Role of inflammation in MLL-AF9 acute myeloid leukemia
The aim of this project is to study the contribution of inflammation in acute myeloid leukemia (AML) development and progression, and to investigate possible treatments. Results will lead to advances in AML knowledge and patient treatment improvement. C57BL/6 and Nes-GFP mice will be used as recipient of MLL-AF9 knock in (MLL-AF9 HE) and control (MLL-AF9 WT) bone marrow (BM) cells (n=610 in total- 450 in Tromsø and 160 in Oslo). Nes-GFP mice will allow us to study the bone marrow microenviroment in through trace of nestin+ cells. 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 not feel well for the first 7-14 days and may lose up to 26% of their body weight, which after transplant, will be mostly regained by Day 14-21 post-irradiation. 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. Inflammation/anti-inflammation will be controlled by treatment with low doses of several drugs. Some mice will be treated with anti-inflammatory drugs together with conventional chemotherapy: 5 daily doses of cytarabine (100mg/kg) + 3 daily doses of doxorubicin (3mg/kg) (Sanchez Aguilera et al., 2014). Chemotherapy is a common treatment in cancer. Chemotherapy kills mainly malignant cells which are usually the most proliferative. Chemotherapy exposure induces multiple signs of illness, including lethargy, hunched posture, rough coat, and weight loss. Measurable weight loss occurs immediately after treatment initiation. Mice will be sick after the administration of chemotherapy, and may lose 26% of their weight. A nadir is reached between days 8 and 10, with a gradual recovery to near starting levels within 2 to 3 weeks, and also it may induce regression of the leukemia or prevent its development (Lombardo and Nichols, 2009; Wunderlich et al., 2013). 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 or moribund stages. The irradiation will be performed in two half doses to reduce the adverse effects. Chemotherapy will be standard in the field of hematopoiesis. Study of the contribution of the inflammatory properties of the bone microenvironment can only be studied in vivo. The number of animals will be kept in the minimum necessary 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.
All projects involving severe procedures must be retrospectively assessed by the Norwegian Food Safety Authority.
All projects involving severe procedures must be retrospectively assessed by the Norwegian Food Safety Authority.
Begrunnelse for etterevalueringen
Acute myeloid leukaemia (AML) is a type of cancer characterized by abnormal growth of white blood cells, and it is the most common hematopoietic malignancy in adults. Gene alterations like chromosomal translocations are common in blood cancer. MLL-AF9 fusion protein results from chromosomal translocation of the mixed lineage leukemia (MLL or KMT2A) and it drives invasive AML characterized by frequent relapses and poor survival.
The purpose of this experiment has been to study the contribution of altered inflammation in AML. Induced inflammation contributes with myeloproliferative neoplasm development in a NRAS-G12D+ mouse model, but those mice do not develop AML. To study the effect of inflammation in a model of AML more like the disease in humans, C57BL/6 or Nes-GFP mice were transplanted with MLL-AF9 knock in or control bone marrow cells. MLL-AF9 knock in mice develop leukemia around six months of age, almost all AML.
Mice were myeloablated through irradiation, and bone marrow was transplanted. After transplantation, mice were treated with different drugs aiming at controlling inflammation. The mice were also treated with standard chemotherapy in combination with anti-inflammatory drugs to test their coadjuvant effect.
The overall aim of the project has been to contribute to finding new therapeutic strategies for the treatment of AML.
This experiment has not led to significant publishable findings as the protocol of treatment with IL-1 receptor antagonist did not show any effect on leukemia development.
MLL-AF9 knock in (MLL-AF9 HE) mice were used as donors for bone marrow transplantation into control C57BL/6 lethally irradiated recipients.
The recipients were afterwards treated with the anti-inflammatory drug Anakinra (IL-1 receptor antagonist) with or without chemotherapy. The experimenters did not observe any significant effect of the drug on the development of leukemia at the dose specified in the application in FOTS.
As an approved change in this project, the experimenters used frozen bone marrow from MLL-AF9 HE mice for transplantation into control C57BL/6 mice treated with succinate or vehicle and observed a worsening of the leukemia disease in succinate-treated mice.
These results have been disseminated and will be submitted for publication.
770 animals were approved for the project, which is divided between FOTS id 19472 and FOTS id 24924 (After transfer to UiT).
114 animals in total were used, 100 animals were reported in the “severe” category.
Animals reported in that category were animals terminated because they reached a humane endpoint after hematopoietic transplantation, or they were affected by severe hematological malignancy as expected from this model.
The responsible researcher states that the research group uses 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.
The research group always uses in vitro and ex vivo experiments when appropriate, before performing in vivo experiments.
However, results must always be confirmed by the gold standard method to study long-term self-renewal of hematopoietic stem cells, i.e. transplantation.
The purpose of this experiment has been to study the contribution of altered inflammation in AML. Induced inflammation contributes with myeloproliferative neoplasm development in a NRAS-G12D+ mouse model, but those mice do not develop AML. To study the effect of inflammation in a model of AML more like the disease in humans, C57BL/6 or Nes-GFP mice were transplanted with MLL-AF9 knock in or control bone marrow cells. MLL-AF9 knock in mice develop leukemia around six months of age, almost all AML.
Mice were myeloablated through irradiation, and bone marrow was transplanted. After transplantation, mice were treated with different drugs aiming at controlling inflammation. The mice were also treated with standard chemotherapy in combination with anti-inflammatory drugs to test their coadjuvant effect.
The overall aim of the project has been to contribute to finding new therapeutic strategies for the treatment of AML.
This experiment has not led to significant publishable findings as the protocol of treatment with IL-1 receptor antagonist did not show any effect on leukemia development.
MLL-AF9 knock in (MLL-AF9 HE) mice were used as donors for bone marrow transplantation into control C57BL/6 lethally irradiated recipients.
The recipients were afterwards treated with the anti-inflammatory drug Anakinra (IL-1 receptor antagonist) with or without chemotherapy. The experimenters did not observe any significant effect of the drug on the development of leukemia at the dose specified in the application in FOTS.
As an approved change in this project, the experimenters used frozen bone marrow from MLL-AF9 HE mice for transplantation into control C57BL/6 mice treated with succinate or vehicle and observed a worsening of the leukemia disease in succinate-treated mice.
These results have been disseminated and will be submitted for publication.
770 animals were approved for the project, which is divided between FOTS id 19472 and FOTS id 24924 (After transfer to UiT).
114 animals in total were used, 100 animals were reported in the “severe” category.
Animals reported in that category were animals terminated because they reached a humane endpoint after hematopoietic transplantation, or they were affected by severe hematological malignancy as expected from this model.
The responsible researcher states that the research group uses 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.
The research group always uses in vitro and ex vivo experiments when appropriate, before performing in vivo experiments.
However, results must always be confirmed by the gold standard method to study long-term self-renewal of hematopoietic stem cells, i.e. transplantation.