Cell death facilitates the removal of infected cells; it can be triggered by many stimuli, which activate numerous biochemical pathways that lead to apoptotic (immunologically silent), as well as non-apoptotic (including pyroptosis or Caspase-1-dependent, and necroptosis or RIPK3/MLKL-dependent) forms of death (1) (Figure 1). Recently we revealed a new role for pyroptosis in hematopoietic stem and progenitor cells (HSPC) during hematopoietic stress induced by viral infection and chemotherapy causing cytopenia, immunosuppression and bone marrow failure (2). We now present preliminary data, currently in press at Cell, that RIPK3-dependent necroptotic cell death limits the self-renewal capacity of LT-HSC in lethally-irradiated recipient mice (Figure 2). This result unveils non-apoptotic cell death as a key biological process restricting HSC self-renewal, and has major clinical implications for improving the engraftment potential of HSC in transplantation settings using bone marrow, G-CSF-mobilized peripheral blood stem cells, cord blood units, and for gene therapy clinical trials. These data will change our view of one of the cornerstones of HSC biology: the self-renewal of HSC and how necroptosis impacts lifelong hematopoiesis and immunity. Our extensive preliminary data also establishes RIPK1 as a negative regulator of RIPK3/MLKL-dependent necroptosis. To access necroptotic cell death pathways, most previous studies utilize caspase inhibitors to block Caspase-8-mediated inhibition of RIPK3. Here we can now investigate necroptosis without treating primary cells or mice with caspase inhibitors, heralding a new approach to understanding necroptosis and the possible interaction of this non-apoptotic cell death pathway with executioner caspases such as caspase-3. This application seeks to use these necroptotic Ripk1-/- chimeric mice to specifically investigate the physiological consequences of necroptosis and its negative regulation by RIPK1 in hematopoieisis. We will investigate the following specific aims to understand how necroptosis affects HSPC:
Aim 1. Define the role of RIPK1 and MLKL in the self-renewal of HSC and recovery from chemotherapy.
Aim 2. Biochemical, morphological, and kinetic analysis of necroptosis ex vivo.
Aim 3. Study the role of RIPK1 and MLKL in viral-infected HSPC.

Public Health Relevance

Bone marrow and peripheral blood stem cell transplantation is a major treatment modality for cancer patients that have undergone hemoablative chemotherapy. Engraftment of autologous or allogeneic hematopoietic stem cells in patients takes several weeks, however, full immunocompetence can take up to 2 years leaving patients vulnerable to life-threatening infection. The mechanisms that restrict the efficient engraftment of hematopoietic stem cells (HSC) and their subsequent expansion to maintain lifelong hematopoiesis and immune competence are still largely undefined. This research investigates the role of necroptosis in restricting the engraftment and self-renewal of HSC.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL124209-05
Application #
9618271
Study Section
Molecular and Cellular Hematology Study Section (MCH)
Program Officer
Yang, Yu-Chung
Project Start
2014-12-01
Project End
2019-02-28
Budget Start
2018-12-01
Budget End
2019-02-28
Support Year
5
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Boston Children's Hospital
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Waight, Jeremy D; Chand, Dhan; Dietrich, Sylvia et al. (2018) Selective Fc?R Co-engagement on APCs Modulates the Activity of Therapeutic Antibodies Targeting T Cell Antigens. Cancer Cell 33:1033-1047.e5
D'Cruz, Akshay A; Speir, Mary; Bliss-Moreau, Meghan et al. (2018) The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils. Sci Signal 11:
Hutton, Melanie L; D'Costa, Kimberley; Rossiter, Amanda E et al. (2017) A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization. Front Cell Infect Microbiol 7:219
Bliss-Moreau, Meghan; Chen, Alyce A; D'Cruz, Akshay A et al. (2017) A motive for killing: effector functions of regulated lytic cell death. Immunol Cell Biol 95:146-151
Zargarian, Sefi; Shlomovitz, Inbar; Erlich, Ziv et al. (2017) Phosphatidylserine externalization, ""necroptotic bodies"" release, and phagocytosis during necroptosis. PLoS Biol 15:e2002711
McArthur, Kate; D'Cruz, Akshay A; Segal, David et al. (2017) Defining a therapeutic window for kinase inhibitors in leukemia to avoid neutropenia. Oncotarget 8:57948-57963
Gerlic, Motti; Croker, Ben A (2017) Myelopoiesis embraces its inner weakness. Nat Immunol 18:953-954
Croker, Ben A; Silke, John; Gerlic, Motti (2015) Fight or flight: regulation of emergency hematopoiesis by pyroptosis and necroptosis. Curr Opin Hematol 22:293-301