Role of succinate in acute myeloid leukemia
The aim of this project is to study the contribution of succinate in acute myeloid leukemia (AML) development and to investigate possible treatments. Results will lead to advances in AML knowledge and potential patient treatment improvement. To explore the role of succinate in various aspects of hematopoetic stem cell (HSC) biology, we will assess the impact of succinate injections in the hematopoietic system of adult healthy and leukemic mice. Mice models knockout for succinate receptor (McCreath et al., 2015, Diabetes) are needed to examine whether Sucnr1 aids AML development and whether it could be a new therapeutic target. 5-fluorouracil (5-FU) challenge in C57BL/6 and Sucnr1 -/- mice will allow us to study whether succinate induces changes in HSC proliferation and self-renewal under stress hematopoiesis (Cheng T et al., 2000, Science). Despite mild toxicity of 5FU (Sanchez-Aguilera et al., 2014, Cell Stem Cell), this is an approved cancer treatment drug regularly used in humans. Our findings will reveal for the first time the contribution of SUCNR1 receptor in bone marrow (BM) microenvironment. Mice will be taken when they show early signs of frailty to prevent unnecessary suffering and a Kaplan-Meier curve will describe survival characteristics. C57BL/6, Sucnr1 -/- and Sucnr1 tomato mice will be used as recipient of control and mutant BM cells from Mx1-Cre NRAS-G12D mice. Recipient mice with a deletion of the Sucnr1 gene in the BM microenvironment will also be used as recipients of transplantation to understand the specific role of this receptor in the BM microenvironment. Conversely, succinate receptor deletion will be performed selectively in the hematopoietic system to understand the cell-autonomous effect in leukemia. Prior to transplantation, recipient mice need to be myeloablated through whole body 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, and simultaneously promotes proliferation of transplanted cells. Mice might suffer from a temporary lose of their body weight up to 26. Animals should be on their way to recovery at day 21 (cut-off weight loss 15%), and recovered at day 30 (cut-off weight loss 10%). All mice will be transplanted after irradiation, done in two half doses to reduce adverse effects. The results will provide a novel platform for more efficient therapies against AML, one of the most aggressive and frequent blood disorder that affects adults and children. It is not the goal of this study to analyze terminal stages.The number of animals will be kept in the minimum necessary (n=1020 in total, done in both locations; Oslo and Tromsø) to obtain statistically meaningful results, using them in the most efficient way possible.
Etterevaluering
We classify the procedures as severe, cf. the Regulation annex B. Humane endpoints are clear and set as early as possible, but the planned procedures will lead to significally reduced welfare for the most affected mice. All projects involving severe procedures must be retrospectively assessed by the Norwegian Food Safety Authority.
Begrunnelse for etterevalueringen
Acute myeloid leukemia (AML) is the most common hematopoietic malignancy in adults, and it is characterized by abnormal growth of white blood cells.
Blood cells express SUCNR1, but its role in healthy and malignant hematopoiesis is unknown.
The purpose of these experiments has been to study the contribution of succinate in acute myeloid leukemia (AML) development.
To explore the role of succinate in various aspects of hematopoietic stem cell (NSC) biology, healthy and leukemic mice received succinate injections.
Both wild-type mice and genetically altered mice were used in the project.
The overall aim of the project has been to contribute to finding new therapeutic strategies for the treatment of AML.
The project was transferred to UiT (FOTS ID 24739) when the group left the animal facility of UiO to move back to AKM UiT.
In vivo experiments performed at UiO and UiT after the transfer of the project, have allowed the experimenters to understand the signaling pathway of succinate receptor in hematopoietic cells and the role of its alterations in malignancy.
Injection of succinate in pre-leukemic Mx1-Cre Nras mice allowed the experimenters to describe the role of extracellular succinate in the promotion of the disease. Using succinate-receptor KO mice, they have identified a new function of succinate and its receptor in the hematopoietic system, both in health and malignancy.
These results may lead to novel targets in the treatment of blood malignancy.
In addition to the parameters proposed in the FOTS application, the experimenters have obtained RNA-seq and single-cell RNA-seq data of FACS-sorted hematopoietic stem and progenitor cells from Sucnr1-KO and WT mice.
These data will be publicly available with the publication of the results.
The total number of animals approved in the project, which has been divided between FOTS id 13407 and FOTS id 24739, was 760.
504 animals were used, including 58 reported in the “severe” category.
Animals which were reported in that category were animals terminated because they reached a humane endpoint after a transplantation experiment, or because they were affected by severe hematological malignancy.
The responsible researcher informs that the experimenters have reduced the number of animals as much as possible by excluding outlier animals before starting in vivo experiments to improve statistic power and prevent repetition.
Transplantation has only been performed by experienced researchers, and animals have been followed up closely with use of score sheets.
The responsible researcher states that it would not be possible to replace the use of animals in this research project. The hematopoietic system is studied in vivo, due to limitations in the knowledge of the stem cell niche and its reproducibility in vitro.
The experimenters use replacement techniques as much as possible, such as replating of CFU-C assays to study long-term hematopoietic potential of hematopoietic stem cells ex vivo.
However, results must always be confirmed by the gold standard method to study long-term self-renewal of hematopoietic stem cells, i.e., transplantation.
Blood cells express SUCNR1, but its role in healthy and malignant hematopoiesis is unknown.
The purpose of these experiments has been to study the contribution of succinate in acute myeloid leukemia (AML) development.
To explore the role of succinate in various aspects of hematopoietic stem cell (NSC) biology, healthy and leukemic mice received succinate injections.
Both wild-type mice and genetically altered mice were used in the project.
The overall aim of the project has been to contribute to finding new therapeutic strategies for the treatment of AML.
The project was transferred to UiT (FOTS ID 24739) when the group left the animal facility of UiO to move back to AKM UiT.
In vivo experiments performed at UiO and UiT after the transfer of the project, have allowed the experimenters to understand the signaling pathway of succinate receptor in hematopoietic cells and the role of its alterations in malignancy.
Injection of succinate in pre-leukemic Mx1-Cre Nras mice allowed the experimenters to describe the role of extracellular succinate in the promotion of the disease. Using succinate-receptor KO mice, they have identified a new function of succinate and its receptor in the hematopoietic system, both in health and malignancy.
These results may lead to novel targets in the treatment of blood malignancy.
In addition to the parameters proposed in the FOTS application, the experimenters have obtained RNA-seq and single-cell RNA-seq data of FACS-sorted hematopoietic stem and progenitor cells from Sucnr1-KO and WT mice.
These data will be publicly available with the publication of the results.
The total number of animals approved in the project, which has been divided between FOTS id 13407 and FOTS id 24739, was 760.
504 animals were used, including 58 reported in the “severe” category.
Animals which were reported in that category were animals terminated because they reached a humane endpoint after a transplantation experiment, or because they were affected by severe hematological malignancy.
The responsible researcher informs that the experimenters have reduced the number of animals as much as possible by excluding outlier animals before starting in vivo experiments to improve statistic power and prevent repetition.
Transplantation has only been performed by experienced researchers, and animals have been followed up closely with use of score sheets.
The responsible researcher states that it would not be possible to replace the use of animals in this research project. The hematopoietic system is studied in vivo, due to limitations in the knowledge of the stem cell niche and its reproducibility in vitro.
The experimenters use replacement techniques as much as possible, such as replating of CFU-C assays to study long-term hematopoietic potential of hematopoietic stem cells ex vivo.
However, results must always be confirmed by the gold standard method to study long-term self-renewal of hematopoietic stem cells, i.e., transplantation.