Anti-inflammatory properties of the bone marrow niche in NRAS-G12D+ leukaemia
This is an application for an extension of the FOTS8252. We will use a number of animals Close to that approved previously; 160. Due to several Challenges including the current pandemic and the working conditions of my Group split in two locations, we need to start, repeat and finish some of our experiments that have been stopped.
We aim to test the contribution of the anti-inflammatory Properties of the bone marrow microenvironment to acute myeloid leukemia (AML). Results will lead to advances in AML knowledge and patient treatment improvement. IL -1 receptor antagonist (IL-1ra) knock-out (KO) and control mice will be used as recipients of mutant Nras G12D and control cells (n=80 mice in total), and changes in disease will be monitored. Recipient mice need to be myeloablated previous to transplantation, through irradiation. Total Whole body irradiation is used in the Field of blood stem cell function and cancer (for more information: http://www.bu.edu/orccommittees/iacuc/policies-and-guidelines/irradiation-of-rodents/). It allows to kill proliferating blood cells without significant damage of resting tissues. Depletion of cells together With factors released allows guidance of transplanted cells and forces their proliferation. This is the only way to study blood stem cell function in vivo. Mice might not feel well for the first 7-14 days and may lose up to 25% of their body weight, which after transplant, will be mostly regained by Day 14-21 post-irradiation. All mice will be transplanted after irradiation.
Besides, our previous observations show an increase of IL-1 signaling through IL-1R after IL-1ra deletion using in IL-1ra KO mice (analyzed in vitro under A015). Activation of IL-1R in hematopoietic stem cells (HCS) drives transcription of nuclear factor- kappa B NF-kB; important for HSC homeostasis (Nagai et al., 2006; Esplin et al.,2011; Schuettpelz et al., 2014; Herman et al., 2016; Pietras et al., 2016). Conversely, aberrant expression of NF-kB takes place in leukemia (Zhou et al., 2015) and other cancers and, targeting NF-kB pathway is used as treatment against different cancers (Braud et al., 2009; Karin et al., 2004). Bortezomib, is a proteasome inhibitor that, ultimately, inhibits NF-kB activity (Hideshima et al., 2009; Xue et al., 2011; Fang et al., 2012; Zhou et al., 2015). Using this therapeutic approach, bortezomib, we intend to rescue IL-1ra KO mice phenotype which show a mild myeloproliferative disease with a differentiation of HSC biased to the myeloid compartment (analyzed in vitro under A015). IL-1ra KO mice will be i.p. injected once per week for a maximum of 4 months.This procedure does not cause any stress or discomfort to the mice. A total of 80 mice will be used for this Experiment.
Total numbers in Tromsø will be 100 and in Oslo 60.
The results will provide a novel platform for more efficient therapies against AML, one of the most aggressive and frequent blood disorders that affects adults and children. It is not the goal of this study to analyze terminal stages. Mice will be used in the most efficient way and numbers will be kept to minimum. The irradiation will be performed in two half doses to reduce the adverse effects.
We aim to test the contribution of the anti-inflammatory Properties of the bone marrow microenvironment to acute myeloid leukemia (AML). Results will lead to advances in AML knowledge and patient treatment improvement. IL -1 receptor antagonist (IL-1ra) knock-out (KO) and control mice will be used as recipients of mutant Nras G12D and control cells (n=80 mice in total), and changes in disease will be monitored. Recipient mice need to be myeloablated previous to transplantation, through irradiation. Total Whole body irradiation is used in the Field of blood stem cell function and cancer (for more information: http://www.bu.edu/orccommittees/iacuc/policies-and-guidelines/irradiation-of-rodents/). It allows to kill proliferating blood cells without significant damage of resting tissues. Depletion of cells together With factors released allows guidance of transplanted cells and forces their proliferation. This is the only way to study blood stem cell function in vivo. Mice might not feel well for the first 7-14 days and may lose up to 25% of their body weight, which after transplant, will be mostly regained by Day 14-21 post-irradiation. All mice will be transplanted after irradiation.
Besides, our previous observations show an increase of IL-1 signaling through IL-1R after IL-1ra deletion using in IL-1ra KO mice (analyzed in vitro under A015). Activation of IL-1R in hematopoietic stem cells (HCS) drives transcription of nuclear factor- kappa B NF-kB; important for HSC homeostasis (Nagai et al., 2006; Esplin et al.,2011; Schuettpelz et al., 2014; Herman et al., 2016; Pietras et al., 2016). Conversely, aberrant expression of NF-kB takes place in leukemia (Zhou et al., 2015) and other cancers and, targeting NF-kB pathway is used as treatment against different cancers (Braud et al., 2009; Karin et al., 2004). Bortezomib, is a proteasome inhibitor that, ultimately, inhibits NF-kB activity (Hideshima et al., 2009; Xue et al., 2011; Fang et al., 2012; Zhou et al., 2015). Using this therapeutic approach, bortezomib, we intend to rescue IL-1ra KO mice phenotype which show a mild myeloproliferative disease with a differentiation of HSC biased to the myeloid compartment (analyzed in vitro under A015). IL-1ra KO mice will be i.p. injected once per week for a maximum of 4 months.This procedure does not cause any stress or discomfort to the mice. A total of 80 mice will be used for this Experiment.
Total numbers in Tromsø will be 100 and in Oslo 60.
The results will provide a novel platform for more efficient therapies against AML, one of the most aggressive and frequent blood disorders that affects adults and children. It is not the goal of this study to analyze terminal stages. Mice will be used in the most efficient way and numbers will be kept to minimum. The irradiation will be performed in two half doses to reduce the adverse effects.
Etterevaluering
We classify the severity of the procedures as severe, cf. the Regulation annex B.
Our evaluation is based on these main points:
Clinical signs of leukemia will probably not appear during the experimental period.
The humane endpoints are based on the observation of a leukaemic transformation in blood samples.
Clinical signs and other relevant parameters will be registered in score sheets adapted to the planned procedures.
Mice will be housed in groups.
However: the mice will go through whole body irradiation for myeloablation. The irradiation will be done in two half doses in order to reduce the adverse effects, but it is unavoidable that the mice become ill from irradiation sickness for 1-2 weeks following the procedure.
Our severity classification is based on the most severe effects likely to be experienced by an individual animal after all refinements have been applied.
The Norwegian Food Safety Authority must retrospectively assess all severe experiments.
Our evaluation is based on these main points:
Clinical signs of leukemia will probably not appear during the experimental period.
The humane endpoints are based on the observation of a leukaemic transformation in blood samples.
Clinical signs and other relevant parameters will be registered in score sheets adapted to the planned procedures.
Mice will be housed in groups.
However: the mice will go through whole body irradiation for myeloablation. The irradiation will be done in two half doses in order to reduce the adverse effects, but it is unavoidable that the mice become ill from irradiation sickness for 1-2 weeks following the procedure.
Our severity classification is based on the most severe effects likely to be experienced by an individual animal after all refinements have been applied.
The Norwegian Food Safety Authority must retrospectively assess all severe experiments.
Begrunnelse for etterevalueringen
The experimenters used 77 IL-1ra KO and control mice either for transplantation assays or treatment with bortezomib (or vehicle).
The purpose of the project has been to study the contribution of an altered anti-inflammatory bone marrow microenvironment to acute myeloid leukemia (AML), with an overall aim to contribute to finding new therapeutic strategies for the treatment of AML.
The study of the hematopoietic system dynamics can only be done in a live organism due to the complex nuances and insufficient scientific knowledge of the bone marrow hematopoietic stem cell niche.
The results obtained so far are crucial to the project and show that 1) deficiency in IL-1ra in the stromal compartment may contribute to Nras-G12D+ leukemia, 2) treatment of IL-1ra-KO mice with bortezomib (nuclear factor- kappa B (NFkB) inhibitor), rescues the myeloproliferative disease observed in IL-1ra KO mice.
NrasG12D expression induces myeloproliferation in C57BL/6 mice which can be followed up by increased myeloid cell number in peripheral blood. To test the contribution of an altered anti-inflammatory microenvironment to leukemia, IL -1 receptor antagonist (IL-1ra) knock-out (KO) and wild type mice (C57BL/6 mice) were used as recipients of mutant Mx1-cre Nras G12D and control cells, and changes in disease progression were monitored in peripheral blood.
Recipient mice were myeloablated previous to transplantation, through irradiation. Total Whole body irradiation was performed and followed by transplantation. Irradiation allowed to kill proliferating blood cells without significant damage of resting tissues. Depletion of cells together with factors released allows guidance of transplanted cells and forces their proliferation. This is the only way to study hematopoietic stem cell function in vivo.
Wild type and IL-1ra KO recipients transplanted with mutant Mx1-cre Nras G12D cells showed higher number of myeloid cells than IL-1ra KO mice transplanted with control cells. Further, IL-1ra KO mice transplanted with mutant Mx1-cre Nras G12D cells showed higher number of myeloid cells compared to wild type mice transplanted with mutant cells. These results suggest that deletion of IL-1ra in the stromal compartment may contribute to Nras-G12D+ leukemia.
Previous observations showed myeloproliferation and an increase of IL-1 signaling through IL-1 receptor (IL-1R1) after IL-1ra deletion, driving activation of transcription of nuclear factor- kappa B (NFkB) in hematopoietic stem cells (HSCs). This project was extended to investigate the therapeutic potential of NFkB inhibition. To do this, the experimenters used bortezomib, a proteasome inhibitor that ultimately inhibits NF-kB activity. With this therapeutic approach, bortezomib, the experimenters aimed at rescuing the phenotype of IL-1ra KO mice showing mild myeloproliferative disease with HSC differentiation biased towards the myeloid compartment (analyzed in vitro under A015). IL-1ra-KO mice treated with bortezomib showed lower number of myeloid cells in peripheral blood.
The experimenters will have to repeat the reciprocal transplants for further characterization of the molecular mechanisms involved. Validation of the results obtained with bortezomib treatment is necessary to draw thorough conclusions.
FOTS id 24009 was an extension of FOTS id 8252.
The Animal Facility at the University of Tromsø closed for remodeling in March 2017, and the research project was moved to the University of Oslo.
Because of the moving, there was not enough time to conclude the experiments in FOTS id 24009, but the experiments were continued in Tromsø under FOTS id 24736.
Whole body irradiation makes the mice ill, and 50 mice were reported with severe negative impact.
Most of these mice regained their weight and recovered 14-21 days post-irradiation. The mice were given wet food to make food and water intake easier.
A cumulative score sheet was used daily for 21 days after irradiation, to carefully minimize the suffering.
Most of the mice treated with bortezomib were reported with moderate severity.
A cumulative score sheet was used daily to safeguard the welfare of the mice in the bortezomib experiment. Given that bortezomib is a proteasome inhibitor that inhibits NFkB activation classified as chemotherapy (targeted therapy), these animals were followed up thoroughly.
The purpose of the project has been to study the contribution of an altered anti-inflammatory bone marrow microenvironment to acute myeloid leukemia (AML), with an overall aim to contribute to finding new therapeutic strategies for the treatment of AML.
The study of the hematopoietic system dynamics can only be done in a live organism due to the complex nuances and insufficient scientific knowledge of the bone marrow hematopoietic stem cell niche.
The results obtained so far are crucial to the project and show that 1) deficiency in IL-1ra in the stromal compartment may contribute to Nras-G12D+ leukemia, 2) treatment of IL-1ra-KO mice with bortezomib (nuclear factor- kappa B (NFkB) inhibitor), rescues the myeloproliferative disease observed in IL-1ra KO mice.
NrasG12D expression induces myeloproliferation in C57BL/6 mice which can be followed up by increased myeloid cell number in peripheral blood. To test the contribution of an altered anti-inflammatory microenvironment to leukemia, IL -1 receptor antagonist (IL-1ra) knock-out (KO) and wild type mice (C57BL/6 mice) were used as recipients of mutant Mx1-cre Nras G12D and control cells, and changes in disease progression were monitored in peripheral blood.
Recipient mice were myeloablated previous to transplantation, through irradiation. Total Whole body irradiation was performed and followed by transplantation. Irradiation allowed to kill proliferating blood cells without significant damage of resting tissues. Depletion of cells together with factors released allows guidance of transplanted cells and forces their proliferation. This is the only way to study hematopoietic stem cell function in vivo.
Wild type and IL-1ra KO recipients transplanted with mutant Mx1-cre Nras G12D cells showed higher number of myeloid cells than IL-1ra KO mice transplanted with control cells. Further, IL-1ra KO mice transplanted with mutant Mx1-cre Nras G12D cells showed higher number of myeloid cells compared to wild type mice transplanted with mutant cells. These results suggest that deletion of IL-1ra in the stromal compartment may contribute to Nras-G12D+ leukemia.
Previous observations showed myeloproliferation and an increase of IL-1 signaling through IL-1 receptor (IL-1R1) after IL-1ra deletion, driving activation of transcription of nuclear factor- kappa B (NFkB) in hematopoietic stem cells (HSCs). This project was extended to investigate the therapeutic potential of NFkB inhibition. To do this, the experimenters used bortezomib, a proteasome inhibitor that ultimately inhibits NF-kB activity. With this therapeutic approach, bortezomib, the experimenters aimed at rescuing the phenotype of IL-1ra KO mice showing mild myeloproliferative disease with HSC differentiation biased towards the myeloid compartment (analyzed in vitro under A015). IL-1ra-KO mice treated with bortezomib showed lower number of myeloid cells in peripheral blood.
The experimenters will have to repeat the reciprocal transplants for further characterization of the molecular mechanisms involved. Validation of the results obtained with bortezomib treatment is necessary to draw thorough conclusions.
FOTS id 24009 was an extension of FOTS id 8252.
The Animal Facility at the University of Tromsø closed for remodeling in March 2017, and the research project was moved to the University of Oslo.
Because of the moving, there was not enough time to conclude the experiments in FOTS id 24009, but the experiments were continued in Tromsø under FOTS id 24736.
Whole body irradiation makes the mice ill, and 50 mice were reported with severe negative impact.
Most of these mice regained their weight and recovered 14-21 days post-irradiation. The mice were given wet food to make food and water intake easier.
A cumulative score sheet was used daily for 21 days after irradiation, to carefully minimize the suffering.
Most of the mice treated with bortezomib were reported with moderate severity.
A cumulative score sheet was used daily to safeguard the welfare of the mice in the bortezomib experiment. Given that bortezomib is a proteasome inhibitor that inhibits NFkB activation classified as chemotherapy (targeted therapy), these animals were followed up thoroughly.