A new humanized in vivo model of KIT D816V+ advanced systemic mastocytosis monitored using a secreted luciferase

Systemic mastocytosis are rare neoplasms characterized by accumulation of mast cells in at least one internal organ. The majority of systemic mastocytosis patients carry KIT D816V mutation, which activates constitutively the KIT receptor. Patient with advanced forms of systemic mastocytosis, such as aggressive systemic mastocytosis or mast cell leukemia, are poorly treated to date. Unfortunately, the lack of in vivo models reflecting KIT D816V+ advanced disease hampers pathophysiological studies and preclinical development of new therapies for such patients. Here, we describe a new in vivo model of KIT D816V+ advanced systemic mastocytosis developed by transplantation of the human ROSAKIT D816V-Gluc mast cell line in NOD-SCID IL-2R g−/− mice, using Gaussia princeps luciferase as a reporter. Intravenous injection of ROSAKIT D816V-Gluc cells led, in 4 weeks, to engraftment in all injected primary recipient mice. Engrafted cells were found at high levels in bone marrow, and at lower levels in spleen, liver and peripheral blood. Disease progression was easily monitored by repeated quantification of Gaussia princeps luciferase activity in peripheral blood. This quantification evidenced a linear relationship between the number of cells injected and the neoplastic mast cell burden in mice. Interestingly, the secondary transplantation of ROSAKIT D816V-Gluc cells increased their engraftment capability. To conclude, this new in vivo model mimics at the best the features of human KIT D816V+ advanced systemic mastocytosis. In addition, it is a unique and convenient tool to study the kinetics of the disease and the potential in vivo activity of new drugs targeting neoplastic mast cells

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes β2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced β3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with β3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.Y.-H.O. received fellowships from Alborada Scholar-ship (University of Cambridge), Trinity-Henry Barlow Scholarship (Universityof Cambridge), and R.O.C. Government Scholarship to Study Abroad (GSSA). A.G.G. received fellowships from the Ramon Areces Foundationand the LaCaixa Foundation. C.K. was supported by Marie Curie Career Inte-gration (H2020-MSCA-IF-2015-70841). S.M.-F. was supported by Red TerCel (ISCIII-Spanish Cell Therapy Network). V.A. is supported by grants from theSpanish Ministerio de Economıa,Industria y Competitividad (MEIC) with co-funding from the Fondo Europeo de Desarrollo Regional (FEDER, ‘‘Una manerade hacer Europa’’) (SAF2016-79490-R), the Instituto de Salud Carlos III (AC16/00091 and AC17/00067), the Fundacio Marato TV3 (122/C/2015), and the Progeria Research Foundation (Established Investigator Award 2014–52). TheCNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovacion y Universidades (MCIU), and the Pro CNIC Foundation,and is a Severo Ochoa Center of Excellence (SEV-2015-0505). This work wassupported by core support grants from the Wellcome Trust and the MRC to theCambridge Stem Cell Institute, MEIC (SAF-2011-30308), Ramon y Cajal Program Grant (RYC-2009-04703), ConSEPOC-Comunidad de Madrid (S2010/BMD-2542), National Health Service Blood and Transplant (United Kingdom), European Union’s Horizon 2020 research (ERC-2014-CoG-64765 and MarieCurie Career Integration grant FP7-PEOPLE-2011-RG-294096), and a Programme Foundation Award from Cancer Research UK to S.M.-F., who wasalso supported in part by an International Early Career Scientist grant fromthe Howard Hughes Medical Institute.S

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging.

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes β2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced β3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with β3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.We thank A.R. Green for advice and support; M. García-Fernández, C. Fielding, C. Kapeni, X. Langa, and other current and former members of the S.M.-F group for help and discussions; A. Barettino and A. Macías (CNIC), D. Pask, T. Hamilton, the Central Biomedical Services and Cambridge NIHR BRC Cell Phenotyping Hub for technical assistance; H. Jolin and A.N.J. McZenzie (MRC Laboratory of Molecular Biology, Cambridge, UK) for help with milliplex analyses. Y.-H.O. received fellowships from Alborada Scholarship (University of Cambridge), Trinity-Henry Barlow Scholarship (University of Cambridge) and R.O.C. Government Scholarship to Study Abroad (GSSA) A.G.G. received fellowships from Ramón Areces and LaCaixa Foundations. C.K. was supported by Marie Curie Career Integration grant H2020-MSCA-IF-2015-70841. S.M.F., by Red TerCel (ISCIII-Spanish Cell Therapy Network). VA is supported by grants from the Spanish Ministerio de Economía, Industria y Competitividad (MEIC) with cofunding from the Fondo Europeo de Desarrollo Regional (FEDER, “Una manera de hacer Europa”) (SAF2016-79490-R), the Instituto de Salud Carlos III (AC16/00091), the Fundació Marató TV3 (122/C/2015), and the Progeria Research Foundation (Established Investigator Award 2014–52). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). This work was supported by core support grants from the Wellcome Trust and the MRC to the Cambridge Stem Cell Institute, the Spanish Ministry of Economy and Competitiveness (SAF-2011-30308), Ramón y Cajal Program grant RYC-2009-04703, ConSEPOC-Comunidad de Madrid S2010/BMD-2542, National 427 Health Service Blood and Transplant (United Kingdom), European Union’s Horizon 428 2020 research (ERC-2014-CoG-64765 and Marie Curie Career Integration grant FP7- 429 PEOPLE-2011-RG-294096) and a Programme Foundation Award from Cancer Research 430 UK to S.M.-F., who was also supported in part by an International Early Career Scientist 431 grant of the Howard Hughes Medical Institute

An activating mutation in the CSF3R gene induces a hereditary chronic neutrophilia

We identify an autosomal mutation in the CSF3R gene in a family with a chronic neutrophilia. This T617N mutation energetically favors dimerization of the granulocyte colony-stimulating factor (G-CSF) receptor transmembrane domain, and thus, strongly promotes constitutive activation of the receptor and hypersensitivity to G-CSF for proliferation and differentiation, which ultimately leads to chronic neutrophilia. Mutant hematopoietic stem cells yield a myeloproliferative-like disorder in xenotransplantation and syngenic mouse bone marrow engraftment assays. The survey of 12 affected individuals during three generations indicates that only one patient had a myelodysplastic syndrome. Our data thus indicate that mutations in the CSF3R gene can be responsible for hereditary neutrophilia mimicking a myeloproliferative disorder

The Chemokine Receptor CXCR4 Strongly Promotes Neuroblastoma Primary Tumour and Metastatic Growth, but not Invasion

Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers

Novel Anti-Metastatic Action of Cidofovir Mediated by Inhibition of E6/E7, CXCR4 and Rho/ROCK Signaling in HPV+ Tumor Cells

Cervical cancer is frequently associated with HPV infection. The expression of E6 and E7 HPV oncoproteins is a key factor in its carcinogenicity and might also influence its virulence, including metastatic conversion. The cellular mechanisms involved in metastatic spread remain elusive, but pro-adhesive receptors and their ligands, such as SDF-1α and CXCR4 are implicated. In the present study, we assessed the possible relationship between SDF-1α/CXCR4 signaling, E6/E7 status and the metastatic process. We found that SDF-1α stimulated the invasion of E6/E7-positive cancer cell lines (HeLa and TC-1) in Matrigel though CXCR4 and subsequent Rho/ROCK activation. In pulmonary metastatic foci generated by TC-1 cells IV injection a high proportion of cells expressed membrane-associated CXCR4. In both cases models (in vitro and in vivo) cell adhesion and invasion was abrogated by CXCR4 immunological blockade supporting a contribution of SDF-1α/CXCR4 to the metastatic process. E6 and E7 silencing using stable knock-down and the approved anti-viral agent, Cidofovir decreased CXCR4 gene expression as well as both, constitutive and SDF-1α-induced cell invasion. In addition, Cidofovir inhibited lung metastasis (both adhesion and invasion) supporting contribution of E6 and E7 oncoproteins to the metastatic process. Finally, potential signals activated downstream SDF-1α/CXCR4 and involved in lung homing of E6/E7-expressing tumor cells were investigated. The contribution of the Rho/ROCK pathway was suggested by the inhibitory effect triggered by Cidofovir and further confirmed using Y-27632 (a small molecule ROCK inhibitor). These data suggest a novel and highly translatable therapeutic approach to cervix cancer, by inhibition of adhesion and invasion of circulating HPV-positive tumor cells, using Cidofovir and/or ROCK inhibition

Etude de la fonction et de la régulation du récepteur CXCR4 dans l'hématopoïèse

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Régulation de l'expression et la fonction de CXCR4 dans les progéniteurs hématopoïétiques humains normaux

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Rôle de l'oxyde nitrique dans la régulation des cellules souches hématopoétiques

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF