Periodic Reporting for period 1 - LEUKEYOLK (Uncovering the origin and mechanisms of Down syndrome-associated leukaemia through human induced pluripotent stem cell-derived haemopoiesis)
Période du rapport: 2019-02-01 au 2021-01-31
Childhood leukaemias manifest in the first few years of life, which suggests an embryonic origin of the diseases and a possible contribution of haemopoietic progenitor cells generated during embryonic life in driving these types of cancers. In this context, the Down syndrome-associated transient myeloproliferative disorder (DS-TMD) is a paradigmatic example of perinatal malignancy, affecting around 10% of DS newborns. TMD is a pre-leukaemic condition characterized by the expansion of immature megakaryoblasts; although most cases resolve spontaneously, nearly 20% later progress to full-blown acute megakaryoblastic leukaemia (AMKL). Since both TMD and AMKL share the same somatic mutations in the transcription factor GATA1, AMKL is thought to derive from the first transient disease upon acquisition of additional mutations. Interestingly, the progression from TMD to AMKL occurs only within the first 4 years of life, suggesting an early developmental derivation of the pathology, which is yet to be precisely determined. Moreover, as GATA1 mutations in a disomic background do not cause leukaemia, there must be a strong interplay between GATA1 and trisomy 21, whose mechanisms are currently poorly understood. For this reason, unravelling the molecular bases of this interaction is paramount to the understanding of the disease. The overall goal of this proposal was to investigate the cellular origin and mechanisms of DS-TMD/AMKL. More generally, these findings could be applicable also to other types of childhood leukaemias. Research on cancer and childhood leukaemias is of broad interest for the EC, as shown by the extensive funding of projects on this topic. Hence, our project further nurtures this research area, contributing to increasing European research competitiveness.
As both the developmental origin of haemopoietic progenitors and their genetic lesions must be taken into consideration to accurately recapitulate DS-TMD/AMKL, we used human patient-specific induced pluripotent stem cells (iPSCs) that represent a unique tool for modelling human development and disorders. Indeed, human pluripotent stem cell differentiation systems are an incredibly useful platform to study human developmental biology in vitro; as such, they allow the interrogation of the cellular and molecular mechanisms that regulate the human blood system development. In this project we worked, among other cell types, with iPSCs derived from DS-TMD patients carrying GATA1s mutations that enabled us to model the onset of this disorder and to investigate its aetiology and molecular mechanisms.
The LEUKEYOLK project had two main objectives: 1. dissecting the cellular origin of DS-TMD/AMKL, a haemopoietic malignancy with an in utero origin and 2. characterising the molecular mechanism of GATA1s-induced DS-associated leukaemia.
To investigate the cellular origin of this leukaemia and to explore its molecular mechanism(s), we generated a ‘toolbox’ composed of isogenic iPSCs, namely lines with trisomy 21 or disomy 21 carrying or not the GATA1s mutation. These cells were differentiated into different haemopoietic progenitors following a specific protocol previously developed by the laboratory that allows recapitulating the orchestrated sequence of signalling events occuring in the embryo. We have extensively optimised the protocol modulating inductive signals in order to obtain haemopoietic progenitors belonging to the different developmental programs. Cells were either further differentiated along the megakaryocytic fate (as megakaryoblasts are the cells greatly expanded by DS-TMD/AMKL) or transplanted in vivo to assess their leukaemogenic potential. Overall, the work performed to address the first objective indicated that trisomy 21/disomy 21 cells with or without GATA1s mutation do not differ in the early stages haemopoietic differentiation and that they might not be leukaemogenic in vivo, pointing to the fact that there might be a haemopoietic stem cell-dependent contribution to the disease.
In order to investigate the mechanism(s) of GATA1s-induced DS leukemogenesis, we decided to focus on the potential involvement of epigenetic regulators. We started by investigating the role of lysine-specific histone demethylase 1 (LSD1). We found that this factor is present in a complex with GATA1 in different types of progenitors of the erythroid lineage and that its inhibition phenocopies the erythroid blockage observed in GATA1-mutant trisomy 21 cells. This suggests that the mutated GATA1 might exert its effect at the epigenetic level by blocking LSD1 activity. Besides LSD1, the integration of collaborative work carried out in the laboratory using a different in vitro model of DS-AMKL suggested the involvement of another epigenetic regulator. Specifically, this factor is downregulated in such model and due to its known biological role, we investigated this player as potential candidate responsible for driving part of the dysregulated megakaryocyte phenotype. We indeed found that this gene’s downregulation induces an accumulation of megakaryocytes in culture that can be reversed upon its overexpression. Moreover, we further connected this gene to DS by proving a possible direct link between trisomy 21 and its influence on the expression level of this factor. Lastly, through bioinformatics analyses performed on published patients’ data, we confirmed that DS-AMKL cells show a transcriptional signature compatible with a perturbed epigenetic regulation, thus corroborating our in vitro findings. In conclusion, the extensive work carried out here highlighted two important and previously unappreciated molecular mechanisms that could contribute in different ways to DS-leukaemogenesis in concert with GATA1s mutation.
In order to disseminate the LEUKEYOLK project and its results, the Researcher presented her data at international scientific conferences (such as the International Conference of the Trisomy 21 Research Society and the Lindau Nobel Laureates meeting), where she was able to meet and discuss with prominent scientists of the field. The Researcher participated also to other national and international meetings (the annual ISSCR and the ISEH conferences). Moreover, the Researcher showed and discussed her work at multiple periodical seminars a) within the Researcher’s own Department, b) through two different networks of researchers of various Universities in Milan (Italy) focused on developmental biology and stem cells and c) through online meetings with fellow scientists from external Institutions (such as the Washington University School of Medicine, St. Louis - USA).
The LEUKEYOLK project had two main objectives: 1. dissecting the cellular origin of DS-TMD/AMKL, a haemopoietic malignancy with an in utero origin and 2. characterising the molecular mechanism of GATA1s-induced DS-associated leukaemia.
To investigate the cellular origin of this leukaemia and to explore its molecular mechanism(s), we generated a ‘toolbox’ composed of isogenic iPSCs, namely lines with trisomy 21 or disomy 21 carrying or not the GATA1s mutation. These cells were differentiated into different haemopoietic progenitors following a specific protocol previously developed by the laboratory that allows recapitulating the orchestrated sequence of signalling events occuring in the embryo. We have extensively optimised the protocol modulating inductive signals in order to obtain haemopoietic progenitors belonging to the different developmental programs. Cells were either further differentiated along the megakaryocytic fate (as megakaryoblasts are the cells greatly expanded by DS-TMD/AMKL) or transplanted in vivo to assess their leukaemogenic potential. Overall, the work performed to address the first objective indicated that trisomy 21/disomy 21 cells with or without GATA1s mutation do not differ in the early stages haemopoietic differentiation and that they might not be leukaemogenic in vivo, pointing to the fact that there might be a haemopoietic stem cell-dependent contribution to the disease.
In order to investigate the mechanism(s) of GATA1s-induced DS leukemogenesis, we decided to focus on the potential involvement of epigenetic regulators. We started by investigating the role of lysine-specific histone demethylase 1 (LSD1). We found that this factor is present in a complex with GATA1 in different types of progenitors of the erythroid lineage and that its inhibition phenocopies the erythroid blockage observed in GATA1-mutant trisomy 21 cells. This suggests that the mutated GATA1 might exert its effect at the epigenetic level by blocking LSD1 activity. Besides LSD1, the integration of collaborative work carried out in the laboratory using a different in vitro model of DS-AMKL suggested the involvement of another epigenetic regulator. Specifically, this factor is downregulated in such model and due to its known biological role, we investigated this player as potential candidate responsible for driving part of the dysregulated megakaryocyte phenotype. We indeed found that this gene’s downregulation induces an accumulation of megakaryocytes in culture that can be reversed upon its overexpression. Moreover, we further connected this gene to DS by proving a possible direct link between trisomy 21 and its influence on the expression level of this factor. Lastly, through bioinformatics analyses performed on published patients’ data, we confirmed that DS-AMKL cells show a transcriptional signature compatible with a perturbed epigenetic regulation, thus corroborating our in vitro findings. In conclusion, the extensive work carried out here highlighted two important and previously unappreciated molecular mechanisms that could contribute in different ways to DS-leukaemogenesis in concert with GATA1s mutation.
In order to disseminate the LEUKEYOLK project and its results, the Researcher presented her data at international scientific conferences (such as the International Conference of the Trisomy 21 Research Society and the Lindau Nobel Laureates meeting), where she was able to meet and discuss with prominent scientists of the field. The Researcher participated also to other national and international meetings (the annual ISSCR and the ISEH conferences). Moreover, the Researcher showed and discussed her work at multiple periodical seminars a) within the Researcher’s own Department, b) through two different networks of researchers of various Universities in Milan (Italy) focused on developmental biology and stem cells and c) through online meetings with fellow scientists from external Institutions (such as the Washington University School of Medicine, St. Louis - USA).
Different genes, whose expression is dysregulated in DS, have been linked to the development of the associated TMD/AMKL. Here we add further pieces to this picture by identifying an epigenetic control of gene expression potentially driving the disease-associated phenotype. Moreover, we propose that the overall outcome of this interaction could depend on the developmental origin of the cell where this dysregulation occurs.
This project has critical implications both for health and disease. Indeed, determining the developmental origin and mechanisms of DS-associated leukaemia could help designing new therapies also for other myeloid malignancies in non-DS syndrome patients and could lead to a paradigm shift in the field of haemopoiesis and leukaemogenesis.
This project has critical implications both for health and disease. Indeed, determining the developmental origin and mechanisms of DS-associated leukaemia could help designing new therapies also for other myeloid malignancies in non-DS syndrome patients and could lead to a paradigm shift in the field of haemopoiesis and leukaemogenesis.