Periodic Reporting for period 4 - ApoptoMDS (Hematopoietic stem cell Apoptosis in bone marrow failure and MyeloDysplastic Syndromes: Friend or foe?)
Okres sprawozdawczy: 2019-12-01 do 2020-05-31
Patients with inherited bone marrow failure syndromes such as Dyskeratosis congenita and Fanconi anemia do not only develop hematopoietic failure with anemia, bleeding tendency and susceptibility to infection, but are also at risk to develop myeloid malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Once the malignancy arises, the prognosis of the patients is poor and strategies are required to prevent rather than to treat malignant transformation. While hematopoietic cells of affected patients are particularly sensitive to cell death signals, cells that are resistant to apoptosis (a form of programmed cell death) are selected during the process of malignant transformation.
We were analyzing the cell death mechanisms in inherited bone marrow failure syndromes and secondary MDS/AML in order to understand whether and how apoptosis contributed to bone marrow failure and how it influenced leukemogenesis. In addition, we were inhibiting apoptosis in hematopoietic cells with the aim to mitigate the hematological phenotype of affected individuals. We hypothesized that inhibition of bone marrow failure, and as a consequence, reduction of compensatory proliferation and selection pressure, was sufficient to protect the hematopoietic system and delay malignant transformation. To investigate these topics, we were using mouse models and analyzing patient-derived samples.
We were analyzing the cell death mechanisms in inherited bone marrow failure syndromes and secondary MDS/AML in order to understand whether and how apoptosis contributed to bone marrow failure and how it influenced leukemogenesis. In addition, we were inhibiting apoptosis in hematopoietic cells with the aim to mitigate the hematological phenotype of affected individuals. We hypothesized that inhibition of bone marrow failure, and as a consequence, reduction of compensatory proliferation and selection pressure, was sufficient to protect the hematopoietic system and delay malignant transformation. To investigate these topics, we were using mouse models and analyzing patient-derived samples.
We successfully generated mouse models for different bone marrow failure syndromes and for syndromes predisposing to leukemia. In addition we were able to analyze primary patient-derived samples.
The most important findings were:
1) For the telomeropathy dyskeratosis congenita (DC), we generated and optimized a mouse model that closely resembled human blood disease. Approx. 45% of mice succumbed due to hematopoietic failure. In line with our hypothesis, we were able to prevent death of half of the animals by inhibiting p53-induced apoptosis by genetic deletion of its pro-apopotic target PUMA (21% vs. 45%). Surviving mice had much better blood counts when Puma was deleted, and telomere attrition was delayed. We thus were able to break the vicious circle and prevent the development of bone marrow failure. Most importantly, our treatment maintained genomic stability and did not increase risk of malignant transformation. In parallel to the mouse experiments performed, we analyzed trephine biopsies from DC patients and found increased levels of p53 and PUMA expression indicating a conserved role of the p53/PUMA axis in murine and human telomeropathies.
2) For the DNA damage repair Fanconi anemia, we decided to work with primary patient material rather than mouse models. Patient samples from different disease stages (bone marrow failure, secondary MDS or AML) could be included and were subjected to RNAseq and WES. We identified few mutations in oncogenes and tumor suprressor genes but significant transcriptomic changes, both in non-malignant and malignant disease stages. While inflammatory signals were strongly increased in the pre-malignant stage (bone marrow failure), these signals were reduced during malignant transformation. The underlying mechanisms still need to be investigated.
3) During the last years, GATA2 deficiency gained strong relevance as predisposition syndrome for MDS and AML. In our ApoptoMDS project, we hypothesized that apoptosis deregulation represents a major pathogenetic driver in GATA2 deficiency. It was thus our aim to manipulate apoptosis signaling (similar as in dyskeratosis congenita). However, we realized that all available mouse models were not appropriate to study these research questions. We therefore first established a mouse model, in which mice first developed bone marrow failure (in 40% of all mice) and then leukemia (in 30% of mice which earlier had developed bone marrow failure). None of the mice that did not develop bone marrow failure showed signs of leukemia which indicates that leukemogenesis is a secondary event after bone marrow failure. We analyzed mechanisms contributing to bone marrow failure and identified increased apoptotic susceptibility as well as reduced proliferation potential of Gata2-haploinsufficient stem and progenitor cells. To understand why some but not all mice succumbed to bone marrow failure, we performed transcriptomic analyses and found two very different transcriptomic programs. Whether this is the cause of the variable outcomes needs to be tested. In addition, we performed WES to identify which mutations contribute to leukemia development. We found especially genes in the NOTCH signaling pathways and DNA damage repair genes to be mutated in Gata2+/- leukemias.
The most important findings were:
1) For the telomeropathy dyskeratosis congenita (DC), we generated and optimized a mouse model that closely resembled human blood disease. Approx. 45% of mice succumbed due to hematopoietic failure. In line with our hypothesis, we were able to prevent death of half of the animals by inhibiting p53-induced apoptosis by genetic deletion of its pro-apopotic target PUMA (21% vs. 45%). Surviving mice had much better blood counts when Puma was deleted, and telomere attrition was delayed. We thus were able to break the vicious circle and prevent the development of bone marrow failure. Most importantly, our treatment maintained genomic stability and did not increase risk of malignant transformation. In parallel to the mouse experiments performed, we analyzed trephine biopsies from DC patients and found increased levels of p53 and PUMA expression indicating a conserved role of the p53/PUMA axis in murine and human telomeropathies.
2) For the DNA damage repair Fanconi anemia, we decided to work with primary patient material rather than mouse models. Patient samples from different disease stages (bone marrow failure, secondary MDS or AML) could be included and were subjected to RNAseq and WES. We identified few mutations in oncogenes and tumor suprressor genes but significant transcriptomic changes, both in non-malignant and malignant disease stages. While inflammatory signals were strongly increased in the pre-malignant stage (bone marrow failure), these signals were reduced during malignant transformation. The underlying mechanisms still need to be investigated.
3) During the last years, GATA2 deficiency gained strong relevance as predisposition syndrome for MDS and AML. In our ApoptoMDS project, we hypothesized that apoptosis deregulation represents a major pathogenetic driver in GATA2 deficiency. It was thus our aim to manipulate apoptosis signaling (similar as in dyskeratosis congenita). However, we realized that all available mouse models were not appropriate to study these research questions. We therefore first established a mouse model, in which mice first developed bone marrow failure (in 40% of all mice) and then leukemia (in 30% of mice which earlier had developed bone marrow failure). None of the mice that did not develop bone marrow failure showed signs of leukemia which indicates that leukemogenesis is a secondary event after bone marrow failure. We analyzed mechanisms contributing to bone marrow failure and identified increased apoptotic susceptibility as well as reduced proliferation potential of Gata2-haploinsufficient stem and progenitor cells. To understand why some but not all mice succumbed to bone marrow failure, we performed transcriptomic analyses and found two very different transcriptomic programs. Whether this is the cause of the variable outcomes needs to be tested. In addition, we performed WES to identify which mutations contribute to leukemia development. We found especially genes in the NOTCH signaling pathways and DNA damage repair genes to be mutated in Gata2+/- leukemias.
"1) Although apoptosis resistance is traditionally regarded a mechanism contributing to malignant transformation, we propose that specifically interfering with p53-initiated apoptosis can be a therapeutic option to delay or prevent hematopoietic failure and possibly secondary leukemia in patients with bone marrow failure syndromes. This was shown on the example of dyskeratosis congenita.
2) The generation of a mouse model reflecting the human syndrome ""GATA2 deficiency"" is very valuable for our research and for collaborations with other researchers. The model will be made available to the community."
2) The generation of a mouse model reflecting the human syndrome ""GATA2 deficiency"" is very valuable for our research and for collaborations with other researchers. The model will be made available to the community."