably to IL-7. Clinically, IL-3 may therefore be useful treatment for X-linked and Jak3-deficient SCID patients who lack bone marrow donors.

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In vitro Effects of Enamel Matrix Proteins on Rat Bone Marrow Cells and Gingival Fibroblasts

Journal of Dental Research ◽

10.1177/154405910408300210 ◽

2004 ◽

Vol 83 (2) ◽

pp. 134-138 ◽

Cited By ~ 73

Author(s):

S. Keila ◽

C.E. Nemcovsky ◽

O. Moses ◽

Z. Artzi ◽

M. Weinreb

Keyword(s):

Bone Marrow ◽

Fold Increase ◽

Matrix Proteins ◽

Enamel Matrix ◽

Gingival Fibroblasts ◽

Enamel Matrix Proteins ◽

Alp Activity ◽

In Vitro Effects ◽

Rat Bone

Emdogain® (EMD), a formulation of Enamel Matrix Proteins (EMP), is used clinically for periodontal regeneration, where it stimulates cementum formation and promotes gingival healing. In this study, we investigated the in vitro effects of EMD on rat bone marrow stromal cells (BMSC) and gingival fibroblasts (GF). EMD (at 25 μg/mL) increased the osteogenic capacity of bone marrow, as evidenced by ~ three-fold increase in BMSC cell number and ~ two-fold increase in alkaline phosphatase (ALP) activity and mineralized nodule formation. The presence of EMD in the initial stages (first 48 hrs) of the culture was crucial for this effect. In contrast, EMD did not induce osteoblastic differentiation of GF (evidenced by lack of mineralization or ALP activity) but increased up to two-fold both their number and the amount of matrix produced. These in vitro data on BMSC and GF could explain the promotive effect of EMD on bone formation and connective tissue regeneration, respectively.

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Targeting the Inflammatory Niche in MDS By Tasquinimod Restores Hematopoietic Support and Suppresses Immune-Checkpoint Expression in Vitro

Blood ◽

10.1182/blood-2021-150455 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2596-2596

Author(s):

Manja Wobus ◽

Ekaterina Balaian ◽

Uta Oelschlaegel ◽

Russell Towers ◽

Kristin Möbus ◽

...

Keyword(s):

Bone Marrow ◽

Mrna Expression ◽

Immunohistochemical Staining ◽

Research Funding ◽

Fold Increase ◽

Low Risk ◽

Hematopoietic Stem ◽

Healthy Donors ◽

The Impact

Abstract Introduction Myelodysplastic syndromes (MDS) belong to the most common hematological neoplasms in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. There is increasing evidence that an aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species. Tasquinimod (TASQ, Active Biotech) is a novel, oral small molecular drug with S100A9 inhibitory activity and it is currently investigated in a phase Ib/IIa trial in relapsed/refractory multiple myeloma (NCT04405167). TASQ has demonstrated anti-angiogenic, antitumor and immunomodulatory properties in a broad range of preclinical solid tumor models; however, little is known about its effects in myeloid malignancies. Aim We investigated the role of S100A9 in cellular models of MDS and the potential of TASQ to target S100A9 within the MDS stroma in vitro. Methods Immunohistochemical staining of S100A9, CD271+ mesenchymal stromal cells (MSCs), CD68+ macrophages and CD66b+ neutrophils in BM tissues from MDS patients and healthy donors was performed with multiplex immunohistochemistry and analyzed with the VECTRA imaging system. MSCs from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) in the presence or absence of TASQ (10µM). Subsequently, TLR4 downstreaming molecules such as IRAK1, gasdermin and NF-kB-p65 were analyzed by Western blot. Moreover, the mRNA expression of further proinflammatory molecules (IL-1b, IL-18, caspase1) and PD-L1 was quantified by real-time PCR. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium. Additionally, PD-1 mRNA expression was quantified in cocultured HSPCs. Results Immunohistochemical staining of BM tissue demonstrated S100A9 expression mainly by CD66b+ neutrophils and with less extent by CD68+ macrophages. In line with this, we could not detect relevant S100A9 mRNA expression in cultured MDS or healthy MSCs in vitro. Exposure of MDS and healthy MSCs with S100A9 induced TLR4 downstream signalling as demonstrated by increased expression of IRAK1 and NF-kB-p65. We further detected a higher expression of gasdermin, an inductor of pyroptosis, in S100A9 exposed MSCs. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation. Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1b and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50%. To evaluate the impact on the hematopoietic support of MSCs, we analysed MSC/HSPC cocultures after treatment with S100A9. We observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs. Interestingly, both the number of CAF-C and CFU could be increased by TASQ pre-treatment. Finally, the PD-1 expression in co-cultured HSPCs was regulated in the same way as its ligand in treated MSCs, nominating this interaction as a potential target of S100A9/TASQ in the MDS BM. Conclusion In summary, we provide evidence that the pathological inflammasome activation in the myelodysplastic bone marrow can be rescued by TASQ at least in part by inhibition of the S100A9 mediated TLR4 downstream signalling including NF-kB-p65 transcription and PD-L1 expression. These effects result in an improved hematopoietic support by MSCs, suggesting a potential efficacy to improve cytopenia in low-risk MDS patients. Disclosures Balaian: Novartis: Honoraria. Törngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. Platzbecker: AbbVie: Honoraria; Takeda: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Geron: Honoraria. Röllig: Novartis: Honoraria, Research Funding; Jazz: Honoraria; Janssen: Honoraria; Bristol-Meyer-Squibb: Honoraria, Research Funding; Amgen: Honoraria; AbbVie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

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PIM Kinase Inhibition Decreases the Proangiogenic Properties of Multiple Myeloma Cells and Affects the Metabolic State of the Vascular Endothelium

Blood ◽

10.1182/blood-2020-134979 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 16-17

Author(s):

Filip Garbicz ◽

Anna Szumera-Ciećkiewicz ◽

Joanna Barankiewicz ◽

Dorota Komar ◽

Michał Pawlak ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Endothelial Cells ◽

Cell Death ◽

Tumor Cell ◽

Cell Lines ◽

Fold Increase ◽

Pim Kinases ◽

Rpmi 8226

The development and progression of multiple myeloma (MM) depend on the formation and perpetual evolution of an immunosuppressive and hypervascular bone marrow microenvironment. MM undergoes an angiogenic switch during its early progression stages and initiates the secretion of proangiogenic proteins, such as VEGFA and Galectin-1. Following their engagement with the VEGF receptor 2 on the surface of the endothelium, quiescent endothelial cells (ECs) rapidly switch to an activated state, thus gaining the ability to create sprouts, migrate and proliferate. However, chronic angiogenic stimulation results in the formation of a dense and leaky network of pathological vessels, which in the case of MM also serves as a major source of prosurvival paracrine signals. Since PIM kinases are known modulators of cytokine signaling, owing to their ability to activate NFκB, JAK/STAT and mTOR pathways, we analyzed the expression pattern of PIM1, PIM2 and PIM3 in multiple myeloma bone marrow samples using immunohistochemistry. We found that both MM cells as well as myeloma-associated ECs exhibit a significantly higher PIM3 expression than their normal bone marrow counterparts. Since the role of PIM kinases in the vascular compartment of the tumor microenvironment is currently unknown, we decided to explore the proangiogenic functions of PIM kinases using in vitro MM and EC model cell lines. 3 MM cell lines (RPMI 8226, MM1.s, U266), immortalized bone marrow ECs (HBMEC-60) and human umbilical vein ECs (HUVECs) were used for the experiments. Primary MM cells were obtained from MACS-separated bone marrow aspirates. Chemical blockade of PIM kinase activity was achieved using the pan-PIM inhibitor SEL24/MEN1703. The compound decreased the viability of MM cell lines with IC50 in the submicromolar range, induced G2 cell cycle arrest and apoptosis. Moreover, SEL24/MEN1703 induced apoptosis in primary MM cells, even when cocultured with the CD138- bone marrow fraction. PIM inhibitor treatment inhibited the phosphorylation of mTOR substrates S6 and 4EBP1, STAT3/5, as well as RelA/p65. Consequently, we observed markedly decreased VEGFA and Gal-1 levels in SEL24/MEN1703-treated MM cells. When cultured together, separated by a permeable transwell membrane, both RPMI 8226 cells, as well as ECs, exhibited a 2-fold increase in proliferation rate. This effect was completely blocked by a 2-day treatment with a PIM inhibitor. Exposure of ECs to recombinant VEGFA (10ng/ul) or MM supernatant resulted in an increase in VEGFR2 Y1175 phosphorylation level and induction of PIM3 expression. Increased MYC activity is a hallmark of VEGF-dependent endothelial activation and is necessary to support the creation of new vessels. Since the PIM3 promoter region contains putative MYC-binding sites (E-boxes), we checked if PIM3 induction depends on MYC in ECs. MYC silencing using siRNA resulted in an 88% lower PIM3 expression than the non-targeting siRNA. One of MYC's main tasks during angiogenesis is the stimulation of cellular ATP synthesis to meet the energy demands created by the dynamic remodeling of the actin cytoskeleton. Surprisingly, PIM inhibition decreased the total ATP content in ECs by 25%, thus disrupting the energetic homeostasis, as evidenced by a 9.6-fold increase in phosphorylated AMPK T172 levels. Furthermore, SEL24/MEN1703-treated ECs were depleted of higher-order actin structures necessary for efficient angiogenesis, such as actin stress fibers, membrane ruffles and lamellipodia. In consequence, PIM kinase inhibition decreased proliferation, migration and formation of new vessel-like structures in Matrigel by ECs. Collectively, our data demonstrate that PIM inhibition induces MM cell death and abolishes important tumor cell-ECs interactions. In addition, we show that PIM3 is overexpressed in MM tumor endothelial cells and PIM inhibition disrupts the activation state in in vitro cultured ECs. Hence, targeting PIM kinases may represent an efficient approach to induce tumor cell death and to block angiogenesis in MM. RNA-sequencing studies on the downstream effectors of PIM3 are currently ongoing in order to unravel the molecular mechanism behind the observed effects. Figure Disclosures Brzózka: Ryvu Therapeutics: Current Employment. Rzymski:Ryvu Therapeutics: Current Employment. Tomirotti:Menarini Ricerche: Current Employment. Lech-Marańda:Roche, Novartis, Takeda, Janssen-Cilag, Amgen, Gilead, AbbVie, Sanofi: Consultancy; Roche, Amgen, Gilead: Speakers Bureau. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

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Pharmacological Inhibition of Nemo-like Kinase Rescues mTOR-Mediated Translation and Primes Progenitors for Leucine-Stimulated Erythroid Expansion in Pre-Clinical Models of Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2019-129570 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 455-455

Author(s):

Mark C Wilkes ◽

Jacqueline D Mercado ◽

Mallika Saxena ◽

Jun Chen ◽

Kavitha Siva ◽

...

Keyword(s):

Bone Marrow ◽

Fold Increase ◽

Healthy Controls ◽

In Vitro Activity ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Mtor Activity

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions or infections from immunosuppressive drugs and hematopoietic stem cell transplantation. Human hematopoietic stem and progenitor cells (HSPCs) from cord blood were transduced with lentiviral shRNA against a number of ribosomal genes associated with DBA, reducing the specific ribosomal protein expression by approximately 50%. During differentiation, these cells demonstrated a DBA-like phenotype with significantly reduced differentiation of erythroid progenitors (over 80%), yet only modest (15-30%) reduction of other hematopoietic lineages. NLK was immunopurifed from differentiating HSPCs and activity was assessed by the extent of in vitro phosphorylation of 3 known NLK substrates NLK, c-Myb and Raptor. As NLK activation requires phosphorylation at Thr298, we also showed that in vitro activity correlated with intracellular NLK phosphorylation by Western blot analysis. Nemo-like Kinase (NLK) was hyperactivated in the erythroid progenitors (but not other lineages), irrespective of the type of ribosomal gene insufficiency. We extended these studies using other sources of HSPCs (fetal liver, whole blood and bone marrow), along with RPS19- and RPL11-insufficient mouse models of the disease, as well as DBA patient samples. NLK was hyperactivated in erythroid progenitors from mice (5.3- and 7.2-fold increase in Raptor phosphorylation in RPS19- and RPL-11 insufficiency respectively) and from humans (7.3- and 9.0-fold in RPS19- and RPL11-insufficiency respectively) as well as HSPCs from three DBA patient (4.8-, 4.1- and 4.2-fold increase above controls). In RPS19-insufficient human HSPCs, genetic silencing of NLK increased erythroid expansion by 2.2-fold (p=0.0065), indicating that aberrant NLK activation contributes to disease pathogenesis. Furthermore, a high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increases erythroid expansion in murine (5.4-fold) and human (6.3-fold) models of DBA without effects on normal erythropoiesis (EC50: 0.7 µM). Identical results were observed in bone marrow CD34+ progenitors from three DBA patients with a 2.3 (p=0.0009), 1.9 (p=0.0007) and 2.1-fold (p=0.0001) increase in CD235+ erythroid progenitor population following NLK inhibition. In erythroid progenitors, RPS19-insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR in vitro activity by 82%. This was restored close to basal levels (93.8% of healthy control) upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. In early clinical trials, not all DBA patients have responded to leucine therapy. We hypothesize that one of the reasons might be due to NLK phosphorylation of Raptor. While leucine treatment increased mTOR activity in both RPS19-insufficient and control cells (164% of healthy controls: p=0.007 and 24% to 42% of healthy controls: p=0.0064), combining leucine with NLK inhibition increased mTOR activity in RPS19-insufficiency from 24% to 142% of control (p=0.0012). This translated to improvements in erythroid expansion of RPS19-insufficient HSPCs from 8.4% to 16.3% with leucine treatment alone, 28.4% with NLK inhibition alone, but 68.6% when leucine and NLK inhibition were combined. This 8.2-fold improvement in erythroid progenitor production indicates that identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Ap2a2 Is Crucial for LT-HSC Quiescence

Blood ◽

10.1182/blood.v128.22.2642.2642 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2642-2642

Author(s):

Stephen B Ting ◽

Sara Rhost ◽

Sarah Ghotb

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Fold Increase ◽

Wild Type ◽

Lacz Reporter ◽

Adult Survivor ◽

Self Renewal ◽

Post Transplant

Abstract Despite the relative rarity of haematopoietic stem cells (HSCs) within the blood system, functional heterogeneity is paramount to their ability to sustain lifelong blood production. The quiescent HSC sits at the functional apex possessed with self-renewal properties and the greatest repopulation output. We previously identified the gene, Ap2a2 as an enhancer of HSC function and its protein as a potential cell fate determinant in HSC asymmetric cell divisions (Ting SB et al., Blood 2012). Mechanistically, we hypothesise Ap2a2 induces a state of HSC quiescence. Using the Tet-On histone H2B-GFP mouse model (Foudi et al., Nat Biotech 2009), we have shown Ap2a2 to be highly and differentially expressed in the predominantly, G0 dormant CD150+48-LSK GFPhigh as opposed to the more cycling GFPlow HSC subpopulation. Competitive transplantation of Ap2a2- versus empty vector-transduced H2B-GFP HSCs results in a three-fold increase of the CD150+48-LSK GFPhigh HSC subpopulation. To further confirm the importance of Ap2a2 in haematopoiesis, we have constructed Ap2a2-LacZ reporter and constitutive Ap2a2 knockout (KO) mouse lines. The Ap2a2 LacZ reporter with b-galactosidase flow cytometry staining of bone marrow subpopulations confirmed high endogenous Ap2a2 expression in the CD150+48-LSK long-term (LT-) versus CD150-48-LSK short-term (ST-) repopulating HSCs. Interim analyses of the constitutive Ap2a2 KO mice have revealed two obvious phenotypes: 14% of Ap2a2-null mice termed "non-survivors" are smaller, paler with failure of fetal liver (FL) development and die between E18.5 and weaning, whilst the remaining 11% are adult viable "survivors". However, at E14.5, Ap2a2-null compared to Ap2a2-wild type fetal livers showed less absolute total FL cells but increased CD150+48-LSM FL HSCs. This was quantitatively correlated via limiting dilution assay assessed at 16 weeks post-transplant with a two-fold increase in Ap2a2-null HSC numbers (1 in 78,917 versus 1 in 150,891, p=0.027). This suggests Ap2a2 has a role in FL HSC differentiation and/or fate with potential impairment of symmetrical versus asymmetrical HSC divisions currently being studied. When E14.5 FL cells were competitively transplanted, the Ap2a2-null HSC had impaired donor reconstitution function measured at 16 weeks post-transplant (19.8% versus 48.6%, p=0.015). Ap2a2-null versus wild-type E14.5 FL cells showed equivalent numbers of primary in vitro methylcellulose colony assays but loss of secondary colonies upon re-plating indicative of loss of in-vitro HSC self-renewal. Importantly, although the Ap2a2 adult "survivors" exhibited normal quantities of bone marrow HSC subpopulations, when functionally assessed, Ap2a2-null adult "survivor" HSCs showed loss of in-vivo HSC self-renewal in secondary transplantation assays. To investigate potential cellular mechanisms, we studied the cell cycle state of Ap2a2-null and wild-type E14.5 FL cells and identified that Ap2a2-null "non-survivors" had a relative loss of quiescent G0, specifically in the LT-HSC (and not seen in the ST-HSC) subpopulation throughout all of (E14.5 to E18.5) FL development. In contrast, the LT-HSC subpopulation in FLs of Ap2a2-null "survivors" had an initial loss of G0 at E14.5 but a compensatory increase in LT-HSC G0 by E18.5. Our preliminary data suggests Ap2a2 is a crucial factor for the quiescent LT-HSC subpopulation, and we propose that both during the highly proliferative fetal liver stage of haematopoiesis and adult HSCs under stress that Ap2a2 maintains a critical balance of dormant ("deep-sleeper") HSCs to ensure global HSC function. Disclosures No relevant conflicts of interest to declare.

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