Analyse von kombinatorischen Leukämogene Effekten von angeborenen ELANE und erworbenen CSF3R/RUNX1 Genmutationen in kongenitale Neutropenie im Humanisierten NSG Maus-Modell
Zusammenfassung der Projektergebnisse
Main conclusions: (1) We established a method of the structural prediction of the damaging effects of CN/AML- associated ELANE mutations, in order to select ELANE mutations with most “damaging” effects for the downstream analyses. (2) We developed an ultra-sensitive deep-sequencing of the CSF3R in CN and CN/AML patients in order to select positions of CSF3R mutations for downstream analysis. (3) We detected an essential role of the gene dosage of mutated RUNX1, depending of the type of mutation: a majority of CN/AML patients with missense RUNX1 mutations acquired additional trisomy 21 with two mutated RUNX1 and one WT RUNX1 alleles. At the same time, patients with truncated RUNX1 mutations had no trisomy 21. This info is essential for the experimental set-up for in vivo analyses. (4) We observed low engraftment of the gene-modified CN patients` HSPCs in NSG mice. Therefore, we performed multiple experimental attempts to improve engraftment: a) we used healthy donor cord blood cells transduced with lentivirus constructs expressing mutated ELANE; b) we developed an efficient method for CRIPSR/Cas9 gene-editing of primary human CD34+ cells with the aim to use this method for the introduction of CSF3R- and RUNX1 mutations in CB CD34+ cells expressing MUT ELANE; c) we further optimized CRISPR/Cas9 gene-editing of CD34+ cells by establishing a method of fluorescent labeling and sorting of CRPSR/Cas9-sgRNA RNP gee-edited cells. (5) First experiments of engraftment of gene-edited CD34+ cells in NSG mice show very promising results, we continue working on it. (6) In parallel to the establishment of the successful engraftment of human CD34+ cells in NSG mice, we established a mouse-mouse model of CN/AML. We used transplantation of the sublethally irradiated mice with CSF3R mutated lin-cells transduced with ELANE and RUNX1 mutation carrying consructs and treated transplanted mice or not with G-CSF. Mice developed AML or CMML within weeks after transplantation. This is the first ever in vivo model of CN/AML with very fast development of leukemia in the recipient mice. (7) We identified marked elevation of the inflammatory and innate immunity signaling pathways downstream of RUNX1 and CSF3R mutations. (8) We further studied a possible mechanism of acquisition of CSF3R and RUNX1 mutations in HSPCs of CN patients and found that HSPCs of CN patients exhibited elevated DNA damage and a strong bias towards lymphoid commitment despite G-CSF treatment. (9) We have successfully established an experimental model of step-wise leukemia development in CN using patients-derived iPSCs. Using iPSC-based model of leukemogenesis we identified deregulated signal transduction pathways along with elevated DNA damage and prolonged DNA repair in HSPCS derived from CN and CN/AML cells. (10) We demonstrated that ELANE Mutations are an ultimate genetic cause of CN.
Projektbezogene Publikationen (Auswahl)
- Severe congenital neutropenias. Nat Rev Dis Primers. 2017;3:17032
Skokowa J, Dale DC, Touw IP, Zeidler C, Welte K
(Siehe online unter https://doi.org/10.1038/nrdp.2017.32) - Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response. Blood Adv. 2019;3:63
Nasri M, Mir P, Dannenmann B, Amend D, Skroblyn T, Xu Y, Schulze-Osthoff K, Klimiankou M, Welte K, Skokowa J
(Siehe online unter https://doi.org/10.1182/bloodadvances.2017015511) - Human iPSC-based model of severe congenital neutropenia reveals elevated UPR and DNA damage in CD34+ cells preceding leukemic transformation. Exp Hematol. 2019;71:51-60
Dannenmann B, Zahabi A, Mir P, Oswald B, Bernhard R, Klimiankou M, Morishima T, Schulze- Osthoff K, Zeidler C, Kanz L, Lachmann N, Moritz T, Welte K, Skokowa J
(Siehe online unter https://doi.org/10.1016/j.exphem.2018.12.006) - Ultra-Sensitive CSF3R Deep Sequencing in Patients With Severe Congenital Neutropenia. Front Immunol. 2019 Feb 28;10:116
Klimiankou M, Uenalan M, Kandabarau S, Nustede R, Steiert I, Mellor-Heineke S, Zeidler C, Skokowa J, Welte K
(Siehe online unter https://doi.org/10.3389/fimmu.2019.00116) - CRISPR/Cas9 Genome Editing of Human-Induced Pluripotent Stem Cells Followed by Granulocytic Differentiation. Methods Mol Biol. 2020;2115:471- 483
Dannenmann B, Nasri M, Welte K, Skokowa J
(Siehe online unter https://doi.org/10.1007/978-1-0716-0290-4_27) - CRISPR/Cas9 mediated ELANE knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients. Haematologica. 2020;105(3):598-609
Nasri M, Ritter M, Mir P, Dannenmann B, Aghaallaei N, Amend D, Makaryan V, Xu Y, Fletcher B, Bernhard R, Steiert I, Hahnel K, Berger J, Koch I, Sailer B, Hipp K, Zeidler C, Klimiankou M, Bajoghli B, Dale DC, Welte K, Skokowa J
(Siehe online unter https://doi.org/10.3324/haematol.2019.221804) - Gene Knockout in Hematopoietic Stem and Progenitor Cells Followed by Granulocytic Differentiation. Methods Mol Biol. 2020;2115:455
Mir P, Ritter M, Welte K, Skokowa J, Klimiankou M
(Siehe online unter https://doi.org/10.1007/978-1-0716-0290-4_26) - New insights into the pathomechanism of cyclic neutropenia. Ann N Y Acad Sci. 2020;1466:83
Mir P, Klimiankou M, Findik B, Hähnel K, Mellor-Heineke S, Zeidler C, Skokowa J, Welte K
(Siehe online unter https://doi.org/10.1111/nyas.14309)
